METHODS AND PRODUCTS FOR PRODUCTION OF WAX ESTERS (U.S. NATIONAL PHASE)

Abstract

The present invention relates to the provision of genetically modified fungal cells, such as yeast cells with an improved ability for producing different fatty acids and specifically fatty acid ethyl esters (FAEE), the main components of biodiesel. An increased in fatty acid production, and hence in FAEE, is obtained in the first place by expressing different heterologous polypeptides in combination with the down-regulation, attenuation, deletion or over-expression of specially selected genes, wherein said genes encode enzymes involved in the fatty acids synthesizing pathway, fatty acid consuming pathways, carbohydrate biosynthesis pathways or enzyme acting as wax ester transporters or a combination thereof. The methods and products of the invention would allow large-scale production of FAEE with carbohydrates as the only externally-supplied substrate.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the development of genetically engineered microorganisms that can produce wax esters in a controllable and economic fashion. More specifically the invention relates to the production of liquid wax esters that can be used for biofuel, lubricants, cosmetics, linoleum, printing inks as well as products related thereto, and for the production of solid wax esters used for candles and polishes as well as products related thereto.

[0003] 2. Description of the Related Art

[0004] Fossil fuels, such as coal, oil, and natural gas, have been powering modern society for more than one century. However, fresh discoveries of deposits are on the wane and demands are increasing. The world's demand of fossil fuels will soon outweigh the current supply. An innovative approach offering some solution comes from the biotechnology industries. Efforts have made biodiesel as one of the most thoroughly developed and promising alternative fuels on the market. It works well in conventional diesel engines, with less hazardous emissions, and is consumed at greater than 3.5 billion gallons per year.

[0005] Biodiesel is generally composed of fatty acid methyl esters (FAMEs) or fatty acid ethyl esters (FAEE), and is mostly derived from vegetable oil or animal fat by chemically transesterification with methanol or ethanol. Despite the fact that ethanol-yielded FAEEs have better performances, for cost reasons methanol is the reagent most frequently used for triglyceride transesterification. The current process has several drawbacks, including energy intensiveness, consuming edible feedstocks, difficulty of removal of the catalyst from the product and treatment of toxic waste-water, as well as geographical and seasonal restrictions.

[0006] To overcome the problems related to the use of catalysts people have been exploring new alternatives such as enzymatic conversion using lipases (EC 3.1.1.3, triacylglycerol hydrolases). Lipases can break down neutral lipids such as triglycerides and perform a transesterification reaction in a solvent system (i.e. tert-butanol). Enzymatic production of biodiesel can be carried out at moderate reaction conditions and at a lower alcohol to oil ratio. The main drawbacks with this kind of enzymatic catalysis are the strong inactivation effect caused by alcohols (i.e. methanol) and the high enzyme costs.

[0007] Both chemical and enzymatic transesterification require the use of toxic, petrochemically-derived alcohols and expensive feedstocks. Thus, transesterification-based biodiesel becomes unsustainable when fossil fuel derived products are used. As a result, the current feedstocks for biodiesel are mainly derived from plant oils like rapeseed oil.

[0008] However such plant oils are inherently limited by supply of water and land, and subsequently, they cannot produce enough biofuel without threatening food supplies and/or native biodiversity. Algae are a promising choice as an alternative feedstock. Nevertheless, there are problems with surface usage and oil extraction from algae based production. Everyone agrees that fuels derived from biomass are one of the best alternatives to fossil fuels. Thus, genetically manipulation of microorganisms to produce fatty acid esters, will substantially contribute to produce environmentally friendlier, sustainable, and cost-effective biodiesels.

[0009] In this regard, it was previously shown that an engineered E. coli strain expressing the wax synthase (WS) from Acinetobacter baylyi ADP1 and ethanol-production genes from Z. mobilis, could produce fatty acid ethyl esters by esterifying exogenously added fatty acids (Kalscheuer, Stolting et al. 2006). The research is an excellent demonstration of feasibility for microbial production of fatty acid esters. Recently, researchers from the Keasling group and the company LS9 Inc. (South San Francisco, USA) developed this idea further by constructing an engineered E. coli that can produce fatty-acid-derived fuels and chemicals from simple sugars and plant-derived biomass, without the need for fatty acid feeding (Steen, Kang et al. 2010). Production of fatty acid derivatives as biofuels has also been reported in recent patent applications WO2009/009391, WO2007136762 and WO2008119082, all owned by LS9 Inc. Briefly, the metabolically engineered E. coli strain was manipulated to be able to produce fatty (acid) esters and derivatives thereof (short and long chain alcohols, hydrocarbons, fatty alcohols, waxes, etc.) through the introduction of several genes encoding for enzymes such as thioesterase, wax synthase, alcohol acyltransferase, alcohol dehydrogenase, and different kinds of fatty alcohol forming acyl-CoA reductases. In U.S. patent publication 2010/0071259, inventors from the same company teach that by adding a mixture of at least two different alcohols to a medium containing the engineered E. coli strain that produces fatty esters, at least two different fatty esters could be produced.

[0010] The afore-mentioned biodiesel producing methods are all based on the use of the bacterium E. coli. However, E. coli is unable to naturally overproduce the two substrates of biodiesel, fatty acids and alcohol (i.e. ethanol), and this organism is not suitable for large-scale production that often involves harsh environmental conditions. Furthermore, E. coli is sensitive to phage contamination often resulting in substantial economic losses. The patents of the prior art successfully teach several strategies to enhance fatty acids biosynthesis in E. coli. Nevertheless, and apart from the drawbacks associated with the use of this host, it should be noted that strategies working in E. coli might not be appropriate when applied in other microorganisms.

[0011] A far better choice of microbial cell factory for industrial production of biodiesel would be the yeast Saccharomyces cerevisiae. This yeast is already widely used in industry, including for large-scale bioethanol production, but also for a range of specialty chemicals. The development of S. cerevisiae as a cell factory for biodiesel production would represent a major contribution as this could represent a plug and play solution where current infrastructures used for production of bioethanol could be used for production of far more valuable biodiesels. In contrast to the insufficient ethanol productivity of E. coli, S. cerevisiae is already a good ethanol producer.

[0012] In fact, production of FAEEs and fatty acid isoamyl esters (FAIEs) has been achieved in recombinant S. cerevisiae with oleic acid addition by expressing the A. baylyi bifunctional WS/DGAT enzyme (Kalscheuer, Luftmann et al. 2004). A recent patent application, namely US patent application 2009/0117629 by Schmidt-dannert and Holtzapple, also describes a method for the production of esters, including isoprenoid wax esters and fatty acid alkyl esters, such as FAME and FAEE, by heterologous expression of Marinobacter hydrocarbonoclasticus wax synthase (WS2) in S. cerevisiae. The invention is however, limited to the use of specific isolated polynucleotides from Marinobacter hydrocarbonoclasticus, and its application in e.g. producing biodiesel). Moreover this method requires exogenous supply of fatty acids as the endogeneous production of fatty acids by yeast is too low to ensure economically viable production of FAEEs.

[0013] A modified strain carrying the genes encoding the wax synthase from Marinobacter hydrocarbonoclasticus could be considered a potential host for biodiesel production in yeasts. Nonetheless, while this product is very suitable for the particular purpose it addresses, it is not the ideal option when the synthesis of other esters is desired. The knowledge of the preferred substrates for each wax synthase allows the use of yeast cells in applications other than biodiesel production. Moreover there is still a need for methods and products allowing large-scale production of fatty acid esters.

[0014] Thus it is an object of the present invention to provide an improved fungal cell factory, such as a yeast cell factory that can be used for fermentation based production of FAEEs, that is not dependent on the addition of exogenous fatty acids to the yeast culture and that possess an increased flux towards fatty acid biosynthesis and where high level production of FAEEs is obtained.

SUMMARY OF THE INVENTION

[0015] The above presented problems have now been solved by providing a fungal cell system for producing fatty acyl ethyl esters (FAEE), said system comprising a fungal cell and an expression vector encoding at least one wax synthase, wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester transporter(s) of said fungal cell. Such a fungal cell system provides for an increased flux towards fatty acid biosynthesis and thereby a high level production of FAEEs. Examples of fungal cells applicable to the present invention can be selected from Saccharomyces, Saccharomyces cerevisae Hansenula polymorpha, Kluyveromyces, Pichia, Candida albicans, Aspergilli, Rhodotorula rubra, Torulopsis, Trichosporon cutaneum, Trichoderma reesei, Apiofrichum curvafum, Yarrowia lipolytica, and Cryptococcus curvatus.

[0016] Accordingly, a primary object of the present invention is to provide an advance in the microorganism fermentation method for producing wax esters, which include, but is not limited to, the liquid waxes used for biofuel, lubricants, cosmetics, linoleum and printing inks, and the solid waxes used for candles, polishes etc. The fungal cell system and the method disclosed herein combine the expression of different wax synthases with metabolic engineering modifications to ensure a high flux to biosynthesize wax esters. The high flux described herein means at least 2-fold increase in the fatty acids flux compared with flux towards fatty acids in the reference yeast.

[0017] In one embodiment, the invention disclosure provides different nucleotide sequences encoding the polypeptides having wax synthase activity with differences in specificity towards different-chain-length substrates. Examples of different wax synthases applicable within the scope of the present invention are Mycobacterium, Rhodococcus, Acinetobacter, Mus Musculus and/or Marinobacter, such as Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter articus 273-4.

[0018] In another embodiment, the present invention provides a wax ester composition in the different production hosts expressing different wax synthases, wherein the wax ester with preferred carbon chain length could be produced according to the method disclosed herein.

[0019] Accordingly, the present invention also relates to a method for producing FAEE, said method comprising providing a fungal cell system as defined herein in a culture broth, adding one or more sources of carbohydrates as an external substrate to said fungal system, and wherein said FAEE are thereafter retrieved by extraction from said culture broth.

[0020] In particular implementations, the produced wax ester includes fatty acid ethyl esters that can be used as biofuels. In such an example, the only externally supplied substrates are carbohydrates, which can be transformed into ethanol and fatty acids, which can then be combined into esters.

[0021] In yet another embodiment, the invention provides a method of overproducing fatty acids. The microorganism can have ACBP (acyl-CoA-binding protein) over-expressed to deregulate the activity of enzymes involved in lipid metabolism (e.g., acetyl-CoA carboxylase).

[0022] In a further embodiment, the invention disclosure provides a method to overproduce fatty acids, in addition to over-expressing ACBP. The ACBP over-expressing microorganism can have one or more pathway modified, e.g., fatty acids synthesizing pathway, fatty acids consuming pathways, wax ester transporters, and engineering of the central carbon metabolism.

[0023] In a preferred embodiment the present invention provides a Saccharomyces cerevisae yeast cell with increased metabolic flux towards fatty acid ester biosynthesis. This in-house developed host cell expresses at least one (Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 or Psychrobacter articus 273-4) specifically selected wax synthase in combination with an over-expressed ACBP.

[0024] In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of the description and should not be regarded as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows the glycolysis pathway in yeast Saccharomyces cerevisiae for producing two direct precursor substrates (ethanol and acyl-CoA) of wax synthase. The glucose, via glycolysis, could be converted to ethanol (1), and acyl-CoA (2), the precursor for fatty acids.

[0026] FIG. 2 shows the different reactions of the fatty acids consuming pathways.

[0027] FIG. 3 shows a wax ester (e.g. FAEEs) biosynthesis pathway catalyzed by heterologous wax synthase in Saccharomyces cerevisiae. The alcohols could be biosynthesized by the production host or heterologous supplemented. The acyl-CoA could be produced via fatty acids biosynthesis by a production host or supplemented heterologously.

[0028] FIG. 4 shows the vector used for gene expression in the invention herein.

[0029] FIG. 5 shows GC-MS analysis of heptadecanoic acid ethyl ester produced by wax synthase expressing S. cerevisiae CB1 with heptadecanoic acid supplemented. The retention time is 15.53 minutes.

[0030] FIG. 6 shows a GC-MS analysis of standard heptadecanoic acid ethyl ester. The retention time is 15.62 minutes.

[0031] FIG. 7 shows the constructed plasmids for expressing WSs from Acinetobacter baylyi, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter articus 273-4.

[0032] FIG. 8 shows an overview of different metabolic engineering strategies for enhancing fatty acid derivative production in yeast. The heterologous enzymes are shown underlined.

[0033] FIG. 9 shows the outline of the gene deletion method.

[0034] FIG. 10 shows biodiesel production in engineered strains.

[0035] FIG. 11 shows a method for chromosomal integration. The chromosomal integration cassette, obtained by fusion PCR, contains wax synthase controlled by TEF1 or PGK1 and a selectable marker (neo) is delivered to the chromosome. Iterative tandem gene duplication is accomplished by selecting in the plates with higher antibiotics.

[0036] FIG. 12 shows the effect of different promoter and biodiesel production in plasmid or chromosome integration based strains.

[0037] FIG. 13 shows the relationship of the concentration of biodiesel production and the concentration of G418.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

[0038] The invention herein relies, unless otherwise indicated, on the use of conventional techniques of biochemistry, molecular biology, microbiology, cell biology, genomics and recombinant technology.

[0039] To facilitate understanding of the invention, a number of terms are defined below. The term "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems

[0040] The term "overproducing" is used herein in reference to the production of FAEE in a host cell and indicates that the host cell is producing more of the FAEE by virtue of the introduction of nucleic acid sequences encoding different polypeptides involved in the host cell's metabolic pathways or as a result of other modifications as compared with the wild-type or unmodified host cell.

[0041] As used herein, the terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.

[0042] As used herein, an "ACBP or acyl-CoA-binding protein" is a small (10 Kd) protein that binds medium- and long-chain acyl-CoA esters with very high affinity and may function as an intracellular carrier of acyl-CoA esters. The majority of the cellular long-chain acyl-CoA esters are presumed to be sequestered with acyl-CoA binding protein (ACBP).

[0043] Although ACBP occurs as a completely independent protein, intact ACB domains have been identified in a number of large, multifunctional proteins in a variety of eukaryotic species ranging from yeasts and plants to reptiles and mammals. In general ACBP is highly conserved in all eukaryotes. The yeasts homologue of ACBP is known as Acb1p. As used herein, an "Acetyl CoA carboxylase" is a biotin-containing enzyme that catalyzes the irreversible reaction in which acetyl-CoA is carboxylated to malonyl-CoA, see FIGS. 1 and 8, which is the precursor of long-chain fatty acyl-CoA. In mammals, two main isoforms of ACC are expressed, ACC1 and ACC2, which differ in both tissue distribution and function. ACC1 is found in the cytoplasm of all cells and encodes acetyl CoA carboxylase in yeast cells.

[0044] As used herein, FAS or a "fatty acid synthases" is an enzymatic system that catalyzes the initiation and elongation of acyl chains and thus plays a key role in fatty acid synthesis from acetyl-CoA and malonyl-CoA. Examples of these enzymes are AccABCD, FabD, FabH, FabG, FabA, FabZ, Fabl, FabK, FabL, FabM, FabB, and FabF. In the yeast Saccharomyces cerevisae, fatty acids are synthesized by a 2.4 Mba multifunctional enzyme complex with two subunits encoded by two unlinked genes FAS1 and FAS 2.

[0045] As used herein, acyl-CoA synthase includes peptides in enzyme classification number EC 2.3.1.86, and are any of various ligases that catalyze the conversion of a fatty acid to acyl-CoA for subsequent (3-oxidation.

[0046] As used herein, "glyceraldehyde-3-phosphate dehydrogenase" (GAPDH) catalyzes the reversible interconversion between 1,3-bisphosphoglycerate and d-glyceraldehyde 3-phosphate using either NAD(H) or NADP(H) as a coenzyme. This is the sixth step of the glycolysis (FIG. 1) and thus serves to break down glucose for energy and carbon molecules.

[0047] NADPH, a product of the pentose phosphate pathway, functions as a reductant in various synthetic (anabolic) pathways including fatty acid synthesis.

[0048] As used herein, an "acetyl-coenzyme A synthetase" is an enzyme that catalyzes the formation of a new chemical bond between acetate and coenzyme A (CoA), which is a key branching molecule for different metabolic pathways.

[0049] As used herein, "(3-oxidation" is the process by which fatty acids in the form of Acyl-CoA molecules are broken down to generate Acetyl-CoA. It is the principal metabolic pathway responsible for the degradation of fatty acids (FIG. 2).

[0050] As used herein a "catalytic motif" is a three-dimensional structural unit formed by a particular sequence of amino acids, found in proteins and which is often linked with a particular function. For nucleic acids is a particular, usually short, nucleotide sequence that forms a recognition site usually, to which other proteins bind.

[0051] A peptide of the present invention may be present in an expression vector. The term "expression vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention, and which is operably linked to additional nucleotides that ensure its expression. Suitable expression vectors include fungal, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, Acinetobacter baylyi, yeast plasmids and any other vectors specific for the hosts of interest. Vectors may be introduced into a host cell using methods that are known in the art such as, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, etc.

[0052] A "fungal cell system" as disclosed herein comprises a fungal cell which has been modified, such as genetically modified, as described herein, and which expresses at least one wax synthase, as exemplified herein. Said wax synthase is introduced into said fungal cell to provide for expression thereof in said fungal cell. The fungal cell system according to the invention hence provides the combination of a modified fungal cell and the expression of a wax synthase in said fungal cell, which allows for an increased metabolic flux towards fatty acid ester biosynthesis in said fungal cell. This advantageous combination is herein referred to as the "fungal cell system".

[0053] As used herein pESC vectors are a series of epitope-tagging vectors designed for expression and functional analysis of eukaryotic genes in the yeast S. cerevisiae. These vectors contain the GAL1 and GAL10 yeast promoters in opposing orientation. With these vectors one or two cloned genes can be introduced into a yeast host strain under the control of a repressible promoter Preferably the expression vector of the present invention is a pESC-derived plasmid in which the original promoter have been replaced. (S. Partow et al. 2010)

[0054] As used herein a "promoter" is a DNA sequence that usually precedes a gene in a DNA polymer and provides a site for initiation of the transcription into mRNA. In the present invention we used promoters derived from transcriptional Enhancer Factor 1 (TEF1) and phosphoglycerate kinase (PGK1). (S. Partow et al. 2010).

[0055] As used herein, sequence identity refers to sequence similarity between two nucleotide sequence or two peptide or protein sequences. The similarity is determined by sequence alignment to determine the functional, structural, and/or evolutionary relationships between the sequences. Gaps in either or both sequences are permitted in making successive alignment.

[0056] By two nucleotide sequence or two peptide or protein sequences having an amino acid sequence at least, for example 95% identical to a reference amino acid sequence, is intended that the amino acid sequence of e.g. the peptides is identical to the reference sequence, except that the amino acid sequence may include up to 5 point mutations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a peptide having an amino acid sequence at least 95% identical to a reference amino acid sequence: up to 5% of the amino acids in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the amino and/or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.

[0057] In the present invention, a local algorithm program is best suited to determine identity. Local algorithm programs, (such as Smith Waterman) compare a subsequence in one sequence with a subsequence in a second sequence, and find the combination of subsequences and the alignment of those subsequences, which yields the highest overall similarity score. Internal gaps, if allowed, are penalized. Local algorithms work well for comparing two multidomain proteins, which have a single domain or just a binding site in common.

[0058] Methods to determine identity and similarity are codified in publicly available programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al (1994)) BLASTP, BLASTN, and FASTA (Altschul, S. F. et al (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. F. et al, Altschul, S. F. et al (1990)). Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used.

Fatty Acids' Synthesizing Pathway

[0059] The fatty acids synthesizing pathway includes fatty acid synthase enzymes selected from the group consisting of ACC1 (encoding acetyl-CoA carboxylase), FAS1/FAS 2 (encoding fatty acid synthase), and ACS1 (acetyl coenzyme A synthase) from any species to encode such proteins.

[0060] Fatty acids (FA) play an important role as building blocks of biodiesel. In S. cerevisiae, FA is mainly synthesized in cytosol and limits biodiesel production. A fatty acid overproducing yeast cell will in turn overproduce fatty acids derived esters, e.g. FAEES (biodiesel). In this embodiment the inventors herein improve the supply of FAEE precursors. Thus an over-expression of the gene coding to acetyl-CoA carboxylase (ACC1) in combination with an increased expression of fatty acid synthetases (FAS1 and FAS 2) yields an increased amount of Malonyl CoA and Fatty Acids, respectively.

[0061] The sources of malonyl-CoA, are generally supposed to be limited, impeding its utility for overproducing FA. The activity of acetyl-CoA carboxylase is highly regulated in S. cerevisiae: (1) Transcription of ACC1 is repressed by inositol and choline, as UASINO site was found in the promoter of ACC1 (Chirala, Zhong et al. 1994); (2) Acetyl-CoA carboxylase activity could be directly inactivated by Snf1p through phosphorylating (Shirra, Patton-Vogt et al. 2001).

[0062] For releasing the tight regulation of the ACC1 at the mRNA and protein level, the promoter of ACC1 is replaced. Furthermore, the inventors herein have found that under control of the constitutively expressed promoter, a release of ACC1 phosphorylation sites would provide a further increase towards FAEE biosynthetic flux. For example Ser659Ala and Ser1157Ala could be substituted (SEQ ID NO 16). Thus the inactivation by Snf1 could be avoided. The resulting strain with hyperactive Acc1p would enhance the FA biosynthesis significantly.

[0063] As previously stated, in addition to up-regulated activity of acetyl-CoA carboxylase, fatty acid synthase (FAS) could be over-activated to reinforce the push of hyperactive acetyl-CoA carboxylase. Therefore, FASI and FAS2 would be over-expressed in the engineered strain with hyperactive Acc1p. The combined manipulations would lead a high flux towards fatty acids biosynthesis.

[0064] Another preferred modification aimed at increasing the pool of fatty acids is the over-expression of acetyl coenzyme A synthase. In S. cerevisiae, cytosolic acetyl-CoA is produced by decarboxylation of pyruvate to acetaldehyde that is then converted further to acetate and acetyl-CoA (FIG. 1; FIG. 8), which is used for the synthesis of malonyl-CoA and FA biosynthesis. The supply of acetyl-CoA may become a shortage when the FA biosynthesis ability is severely reinforced. The step for biosynthesizing acetyl-CoA is catalyzed by acetyl-coenzyme A synthetase, which is encoded by two genes, ACS1 and ACS2, in S. cerevisiae. Compared to ACS2, ACS1 has been reported to show a considerably higher activity and therefore in this invention, ACS1 has been chosen to be over-expressed.

[0065] The genetically modified yeast cell of the present invention will provide for increased production of FA that is at least 2-fold higher than the amount of the ester produced by a control yeast that is not genetically modified as described herein.

Fatty Acids Consuming Pathways

[0066] Fatty acids are the precursor of Acyl CoA and the production host is engineered to produce fatty acid esters from acyl-CoA and ethanol. That's why it is important to improve the pool of fatty acids by down-regulating the fatty acid-consuming pathways. Most of the fatty acids are stored in the form of neutral lipids such as triacylglycerols (TAG) and steryl esters, which can constitute up to 70% of the total lipid content of the cell. In S. cerevisiae, TAG can be synthesized through two different pathways. As shown in FIG. 8, one is an acyl-CoA-dependent reaction that is catalyzed by acyl-CoA:diacylglycerol acyltransferase (encoded by DGA1 gene); another is phospholipids (PL) dependent reaction that is catalyzed by lecithin:cholesterol acyltransferase (encoded by the LRO1 gene). Steryl esters are formed from sterols through the action of the enzyme acyl-CoA:sterol acyltransferase (ASAT) which is encoded by ARE1 and ARE2 genes in S. cerevisiae.

[0067] Previous studies have shown that the quadruple mutant, S. cerevisiae H1246, in which DGA1, LRO1, ARE1 and ARE2 were disrupted, was no longer capable of producing any TAG or steryl esters and had no apparent growth defects under standard conditions (Sandager, Gustaysson et al. 2002). In stationary phase, the quadruple disrupted strain has a 2.5-fold increase in fatty acids. Using Cre-loxP system, the four genes, DGA1, LRO1, ARE1 and ARE2, were disrupted sequentially. The mutant would decrease or abolish the amount of FA converted to neutral lipid production.

[0068] The stored neutral lipids could be hydrolyzed at any moment to yield fatty acids. The liberated fatty acids and free fatty acids could in turn be oxidized to generate energy by β-oxidation. S. cerevisiae has only one peroxisomal acyl-CoA oxidase, Pox1p, which is regarded as being the main enzymatic step controlling the flux through the β-oxidation. Knocking out the endogenous PDX1 gene to block fatty acid β-oxidation would be beneficial for the accumulation of lipid.

[0069] Suitable modifications allowing this particular embodiment include deletion of the afore-mentioned key genes: DGA1, LRO1, ARE1, ARE2 and PDX1. Thus, a yeast cell with all non-essential fatty acid conversion reactions deleted or attenuated, specifically those related with R-oxidation, synthesis of phospholipids, triacylglycerol and sterol esters, would show a higher production of fatty acids and hence an over-production of FAEE. On the other hand, as reported by several authors a decrease in β-oxidation flux would increase lipid accumulation (Slocombe, Cornah et al. 2009; Steen, Kang et al. 2010).

Carbohydrate Biosynthesis Pathways

[0070] The modified yeast cell with e.g. an enhanced ability to overproduce fatty acids, should need much more NADPH, as two molecules of NADPH are required for each step in the elongation of the growing FA acyl chain (FIG. 1).

[0071] Basically, the availability of intracellular NADPH is enhanced by engineering the production host to express an NADH:NADPH transhydrogenase. The expression of one or more NADH:NADPH transhydrogenases results in an increased conversion of the NADH produced in glycolysis to NADPH. Specifically, the authors herein have designed a novel yeast strain expressing a heterologous NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN, coded by gapN gene) (FIG. 8), for augmenting the production of fatty acid derivatives. Heterologous expression of gapN, from Streptococcus mutants in yeast provides a further push on FA biosynthesis ability, meanwhile it also lead to a higher ethanol yield, which is another precursor for biodiesel (FIG. 8).

Establishment of FAEE Biosynthesis Pathway

[0072] One known method for producing fatty acid esters includes increasing the expression of ester synthases such as wax synthases (EC 2.3.1.75) (FIG. 2). A further increase might be obtained by increasing wax synthase's substrate availability e.g. overproducing fatty acids as suggested above.

[0073] A wild-type yeast cell does not have the metabolic machinery for producing FAEEs from fatty acids. Wax synthases, the enzymes catalyzing these reactions, are characteristics of organisms such as Mycobacterium, Rhodococcus, Acinetobacter, and Marinobacter strains that grow in environments where a carbon source was abundant relative to other nutrients such as phosphorous and nitrogen. The wax synthase sequence usually contains the catalytic motif HHXXXDG, which is reported to be crucial for enzymatic activity.

[0074] Wax synthase activity has never previously been described for yeast. In yeast, it was only shown that polypeptides Eht1p and Eeb1p have medium chain (C4-C8) fatty acid ethyl ester-synthesizing and -degrading activity. However, Kalscheuer et al. (2004) showed for the first time that low wax synthase activity could be detected in wild-type S. cerevisiae G175 using palmitoyl-CoA and 1-Hexadecanol as substrates. But no homologous sequence was detected in yeast. In addition, GC/MS analysis of total lipid extracts from wild-type S. cerevisiae showed that FAEEs were absent, even when the medium is supplied with fatty acids (oleic acid).

[0075] Therefore, the ability to synthesize long chain fatty acid ethyl esters may exist in yeast and it may be generated by the unspecific activity of Eht1p and Eeb1p, but the activity is very poor, which is not enough to form FAEEs (i.e. long chain fatty acid ethyl ester) from fatty acids in a wild-type yeast.

[0076] In a particular embodiment the inventors herein propose the use of a microbial wax ester synthase/acyltransferase (WS/DGAT) from Acinetobacter baylyi ADP1 as this enzyme is known to have the activity for short-chain alcohols and the ability to form FAEEs. It is obtained by expressing the atfA gene. This wax synthase may have sequence similarity with the nucleotide sequence of SEQ ID NO 1 (see attached Sequence Listing). However, the wax synthase from Acinetobacter baylyi ADP1 is a rather unspecific enzyme with broad spectra of possible substrates, and it was in fact bifunctional in vivo, also acting as a diacylglycerol acyltransferase (DGAT).

[0077] Genes with high homologies to the Acinetobacter baylyi ADP1 wax synthase have also been identified in other species. There are three unrelated families of wax synthase found in higher plants, mammals and bacteria. The wax synthase of plants shows no activity for short-chain alcohols.

[0078] Several heterologous wax synthases from other organisms were evaluated (Example 3). As suspected, most wax synthase had the highest activity for acyl-CoAs and alcohols with a chain length from 14 to 18, with a much lower specificity for ethanol. All the detected wax synthase (i.e. wax synthase from Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter articus 273-4) have varied activity for ethanol and could lead to the formation of FAEEs. However the enzyme with highest activity for synthesizing FAEEs was found to be wax synthase from Marinobacter hydrocarbonoclasticus DSM 8798 (named CB2 in the present application). According to the present invention a yeast cell able to efficiently produce FAEEs that could directly be used as biodiesel should express a wax synthase from M. hydrocarbonoclasticus or wax synthase from Psychrobacter articus (Table 3).

[0079] Overexpression of native EEb1gene coding for EeB1p, with the ability to synthesize medium chain fatty acid ethyl ester in combination with expression of selected WSs might lead to favorable results with regard to the synthesis of specific FAEEs. For this purpose, a wax synthase with the best-adapted substrate specificity should be chosen.

[0080] On the other hand, the standardization of methods of molecular evolution or protein fusion could help improving the preference of existing WSs for certain substrates, e.g. using error prone PCR, gene shuffling or more directed protein engineering of the WSs. For example it could lead to the identification of WSs with higher specificity for ethanol. The selection of WSs with high activity for ethanol is of course, of crucial importance for designing an effective biodiesel producer as biodiesel is generally composed of fatty acid ethyl esters (FAEE). Said fatty acids have generally a chain-length from 14 to 20 carbon atoms, within the optimal operating range for acyl-CoAs. A recombinant yeast cell expressing e.g a M. hydrocarbonoclasticus is a good choice for designing a FAEEs producer because of its high preference for ethanol.

[0081] The identified broad spectra of possible substrates of different WSs as shown in Table 3 of the invention herein (see below) allows for many biotechnological applications including but not limited to biodiesel production. Depending on the substrate specificity of the wax synthase (WS) enzymes, various mixtures of ester isomers and chain lengths can be generated. These esters relates to liquid wax esters that can be used for biofuel, lubricants, cosmetics, linoleum, printing inks as well as products related thereto, and solid wax esters used for candles, polishes as well as products related thereto. Another exemplary biotechnological application of wax synthase is spermaceti production. Spermaceti is mainly composed by cetyl palmitate and cetyl myristate, and is widely used in cosmetics, pharmacy and also in candles.

[0082] A wax synthase polypeptide of the present invention may be isolated and obtained from other sources including microorganisms isolated from nature. People skilled in the art know how to screen a genomic or cDNA library for this purpose. Once a polynucleotide sequence encoding a polypeptide has been detected it can be isolated or cloned by utilizing techniques, which are well known to those of ordinary skill in the art.

[0083] Here again we have used plasmid pSP-GM2, derived from pESC, which is a common plasmid with high copy number. The original weaker promoters in pESC were exchanged by two strong promoter TEF1 and PGK1, respectively, to construct pSP-GM2. The high copy number and the strong driven by TEF1 ensures high-level expression of the WS. A polynucleotide encoding a wax synthase polypeptide of the present invention may be present in the yeast cell as a vector or integrated into a chromosome (S. Partow et al.).

Enzyme Acting as Wax Ester Transporters.

[0084] As mentioned herein, the engineered cell expressing a wax synthase would be able to synthesize fatty acid esters e.g. FAEEs. The transfer of esters to the fermentation medium is dependent on their composition. It decreases drastically with increasing chain length, e.g. from 100% for ethyl hexanoate, to 54-68% for ethyl octanoate and 8-17% for ethyl decanoate. A wax ester transporter would facilitate the release of esters to the fermentation medium.

[0085] In one embodiment the invention herein uses a plant wax ester transporter (Pighin, Zheng et al. 2004). For example, Cer5 from Arabidopsis facilitates the export of very long chain aldehydes, ketones, alcohols, alkanes, esters and other possible fatty acids derivatives.

Strain and Polypeptide Characterizations

[0086] The wax synthase activity is an important parameter. It is measured according to previous publications (Kalscheuer et al., 2004). Basically, crude extracts are prepared from S. cerevisiae strains and added into a reaction system containing [1-14C] palmitoyl-CoA and alcohols with specific chain. The test assays are incubated at 35° C. for 30 min, and stopped by extraction with chloroform/methanol. The extracts are separated by TLC.

[0087] Spots corresponding to waxes are scraped from the plates, and radioactivity is measured by scintillation counting.

[0088] The FAEEs, are detected by GC-MS. Briefly, total lipids are first extracted from S. cerevisiae strains, and then run on a TLC plate. Spots corresponding to FAEEs are scraped from the plates, and resolved in chloroform/methanol, which is then measured by GC-MS.

[0089] The genetically modified yeast cells hereby disclosed may be included in a composition further comprising additional components selected from, but not limited to, the group consisting of: buffers; stabilizers; protease-inhibiting agents; hydrolytic enzymes, saccharolytic enzymes; cell membrane- and/or cell wall-preserving compounds, nutritional media appropriate to the cell; and the like.

[0090] For expressing the heterologous sequences, the yeast cells are cultured in a medium supplemented with carbohydrate as the only externally supplied source. Compounds included in this group, but not limited to, are glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose

[0091] In this invention instead of providing the alcohol in the fermentation media as is known in the art e.g. when E. coli is used as biodiesel factory, Applicant has developed a genetically engineered microorganism that can produce wax esters in a controllable and economic fashion without the need of fatty acids or ethanol supplementation. In specific embodiments the carbohydrate concentration in the culture medium is between 20 g/l and 50 g/l. Additional components of the culture media are yeast nitrogen base and CSM-Ura.

[0092] Accordingly, the present invention relates to a fungal cell system for producing fatty acyl ethyl esters (FAEE), said system comprising a fungal cell, and an expression vector encoding at least one wax synthase, wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester transporter(s) of said fungal cell. The invention also relates to a fungal cell which is a yeast cell.

[0093] When herein down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) is referred to, this means that the expression/translation/transcription level of the gene or the gene product has been altered in some manner. The manipulation herein could be achieved by medium supplementation, genetic engineering, or synthetic biology. Regulated genes include genes that could be translated into protein, as well as genes that are transcribed into types of RNA that are not translated into protein. Gene regulation could be made by altering the structural or control region, introducing more copy number, deactivating the corresponding repressor gene or activating the inducible gene, increasing the RNA stability of the gene, and combinations thereof.

[0094] Fatty acid ethyl esters (FAEEs) are esterification products of ethanol and fatty acids. Biodiesel is one kind of mixture of wax esters (FAEEs). The biosynthesis of FAEE is catalyzed by wax ester synthase, also called wax synthase (WS). The chain-length and degree of un-saturation and branching of the fatty acid may vary. Generally, this site of the ester is at least 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length and can be mono-, di-, or tri-unsaturated.

[0095] The present invention provides genetically modified yeast cells that have at least one heterologous polynucleotide encoding a polypeptide involved in a FAEE biosynthesis pathway. The present invention also relates to other genetically modified fungal cells, as exemplified herein, that have at least one heterologous polynucleotide encoding a polypeptide involved in a FAEE biosynthesis pathway.

[0096] A fungal cell used in the context of the present invention can be selected from the group of fungal cells consisting of Saccharomyces, Saccharomyces cerevisae Hansenula polymorpha, Kluyveromyces, Pichia, Candida albicans, Aspergilli, Rhodotorula rubra, Torulopsis, Trichosporon cutaneum, Trichoderma reesei, Apiofrichum curvafum, Yarrowia lipolytica, and Cryptococcus curvatus. An example of a fungal cell that can be used is Saccharomyces cerevisiae CEN.PK113-5D (van Dijken, J. P., et al., 2000). A modification of the metabolism of a fungal cell according to the present invention can be genetic and effectuated by the introduction of one or more exogenous expression vector(s) into said fungal cell. In the context of the invention, said one or more exogenous expression vector(s) can be a plasmid, or another carrier such as exemplified herein. The vector also comprises a structural gene for selection of transformed cells, such as URA3, HIS3.

[0097] In aspects of the invention, said genetic modification of said fungal cell provides for an increased supply of fatty acyls to the metabolism of said fungal cell. Furthermore, said fungal cell can be genetically modified to stimulate overproduction of fatty acids, as further described herein.

[0098] In aspects of the invention, a modification to a fungal cell system as defined herein is performed to any one or more of the following genes, or its expression products: ACB1 (ACBP, acyl-CoA-binding protein), ACC1 (Acetyl-CoA carboxylase), FAS1, FAS2 (Fatty acid synthase), gapN (NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase), ACS1 (Acetyl-CoA synthetase), DGA1 (Acyl-CoA:diacylglycerol acyltransferas), LRO1 (Lecithin: cholesterol acyltransferase), ARE1, ARE2 (Acyl-CoA:sterol acyltransferase), and PDX1 (Peroxisomal acyl-CoA oxidase).

[0099] In other aspects, optionally in combination with other modifications, said modification to said fungal cell is performed by a knockout/deletion of one or more of the genes DGA1, LRO1, ARE1, ARE2, and PDX1.

[0100] According to the invention, a modification to a fungal cell as described herein can also be performed by overexpressing one or more gene product(s) by the introduction of one or more expression vector(s) encoding said one or more gene product(s), said one or more gene product(s) being selected from the group consisting of: acyl-CoA-binding protein, Acetyl-CoA carboxylase (ACC1), NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase, Fatty acid synthases (FAS1, FAS2) and Acetyl-CoA synthetase (ACS1).

[0101] According to the invention, a modification can also provide for an overexpression of ACC1 in combination with an increased expression of FAS1 and FAS2. In some aspects of the invention. the modification of ACC1 is performed by the introduction of an expression vector and an increased expression of FAS1/FAS2 is performed by replacing the promoter thereof (the promoter of FAS1/FAS2). In one aspect of the invention, the ACC1 gene is modified by virtue Ser659Ala and Ser1157Ala of said ACC1 gene being replaced (SEQ ID NO:16).

[0102] The invention also provides for a fungal cell system as defined herein, wherein said wax synthase encoded by said expression vector is heterologous. In this context, a "heterologous" wax synthase refers to a wax synthase originating from a different organism than the fungal cell used in the fungal cell system.

[0103] A fungal cell system as defined herein can comprise a wax synthase obtained from one or more of the species Mycobacterium, Rhodococcus, Acinetobacter, Mus Musculus and/or Marinobacter. Furthermore, more specifically, said at least one wax synthase can be selected from the group consisting of Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter articus 273-4. A gene expressing said wax synthase used herein can be codon optimized and comprise a nucleic acid sequence encoded by any one of SEQ ID NO:1, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and/or SEQ ID NO 7. Also encompassed by the present invention are nucleic acid sequences having at least 80% identity with the presented sequence, such as approximately at least 80, 82, 85, 87, 90, 92, 95, 97, or 99% identity with the presented sequence. A nucleotide sequence disclosed herein can be from a natural species, a mutated version of a naturally occurring wax synthase, or a redesigned enzyme produced by protein engineering, the wax synthases being mutated or redesigned still maintaining their activity when expressed. The present invention also relates to a wax synthase having at least 80%, such as at least 80, 82, 85, 87, 90, 92, 95, 97, or 99% in sequence identity with an amino acid sequence corresponding to any of the wax synthases presented herein.

[0104] A wax synthase of the fungal cell system according to the present invention can be encoded by one or more of the following expression vectors pSP-B1, pSP-B2, pSP-B3, pSP-B4 and/or pSP-B5. These expression vectors are further defined herein, e.g. in the experimental section, and in FIG. 7. The nucleic acid sequences of the prior mentioned vectors are SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, and SEQ ID NO 35. The present invention also relates to expression vectors pSP-B1, pSP-B2, pSP-B3, pSP-B4 and/or pSP-B5, wherein certain parts thereof have been slightly modified or parts have been removed, said expression vectors still retaining their activity, as well as expression vectors comprising any one of the sequences SEQ ID NO:30-35.

[0105] According to the invention, said expression vector encoding said one or more wax synthase(s) can be an episomal plasmid (single copy plasmids) or a high-copy plasmid. A single copy plasmid is defined as a plasmid that exists only as one or a few copies in each host. A high copy plasmid is a plasmid which will provide for a longer expression in the host as it will be present in more copies than the single copy plasmid.

[0106] In the context of the present invention, said expression vector encoding said wax synthase can provide for chromosomal integration into the chromosome of said fungal cell. Such an event is further illustrated in FIGS. 9 and 10 and in the experimental section (Example 7).

[0107] To a fungal cell system as defined herein, carbohydrates can be supplied as an external substrate to said fungal cell system for the production of FAEE. Said carbohydrates can be selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose.

[0108] In some aspects of the invention, additionally either or both of the genes Eht1p and Eeb1p of said fungal cell, are overexpressed by said fungal cell. It was shown that Eht1p and Eeb1p have medium chain fatty acid ethyl ester (including ethyl hexanoate)-synthesizing and -degrading activity (Lilly, M., F. Bauer, M. Lambrechts, J. Swiegers, D. Cozzolino, and I. Pretorius. 2006. The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast 23:641-659.). Eht1 preferred short-chain substrates (highest production was for ethyl butanoate), whereas Eeb1 preferred longer chain substrates (highest production was for ethyl octanoate) (Saerens, S., K. Verstrepen, S. Van Laere, A. Voet, P. Van Dijck, F. Delvaux, and J. Thevelein. 2006. The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. Journal of Biological Chemistry 281:4446.).

[0109] The present invention also relates to the use of an fatty acyl ester, such as a fatty acyl ethyl ester (FAEE), produced by a fungal cell system as defined herein as a component in a biofuel, such as biodiesel, a lubricant, cosmetic, linoleum, printing ink, and/or a solid wax ester used for candles and/or polishes.

[0110] The present invention also relates to a composition comprising a fungal cell system as defined herein, said composition further comprising at least one additional component selected from the group consisting of: buffers; stabilizers; protease-inhibiting agents; hydrolytic enzymes, saccharolytic enzymes; cell membrane- and/or cell wall-preserving compounds, nutritional media appropriate to the cell; and the like.

[0111] The present invention also relates to a method for producing fatty acyl ethyl esters (FAEE), said method comprising:

[0112] a) providing a fungal cell system as defined herein in a culture broth,

[0113] b) adding one or more sources of carbohydrates as an external substrate to said fungal cell system;

[0114] c) and wherein said FAEE are thereafter retrieved by extraction from said culture broth.

[0115] Said carbohydrates can be selected from the group consisting of: glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose. Considering the importance of developing second generation processes based on biomass, it will be a promising advantage how the biofuels can be produced from xylose, cellulose, and hemicellulose, which yeast does not naturally consume.

[0116] The invention also relates to a composition comprising a fungal cell which metabolism is modified thereby possessing an increased flux towards fatty acid biosynthesis; and one or more expression vectors encoding one or more wax synthase(s). Such a fungal cell can be any fungal cell as described herein, i.e. Saccharomyces, Saccharomyces cerevisae Hansenula polymorpha, Kluyveromyces, Pichia, Candida albicans, Aspergilli, Rhodotorula rubra, Torulopsis, Trichosporon cutaneum, Trichoderma reesei, Apiofrichum curvafum, Yarrowia lipolytica, and Cryptococcus curvatus. Furthermore, said wax synthase in such a composition can be selected from Mycobacterium, Rhodococcus, Acinetobacter, Mus Musculus and/or Marinobacter. A modification of such a fungal cell can be performed in any manner exemplified herein, such as by down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester transporter(s) Such a fungal cell can be used for producing biofuel esters, such as biodiesel, lubricants, cosmetics, linoleum and printing inks, and/or the solid waxes used for candles and polishes.

[0117] The present invention also relates to a yeast cell having an increased metabolic flux towards fatty acid ester biosynthesis, said yeast cell expressing at least one wax synthase selected from the group consisting of Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 and Psychrobacter articus 273-4 in combination with over-expressing the protein ACBP (acyl-CoA-binding protein). Said yeast cell can for example be Saccharomyces cerevisae

Preferred Embodiments

[0118] In a preferred embodiment the invention teaches a method for increasing fatty acid production in yeast cells via over-expression of ACBP.

[0119] Saccharomyce cerevisae is the preferred host for carrying out the invention, as it is a popular host in basic and applied research apart from being a good ethanol producer, a precursor of esters and specifically of fatty acid ethyl esters. Nevertheless as previously mentioned herein other fungal cells allowing the present invention are selected from the group consisting of other Saccharomyces species as well as other fungi such as, but not limited to, Hansenula polymorpha, Kluyveromyces, Pichia, Candida albicans, Aspergilli, Rhodotorula rubra, Torulopsis, Trichosporon cutaneum, Trichoderma reesei, Apiofrichum curvafum, Yarrowia lipolytica, Cryptococcus curvatus.

[0120] In S. cerevisae, fatty acids act as a feedback inhibitor of acetyl CoA carboxylase, and also as an inhibitor of fatty acid oxidation in response to increased fatty acid availability. On the other hand we know that the regulatory properties of fatty acids are mediated through their activation to Acyl CoA. This means that in S. cerevisiae, fatty acid biosynthesis is inhibited by its product, the acyl-CoA.

[0121] Acyl CoA binding protein (ACBP) can attenuate the inhibitory effect of Acyl CoA by binding long- and medium-chain acyl-CoA esters with very high affinity. Owing to the high affinity of ACBP for Acyl CoA, the intracellular free Acyl CoA concentration is predicted to be very low. It has been demonstrated that overexpression of Acb1p and bovine ACBP in S. cerevisiae increased the total acyl-CoA pool size. The inventors herein have developed a yeast cell in which ACBP (acyl CoA binding protein) is over-expressed so that to down-regulate the activity of enzymes involved in the lipid metabolism and in this specific case for deregulating acetyl-CoA carboxylase. Increased ACBP expression is translated in low free Acyl CoA levels and more fatty acid availability.

[0122] As previously explained, the present invention adopted a pESC derived plasmid as the expression vector. The plasmid pSP-GM2 shown in FIG. 4, can express two genes simultaneously. In this specific modification, Acb1 is also ligated to plasmid pSP-GM2 and under control of promoter PGK1 (S. Partow et al., 2010).

[0123] Over-expressing ACBP as used herein means altering the rate of transcription, Post-transcription, or translation of the gene encoding the protein as compared with the same rates in the yeast cell without modification.

[0124] Methods of testing for over-expression are well known in the art, for example transcribed RNA levels can be assessed using rtPCR, and protein levels can be assessed using SDS page gel analysis.

[0125] In a further preferred embodiment the invention teaches a system and a method in which a further increase in fatty acid production is obtained by down-regulating, attenuating, deleting or over-expressing additional genes encoding enzymes involved in the fatty acids synthesizing pathway, fatty acid consuming pathways, carbohydrate biosynthesis pathways or enzyme acting as wax ester transporters or a combination thereof. In this regard the genetically modified yeast cell of the present invention may include other modifications in addition to an over-expressed ACBP, but preferably it should contain genes encoding a combination of different wax synthases with high specificity for short-chain alcohols.

[0126] Basically, the genetic modifications may increase the level of enzymes involved in the different biosynthetic pathways, reduce feedback inhibition at different locations in the biosynthesis pathways, affect the availability of different substrates and cofactors used in said pathways, affect expression of genes coding those enzymes, etc.

[0127] Polypeptides according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it is possible to use recombinant techniques to express the genes in the selected suitable host.

EXPERIMENTAL SECTION

Example 1

Construction of Biodiesel Production Host Saccharomyces cerevisiae CB1

[0128] In this experiment the wax ester synthase from A. baylyi ADP1 was expressed in a laboratory strain Saccharomyces cerevisiae CEN.PK113-5D (MAT-alpha ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2) to create biodiesel producer, Saccharomyces cerevisiae CB1.

[0129] Briefly, cloning and DNA manipulations were all carried out in E .coli DH5α and were performed by standard procedures (Sambrook and Russell 2001). The sequence of the gene atfA with the reported wax synthase from Acinetobacter baylyi ADP1 was optimized for expression in a yeast host. The optimized sequence is given as SEQ ID NO 1, which was based on the published gene sequence (Gene bank accession no. AF529086). It was synthesized and provided by the DNA2.0 Company (Menlo Park, Calif.). SEQ ID NO 1 was amplified using the following oligonucleotides: 5'-CGGGATCCCGCTCGAGATGCGTCCATT-3' (SEQ ID NO 2) introducing BamHI restriction site (underlined) and; 5'-GGGGTACCCCAAGCTTGGGTTAGTTTGCAG-3' (SEQ ID NO 3) introducing HindIII restriction site (underlined). The BamHI/HindIII digested DNA sequence was ligated into vector pSP-GM2 (FIG. 4) and under control of the constitutively expressed promoter TEF1, which gave plasmid pSP-B1 (FIG. 7A). The cloned sequences were verified by sequencing. The plasmids pSP-GM2 and pSP-B1 were transformed into S. cerevisiae CEN.PK113-5D. The resulting strains were named S. cerevisiae CB0 and S. cerevisiae CB1, respectively. Synthetic minimal dropout (SD) medium lacking uracil was used to select for transformants.

Example 2

Characteristics of the Recombinant Host

[0130] The inoculated transformants S. cerevisiae CB0 and S. cerevisiae CB1 were cultured to late exponential growth period in 100 mL SD medium lacking uracil and containing 2% (w/v) glucose at 30° C. The cultures were then harvested. Cell-free extracts were prepared using a previously reported fast prep method for enzyme analysis (Hou, Vemuri et al. 2009). The lipid analysis were extracted from the lyophilized cell pellets using the reported method (Gu, Valianpour et al. 2004).

[0131] The wax synthase activities in the transformants were testified in vitro using [1-14C] palmitoyl-CoA and 1-hexadecanol or ethanol as the substrates. Table 1 summarizes the results of enzyme analysis. A low wax synthase activity could be detected in negative control S. cerevisiae CB0 using 1-hexadecanol or ethanol as the substrates. In contrast, a significant high wax synthase activity was detected in S. cerevisiae CB1.

[0132] The lipid extraction was analyzed with Gas Chromatography/Mass spectroscopy (GC/MS). No FAEEs were detected in the negative control S. cerevisiae CB0 even when the cultured medium was supplemented with 0.1% (w/v) free fatty acids, heptadecanoic acid. In contrast, S. cerevisiae CB1 could produced FAEEs to a titer of 5.0 mg/L. The heptadecanoic acid ethyl ester was produced by S. cerevisiae CB1 when the cultured medium was supplemented with 0.1% (w/v) free fatty acids, heptadecanoic acid (C17), which doesn't synthesized by yeast itself. Taking heptadecanoic acid ethyl ester as an example for the GC/MS results, it eluted at around 15.6 min, and the parent ion mass spectrum of m/z 298 was clearly observed (FIG. 5). Additionally, structural confirmation was received by daughter ion scans of m/z 298 (FIG. 5). The spectrums are the same as in standard heptadecanoic acid ethyl ester (FIG. 6).

TABLE-US-00001 TABLE 1 WS activities in crude extracts of different recombinant S. cerevisiae Wax synthase activity^a (pmol [mg cell extract min^-1]) With palmitoyl-CoA and Strain hexadecanol With palmitoyl-CoA and ethanol CB0 0.9 ± 0.2 0.67 ± 0.15 CB1 41.6 ± 2.21 4.9 ± 0.55 ^aData are mean values of two independent experiments ± SD.

Example 3

Evaluation of the Substrate Preference of Different WSs in Yeast

TABLE-US-00002

[0133] TABLE 2 Specific oligonucleotides used for PCR amplification of the synthesized WS sequences Primer Sequence 5'→3' Upstream Downstream WS from CGGGATCCCGCTC GGGGTACCCCAAGCTTG Marinobacter GAGATGAAGAGATT GGTTACTTTCTAGTACG hydrocarbo- AGG (SEQ ID NO 9) noclasticus (SEQ ID NO 8) DSM 8798 WS from CGGGATCCCGCTC GGGGTACCCCAAGCTTG Rhodococcus GAGTTGACCGACG GGTTAGCTAGCCACCACC opacus PD630 TGATTAC (SEQ ID NO 11) (SEQ ID NO 10) WS from Mus CGGGATCCCGCTC GGGGTACCCCAAGCTTG musculus GAGATGTTCTGGCC GGTTAAACAATGACCAAC C57BL/6 AACC (SEQ ID NO 13) (SEQ ID NO 12) WS from CGGGATCCCGCTC GGGGTACCCCAAGCTT Psychrobacter GAGATGAGATTACT GGGTTAAGGGGCCAACT articus 273-4 GACCGCTGT (SEQ ID NO 15) (SEQ ID NO 14)

[0134] In this example, except for the wax synthase from Acinetobacter baylyi ADP1, four other putative WSs from Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter articus 273-4 were optimized for expression in a yeast host. The optimized sequences could be seen in SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7. Then they were synthesized by the DNA2.0 Company (Menlo Park, Calif.). These synthesized sequences were PCR amplified by using specific oligonucleotides introducing BamHI and HindIII restriction sites (Table 2 above). The BamHI/HindIII digested DNA sequences were ligated into pSP-GM2 (FIG. 4), respectively, and under control of the constitutively expressed promoter TEF1, which resulted in the plasmids pSP-B2, pSP-B3, pSP-B4 and pSP-B5 (FIG. 7B, 7C, 7D, 7E). Cloned sequences were verified by sequencing. The plasmids pSP-B2, pSP-B3, pSP-B4 and pSP-B5 were transformed into S. cerevisiae CEN.PK113-5D to construct S. cerevisiae CB2, CB3, CB4 and CB5, respectively. Synthetic minimal dropout (SD) medium lacking uracil was used to select for transformants.

[0135] The cell-free extracts from the constructed recombinant S. cerevisiae CB1, CB2, CB3, CB4 and CB5 were prepared as the method described in Example 2. The wax synthase activities in the transformants were testified in vitro using alcohols with various chain lengths as substrates. Table 3 summarizes the results of enzyme analysis.

[0136] The substrate profiles in Table 3 show that CB2 and CB5 catalyzed ethanol with a higher activity, which could reduce the formation of byproducts and drive the carbon flux toward the target ethyl esters. Actually, CB2 and CB5 could produce FAEEs at yield of 6.3 mg/L and 2.3 mg/L, which are clearly higher than other wax synthase expressing yeast. Moreover CB2 catalyzed cetyl alcohol (1-Hexadecanol) with a higher activity, which is the choice for constructing the spermaceti producing yeast. Our findings clearly show that the substrate preferences of the different WSs are the instructions for producing certain wax esters.

TABLE-US-00003 TABLE 3 Acyl acceptor specificities with different alcohols in crude extracts of different recombinant S. cerevisiae Wax synthase activity^a (pmol [mg cell extract min^-1]) Acyl acceptor CB1 CB2 CB3 CB4 CB5 Ethanol 4.6 ± 0.55 8.1 ± 1.87 2.7 ± 0.37 3.8 ± 0.51 5.9 ± 0.83 Butanol 10.8 ± 1.60 14.6 ± 1.75 6.8 ± 0.82 3.5 ± 0.53 4.2 ± 0.46 1-Hexanol 17.3 ± 2.04 33.8 ± 3.77 16.1 ± 2.29 10.2 ± 1.59 18.7 ± 2.19 1-Octanol 23.0 ± 2.39 45.7 ± 4.51 32.3 ± 3.84 22.3 ± 2.44 17.7 ± 1.67 1-Decanol 19.7 ± 3.11 41.1 ± 4.13 37.3 ± 3.90 33.5 ± 2.22 27.5 ± 2.50 1-Dodecanol 31.8 ± 3.48 48.4 ± 4.56 36.7 ± 3.78 44.2 ± 3.07 42.8 ± 3.11 1-Tetradecanol 45.0 ± 4.72 49.7 ± 4.38 33.5 ± 3.66 35.1 ± 2.87 36.5 ± 3.03 1-Hexadecanol 41.6 ± 2.21 49.0 ± 3.65 28.9 ± 3.29 35.5 ± 2.91 39.1 ± 2.72 ^aData are mean values of two independent experiments ± SD.

Example 4

Metabolic Engineering Strategy for Enhancing Fatty Acid Biosynthesis-Expression of Heterologous NADP+ Dependent Glyceraldehyde-3-Phosphate Dehydrogenase

[0137] In this invention supplying more NADPH is taken as an example to illustrate the metabolic engineering strategy for enhancing fatty acid biosynthesis. To make more NADPH, the heterologous expression of NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase (gapN, from Streptococcus mutants) is used. The heterologous reaction is listed in FIG. 8.

[0138] The sequence of gapN (from Streptococcus mutants) was optimized for expression in a yeast host (SEQ ID NO 17) and was synthesized by DNA2.0 Company (Menlo Park, Calif.). The synthesized sequence was PCR amplified by using specific oligonucleotides: 5'-AAACAA GCGGCCGCACTAGTTTGACAAAAC-3' (SEQ ID NO 18) introducing NotI restriction site (underlined) and 5'-TTAATTAAGAGCTCAGATCTTTATTTGATATCAA-3'(SEQ ID NO 19) introducing SacI restriction site (underlined). The NotI/SacI digested DNA sequences were ligated into pSP-GM2, and transformed into host S. cerevisiae strain.

Example 5

Summary of Modifications Useful for Making Yeast with Increased FA Supply for Producing Wax Esters

[0139] Table 4. is a summary of modifications to construct engineered yeast cells that can efficiently biosynthesis FA for producing wax esters. The modifications can be combined together.

TABLE-US-00004 Enzyme Sources Gene Wax synthase Acinetobacter baylyi ADP1 atfA Marinobacter WS2 hydrocarbonoclasticus DSM 8798 Rhodococcus opacus PD630 atf1 Mus musculus C57BL/6 AY611031 and AY611032 Psychrobacter articus YP_263530 ACBP (acyl-CoA- Saccharomyces cerevisiae Acb1 binding protein) CEN.PK113-5D Acetyl-CoA carboxylase Saccharomyces cerevisiae Desensitized CEN.PK113-5D ACC1 Fatty acid synthase Saccharomyces cerevisiae FAS1, FAS2 CEN.PK113-5D NADP⁺dependent Streptococcus mutants gapN glyceraldehyde-3- phosphate dehydrogenase Acetyl-CoA synthetase Saccharomyces cerevisiae ACS1 CEN.PK113 Acyl-CoA:diacylglycerol Saccharomyces cerevisiae DGA1 acyltransferase CEN.PK113 Lecithin:cholesterol Saccharomyces cerevisiae LRO1 acyltransferase CEN.PK113 Acyl-CoA:sterol Saccharomyces cerevisiae ARE1, ARE2 acyltransferase CEN.PK113 Peroxisomal Saccharomyces cerevisiae POX1 acyl-CoA oxidase CEN.PK113

Example 6

Fermentation

[0140] After combination of the above engineering strategies, the engineered host yeast holds the ability with increased flux towards FA biosynthesis. After combined wax synthase expression, it produces wax ester without the need for addition of exogenous fatty acids to the culture. In such an example, the engineered wax synthase expressing S. cerevisiae with an increased flux towards FA biosynthesis allow for high level production of biodiesel (FAEEs) from the only externally supplied substrate, carbohydrates. For large-scale biodiesel production, the engineered S. cerevisiae is cultured in 5 L fermentor. Glucose is continuously fed into the medium, in which maintained a high ratio of C/N. Meanwhile, dodecane (10%, v/v) was overlayed the medium to potentially prevent FAEEs evaporation and facilitate in-situ product capture.

Example 7

Plasmids Construction for Evaluation of Five Wax Ester Synthases on FAEE Production

[0141] Briefly, cloning and DNA manipulations were all carried out in E .coli DH5α and were performed by standard procedures (Sambrook and Russell 2001). The five sequences of the wax synthase from different species were optimized for expression in a yeast host. Then they were synthesized and provided by the DNA2.0 Company (Menlo Park, Calif.). These five different sequences were amplified using the ligonucleotides primers, respectively (table 2). The five BamHI/HindIII digested DNA sequences were, respectively, ligated into vector pSP-GM2 and under control of the constitutively expressed promoter TEF1, which gave five different plasmids. These plasmids were transformed into Saccharomyces cerevisiae CEN.PK113-5D (MAT-alpha ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2) to create five biodiesel producers. The method for yeast transformation is the standard LiAc/SS Carrier DNA/PEG method (Xiao 2006). Synthetic minimal dropout (SD) medium lacking uracil was used to select for transformants.

Gene Deletions:

[0142] Shown in FIG. 9, the five genes (DGA1, LRO1, ARE1, ARE2, PDX1) were deleted subsequently in Saccharomyces cerevisiae CEN.PK113-5D using the loop-out method with the help of loxP-KanMX-IoxP cassette (Xiao 2006).

General Description and Method for the Chromosomal Integration

[0143] Wax synthase from Marinobacter hydrocarbonoclasticus DSM 8798 is suggested to have the highest activity for biodiesel production and chosen as the working enzyme. In the deletion strains, the related genes were introduced and constructed the following strains. The ability of biodiesel production was shown in FIG. 10, and the genotypes of strains were listed in Table 5. The overexpressed ACC1 were released its phosphorylation sites (Ser659Ala and Ser1157Ala), as shown in SEQ ID NO 16.

TABLE-US-00005 TABLE 5 List of strains and their genotypes. Strain Genetype or relevant Characteristics SJ03 ΔDGA1, ΔLRO1, ΔARE1, ΔARE2, with wax synthase (Marinobacter) overexpressed from plasmid pSP-GM2 SJ04 ΔDGA1, ΔLRO1, ΔARE1, ΔARE2, with wax synthase (Marinobacter) and Acetyl-CoA carboxylase overexpressed from plasmid pSP-GM2 SJ05 ΔPOX1, with wax synthase (Marinobacter) overexpressed from plasmid pSP-GM2 SJ06 ΔPOX1, with wax synthase (Marinobacter) and Acetyl-CoA carboxylase overexpressed from plasmid pSP-GM2 SJ07 ΔDGA1, ΔLRO1, ΔARE1, ΔARE2, ΔPOX1, with wax synthase (Marinobacter) overexpressed from plasmid pSP-GM2 SJ08 ΔDGA1, ΔLRO1, ΔARE1, ΔARE2, ΔPOX1, with wax synthase (Marinobacter) and Acetyl-CoA carboxylase overexpressed from plasmid pSP-GM2

Example 8

[0144] Although plasmids based methods have been used for biodiesel production, the plasmid is not genetic stable, which contributed the loss in productivity. In this work, we developed a plasmid-free method with high genetic stability and high gene copy expression for biodiesel production. Shown in FIG. 11, the wax synthase and bacterial neo gene (Neo, G418 resistance gene) were fused together, and integrated into delta sequence of chromosome by yeast transformation. The copies of delta sequence occur in multiple places throughout the yeast genome, and, under the selection of increasing concentration of G418, clones with multiple copies of the inserted gene can be generated. Finally, pathway copy number is stabilized by RAD52 knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities.

[0145] The WS (wax synthase) from Marinobacter hydrocarbonoclasticus DSM 8798 were evaluated under control of two different strong promoters, TEF1 and PGK1. Amplified by primer 1 and primer 2 (Table 6), the BamHI/HindIII digested WS sequence was ligated into vector pSP-GM2 and under control of the constitutively expressed promoter TEF1, which gave plasmid pSP-B2. Using plasmid pSP-B2 as the template, the WS sequence with TEF1 promoter and CYC1 terminator could be amplified with primer 3 and primer 4. The neo gene was amplified from plasmid pJEF1105 (Wang, Wang et al. 1996) with primer 5 and primer 6. Shown in FIG. 11, the 5' end of primer 3 and primer 6 are homolog to the delta sequence, which would facilitate the integration; the 5' end of primer 4 is homolog to neo gene and the 5' end of primer 5 is homolog to CYC1 terminator, which would facilitate sequence fusion. The two DNA sequences, TEF1 controlled WS (PCR product 1, FIG. 11) and neo gene (PCR product 2, FIG. 11), could be fused together as one by PCR amplification taken these two sequences as the template and primer 3 and 6 as the PCR primers. The fused DNA fragment (PCR product 3, FIG. 11) be transformed into yeast and selected on the plats with G418 concentration. Similarly, DNA fragment that contained PGK1 controlled WS and neo gene was also constructed and integrated into yeast.

[0146] Shown in FIG. 12, the initial results suggests PGK1 controlled WS have a higher productivity and chosen as the choice for selection on the plates with increasing G418 concentration. The colonies selected from the plate with higher concentration of G418 should contain higher copy number of WS, and contribute to a higher biodiesel production. FIG. 13 shows the relationship of the concentration of biodiesel production and the concentration of G418. Yield increased remarkably as more G418 was used in the chromosomal evolution until yield stopped increase when the supply of precursors limited the function of wax synthase. The chromosome integration constructed a stable pathway and the production is comparable or higher than those achievable using multicopy plasmids.

TABLE-US-00006 TABLE 6 primers list Primer Sequence 5'→3' Primer 1 CGGGATCCCGCTCGAGATGAAGAGATTAGG (SEQ ID NO: 20) Primer 2 GGGGTACCCCAAGCTTGGGTTACTTTCTAGTACG (SEQ ID NO: 21) Primer 3 GTTGGGATTCCATTGTTGATAAAGGCGcacacaccatagcttcaaaatgtttc (SEQ ID NO: 22) Primer 4 GTGCAATGTAgatcttcgagcgtcccaaaacc (SEQ ID NO: 23) Primer 5 GacgctcgaagatcTACATTGCACAAGATAAAAATATATCATCATGAACAAT (SEQ ID NO: 24) Primer 6 GCCTTTATCAACAATGGAATCCCAACCGCCGTCCCGTCAAGTC (SEQ ID NO: 25) Primer 7 ACAACAAATATAAAACAAGCGGCCGCACTATGAAGAGATTAGGTACT C (SEQ ID NO: 26) Primer 8 GGCGAAGAATTGTTAATTAAGAGCTCGGTACCCCAAGCTTGGGTTA (SEQ ID NO: 27) Primer 9 GTTGGGATTCCATTGTTGATAAAGGCGGAAGTACCTTCAAAGAATGG GGTC (SEQ ID NO: 28) Primer 10 CTTGTGCAATGTAGAGCGACCTCATGCTATACCTGAG (SEQ ID NO: 29) Primer 11 ATGAGGTCGCTCTACATTGCACAAGATAAAAATATATCATCATGAAC (SEQ ID NO: 30)

Analysis:

[0147] The inoculated transformants of S. cerevisiae were cultured to late exponential growth period in 100 mL SD medium lacking uracil and containing 2% (w/v) glucose at 30° C. The cultures were then harvested. Cell-free extracts were prepared using a previously reported fast prep method for enzyme analysis (Hou, Vemuri et al. 2009). The wax synthase activities in the transformants were testified in vitro using [1-14C] palmitoyl-CoA and 1-hexadecanol or ethanol as the substrates (Kalscheuer, Luftmann et al. 2004). ACCase (Acetyl-CoA carboxylase) activity was measured under a fume hood as the incorporation of radioactivity from NaH₁₄CO₃ into an acid-stable product, as described previously (Diacovich, Peir et al. 2002). The total lipid were extracted from the lyophilized cell pellets using the reported method (Gu, Valianpour et al. 2004). The putative FAEEs in the total lipid were purified by preparative TLC and detected by GC-MS (Kalscheuer, Luftmann et al. 2004).

REFERENCES

[0148] Chirala, S. S., Q. Zhong, et al. (1994). "Analysis of FAS3/ACC regulatory region of Saccharomyces cerevisiae: identification of a functional UASINO and sequences responsible for fatty acid mediated repression." Nucl. Acids Res. 22(3): 412-418.

[0149] Gu, Z., F. Valianpour, et al. (2004). "Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome." Molecular Microbiology 51(1): 149-158.

[0150] Kalscheuer, R., H. Luftmann, et al. (2004). "Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase." Appl. Environ. Microbiol. 70(12): 7119-7125.

[0151] Kalscheuer, R., T. Stolting, et al. (2006). "Microdiesel: Escherichia coli engineered for fuel production." Microbiology 152(9): 2529-2536.

[0152] Sandager, L., M. Gustaysson, et al. (2002). "Storage lipid synthesis is non-essential in yeast." Journal of Biological Chemistry 277(8): 6478.

[0153] Shirra, M. K., J. Patton-Vogt, et al. (2001). "Inhibition of Acetyl Coenzyme A Carboxylase Activity Restores Expression of the INO1 Gene in a snf1 Mutant Strain of Saccharomyces cerevisiae." Mol. Cell. Biol. 21(17): 5710-5722.

[0154] Slocombe, S. P., J. Cornah, et al. (2009). "Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways." Plant Biotechnology Journal 7(7): 694-703.

[0155] Steen, E., Y. Kang, et al. (2010). "Microbial production of fatty-acid-derived fuels and chemicals from plant biomass." Nature 463(7280): 559-562.

[0156] Pighin, J. A., H. Zheng, et al. (2004). "Plant Cuticular Lipid Export Requires an ABC Transporter." Science 306(5696): 702-704.

[0157] Diacovich, L., S. Peir, et al. (2002). "Kinetic and structural analysis of a new group of acyl-CoA carboxylases found in Streptomyces coelicolor A3 (2)." Journal of Biological Chemistry 277(34): 31228.

[0158] Gu, Z., F. Valianpour, et al. (2004). "Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome." Molecular Microbiology 51(1): 149-158.

[0159] Hou, J., G. Vemuri, et al. (2009). "Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae." Applied microbiology and biotechnology 82(5): 909-919.

[0160] Kalscheuer, R., H. Luftmann, et al. (2004). "Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase." Appl. Environ. Microbiol. 70(12): 7119-7125.

[0161] Sambrook, J. and D. W. Russell (2001). "Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.".

[0162] Wang, X., Z. Wang, et al. (1996). "G418 selection and stability of cloned genes integrated at chromosomal delta sequences of Saccharomyces cerevisiae." Biotechnology and Bioengineering 49(1): 45-51.

[0163] Xiao, W. (2006). Yeast Protocols, Humana Press, Totowa, N.J. S. Partow; V. Siewers; S. Bjorn; J. Nielsen; J. Maury (2010) Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast 27:955-964

[0164] van Dijken, J. P., et al., 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26, 706-714.

Sequence CWU 1

1

3511377DNAArtificial sequenceArtificial Sequence, optimized from wax synthase of Acinetobacter baylyi ADP1 1atgcgtccat tacatccaat tgatttcatc ttcctatctt tagaaaagag acagcaacca 60atgcatgtag gcgggctttt tctcttccaa attcctgaca atgctccaga cacattcatc 120caagacttgg ttaatgatat cagaatttcc aagtcaatac ctgttccacc tttcaataac 180aaactcaacg gcttgttctg ggacgaggat gaagagttcg atctggatca tcactttcga 240catatcgcat taccacaccc tggtagaatc agggagttac tcatctacat ttcccaagag 300cattcaacat tacttgatag agccaagcca ttgtggacat gcaacataat cgaaggtata 360gaaggcaata gatttgccat gtacttcaag atacatcacg ctatggttga tggagtcgct 420ggaatgagac taatcgagaa gagcctttct cacgatgtta cagaaaagtc aattgtacca 480ccttggtgtg tagaaggtaa acgagctaag cgtttacgtg aaccaaaaac cggaaagatc 540aaaaagatta tgtctggtat caaatctcaa cttcaagcca cgcctactgt cattcaagag 600ttgtctcaaa cagtgttcaa agacattggt agaaacccag atcacgtgtc ctcatttcaa 660gcgccatgtt ctattctgaa ccaaagagta tccagtagta gaagatttgc agcacagtct 720tttgatcttg ataggttcag aaacattgca aagtctctga acgtcaccat aaacgacgtg 780gttctagctg tttgctctgg ggcactgaga gcttatctaa tgtcacataa cagcttgcca 840tcaaaaccat tgattgcgat ggttcctgcc tctatacgta atgatgattc agatgtcagt 900aacagaataa caatgatcct tgccaaccta gctactcata aggatgatcc tttgcagaga 960ttagaaatca ttagaagatc agtgcaaaac tcaaagcaga gattcaaaag gatgaccagt 1020gatcaaatct tgaattactc tgcagtggta tacggtccag ctggtctgaa tatcatatca 1080ggaatgatgc caaaaagaca agcctttaac ttagttatct ccaatgtacc tggtccacga 1140gaacctctct actggaacgg agctaagttg gatgcacttt acccagcctc tatcgtttta 1200gatggtcagg ctttgaacat tacaatgact agttatctag acaagctaga agttgggttg 1260attgcgtgta gaaatgccct acctagaatg cagaatttgc tgactcactt agaagaggag 1320attcaactct ttgaaggcgt catcgcaaaa caagaggata tcaaaactgc aaactaa 1377227DNAArtificial sequenceprimer 2cgggatcccg ctcgagatgc gtccatt 27330DNAArtificial sequenceprimer 3ggggtacccc aagcttgggt tagtttgcag 3041422DNAArtificial sequenceArtificial Sequence, optimized from wax synthase of Marinobacter hydrocarbonoclasticus DSM 8798 4atgaagagat taggtactct agacgctagt tggcttgcag tcgaatccga agatacgcca 60atgcacgtgg gcactctcca aatcttctca ttaccagaag gtgctccaga gacatttcta 120cgtgatatgg ttacaaggat gaaagaggca ggagatgttg ccccaccatg gggttacaag 180ctcgcatggt ccggtttcct tggcagggtt attgctcctg cctggaaggt agacaaagat 240atcgatttgg attatcatgt ccgacatagt gcattgccaa gaccaggtgg tgaaagagag 300ctagggatac ttgtttctag attacactcc aaccctttag atttctctag accactatgg 360gaatgccatg tcattgaagg tcttgaaaac aacagatttg cactgtatac taagatgcat 420cactctatga ttgatgggat atctggagta agattgatgc aaagagtatt gaccactgac 480ccagagagat gtaacatgcc tcctccatgg acagttagac ctcaccagag aagaggagct 540aaaacagata aagaggcttc tgtgcctgct gcggtttctc aagcaatgga cgccttgaag 600ctccaagcgg atatggcccc tagactatgg caagctggca atcgtctagt acattctgtc 660agacaccctg aggatggctt aacagctcca ttcaccggtc cagtgtctgt ccttaaccat 720agagttacag cgcagagaag attcgctact caacactacc aactagatag attgaaaaac 780ttagcgcatg ccagtggtgg ttcactgaat gatatagtgc tttacttatg tggtactgcc 840ttgagaaggt ttttggctga gcagaataac ttgcctgaca cacctttaac ggcaggaatt 900ccagtgaata tcagaccagc tgatgacgaa ggcaccggaa cacaaatctc attcatgatt 960gctagtttgg ctactgacga agctgatcct ctcaatagat tacaacagat caaaacctca 1020acacgaaggg cgaaggagca tctccaaaag ttgcctaagt cagcactaac acaatacaca 1080atgctgctga tgtcacctta catcttacaa ttgatgagcg gattgggagg tagaatgagg 1140ccagttttca atgttactat aagcaatgtc cctgggcctg aggggacatt gtattacgaa 1200ggagctagat tggaagccat gtacccagtt tcccttatcg cccacggtgg tgccttgaac 1260atcacatgcc tgtcttacgc tggctccctt aactttgggt ttaccggttg tcgtgatact 1320ttaccatcaa tgcaaaagtt agcagtctat actggtgaag cattggatga actcgaatct 1380ctaattctgc caccaaagaa gcgtgcccgt actagaaagt aa 142251422DNAArtificial sequenceArtificial Sequence, optimized from wax synthase of Rhodococcus opacus PD630 5ttgaccgacg tgattaccac aaaccaaaga tacatgactc agaccgattt catgtcttgg 60agaatggagg aagatccaat actgagaagc acgatcgttg cagtggccct gttggacaga 120aggcctgatc aaagtagatt tgttgatatg atgagaagag ctgtcgatct agttccattg 180tttcgtagaa ccgccattga agatccactc ggcttggctc ctccaagatg ggccgatgat 240agagattttg acctatcatg gcatctaaga cgatatactt tagcggaacc taggacttgg 300gacggcgtcc tagatttcgc acgtactgca gagatgacag cttttgataa acgtagacct 360ttgtgggagt tcacaatctt agatggtctt aatgatggta gatcagcgtt ggttatgaag 420gttcaccatt cactcacgga tggtgtctct ggtatgcaaa ttgccagaga aatcgtggac 480tttactagag aaggtacgcc acgaccagga cgtacagata gagctacagc tgttcctcat 540ggaggctctt ctagacctcc ttctagactt agttggtata gagatacagc tgcagacgta 600acacaccgag ctgcgaacat cttgggtaga aattctgtta ggctagttag agcgccacgt 660gctacatgga gagaagccac tgcgttagct ggttccactt taagattaac cagaccagtt 720gtttccacat tgtcaccagt gatgactaag agatcaacaa gacgacattg tgctgtcatc 780gacgtccctg tagaagctct cgcacaggct gcagcagccg cagctgggtc tatcaatgac 840gctttccttg ctgcagtcct gttgggtatg gcaaagtacc atagacttca tggtgccgaa 900atcagagaat tacgtatgac tttaccaata tctttaagga cagaaacaga tccattaggt 960gggaatagaa tttccctagc cagattcgct ttgcctactg atattgatga tccagctgag 1020ttgatgagga gggtacacgc tactgtagat gcatggagaa gagaaccagc aataccattt 1080tcccctatga ttgctggtgc cgtaaactta cttcctgcct caactttagg gaacatgttg 1140aaacacgttg actttgtagc atctaacgtc gctggctcac cagttcctct attcatagcc 1200ggatcagaga tcctacatta ctacgcgttc tcaccaactc ttggatctgc attcaatgtt 1260acgctgatga gttacaccac tcaatgctgt gtcgggataa acgctgatac agacgctgta 1320cctgatcttg ccacactgac cgaaagtttg gcagatggat tcagagccgt tttgggctta 1380tgtgctaaga ctacagacac aagagtggtg gtggctagct aa 142261002DNAArtificial sequenceArtificial Sequence, optimized from wax synthase of Mus musculus C57BL/6 6atgttctggc caaccaaaaa ggatttgaaa actgcaatgg aagtatttgc tctcttccaa 60tgggcccttt ccgctttggt aattgtcact actgtgatca tagtcaattt gtatcttgtt 120gtgtttacat catattggcc agtgacggtc ttaatgttga catggttagc attcgattgg 180aaaacaccag aaagaggtgg caggagattc acatgtgtcc gtaagtggag attgtggaag 240cactactctg attacttccc tttgaaaatg gttaagacta aggacatatc accagataga 300aactacatct tagtatgtca tccacatggt cttatggcac attcatgttt cggacatttc 360gccacagata caactggatt cagtaagact tttcctggta tcactcctta catgctaaca 420ttaggcgcct ttttctgggt tccattcctt agagactatg ttatgtccac tggctcatgc 480tctgtgtcca gaagctcaat ggacttcctc ctaacacaaa aaggaactgg aaacatgttg 540gttgtagttg taggtggttt agctgagtgt cgttactcta cgccaggctc tacaaccctg 600tttttgaaaa agagacaggg tttcgtgaga actgcgttga agcatggtgt ttctctgatc 660ccagcttacg ctttcgggga aactgatctc tacgatcaac acatattcac accaggtggt 720tttgtcaata gatttcagaa atggtttcaa aagatggtac acatctaccc atgcgctttc 780tatggcagag ggctcaccaa aaactcatgg gggctactac cttattcaca gcctgttacc 840acagtggttg gagaaccttt acctctgcca aagattgaaa acccttccga agagattgtt 900gcgaagtacc atacactgta catcgatgca cttaggaagc tattcgacca acacaaaact 960aagtttggta ttagtgaaac ccaagagttg gtcattgttt aa 100271428DNAArtificial sequenceArtificial Sequence, optimized from wax synthase of Psychrobacter articus 273-4 7atgagattac tgaccgctgt cgatcaactc tttctattgt tggagagtag aaagcaccca 60atgcacgttg gtggactgtt cctattcgag cttccagaga atgctgacat tagtttcgtt 120caccagcttg ttaagcaaat gcaagattcc gacgtaccac caacattccc attcaatcag 180gttctggaac acatgatgtt ttggaaggag gacaaaaact ttgacgtaga acatcatcta 240caccatgtgg ctttaccaaa acctgccaga gttagagaat tactcatgta cgtttccagg 300gaacatggga ggttgctcga tagagcaatg ccactatggg agtgccatgt gatcgaaggt 360attcaaccag agactgaagg ttctccagag agattcgcat tgtatttcaa gattcatcat 420tccttagtcg atggtatcgc cgctatgagg ttggtgaaaa agtcattatc acagtcacca 480aacgaaccag ttacccttcc aatctggtct ttgatggctc accatagaaa ccaaatcgat 540gccatcttcc caaaggaaag atcagccttg cgtatcttaa aggaacaagt ttctacaatc 600aagcctgtgt ttactgaact cttgaataac ttcaaaaact acaatgacga tagttacgtc 660agcacttttg acgctcctag atcaatcctt aaccgtagaa tttctgcctc aagacgtatt 720gcagcgcagt catacgatat caaaagattc aatgacatag cggagagaat caacatttcc 780aaaaacgatg tggttttggc agtatgttcc ggtgctatta gaagatacct tatctctatg 840gatgctttac catcaaaacc tctgatagca ttcgttccta tgtctttgcg aactgatgat 900agtatagctg gaaaccaatt gagttttgta ctagcgaatc tgggcacaca tttggatgat 960ccattatcta gaatcaagct cattcatcgt agcatgaaca actctaagag aagattcaga 1020aggatgaacc aagcacaagt tatcaattac tccatagtat cttacgcatg ggaaggcatt 1080aacttggcca ctgatctttt ccctaaaaag caagccttta acttaatcat ctctaacgtc 1140ccaggctcag aaaaaccttt gtattggaat ggtgcaagat tagaatcact atatcctgct 1200tcaatcgtgt ttaacggaca agctatgaat atcacgcttg catcttactt ggacaagatg 1260gaattcggta taactgcttg ttctaaagct ctacctcatg tccaagatat gttgatgctt 1320attgaggaag agctacaact gctggaatct gttagcaagg aactagaatt caatgggatt 1380acagtaaaag ataagtcaga gaaaaagctg aaaaagttgg ccccttaa 1428830DNAArtificial sequenceprimer 8cgggatcccg ctcgagatga agagattagg 30934DNAArtificial sequenceprimer 9ggggtacccc aagcttgggt tactttctag tacg 341033DNAArtificial sequenceprimer 10cgggatcccg ctcgagttga ccgacgtgat tac 331135DNAArtificial sequenceprimer 11ggggtacccc aagcttgggt tagctagcca ccacc 351231DNAArtificial sequenceprimer 12cgggatcccg ctcgagatgt tctggccaac c 311335DNAArtificial sequenceprimer 13ggggtacccc aagcttgggt taaacaatga ccaac 351436DNAArtificial sequenceprimer 14cgggatcccg ctcgagatga gattactgac cgctgt 361533DNAArtificial sequenceprimer 15ggggtacccc aagcttgggt taaggggcca act 33162233PRTArtificial sequencemodified ACC1 gene 16Met Ser Glu Glu Ser Leu Phe Glu Ser Ser Pro Gln Lys Met Glu Tyr 1 5 10 15 Glu Ile Thr Asn Tyr Ser Glu Arg His Thr Glu Leu Pro Gly His Phe 20 25 30 Ile Gly Leu Asn Thr Val Asp Lys Leu Glu Glu Ser Pro Leu Arg Asp 35 40 45 Phe Val Lys Ser His Gly Gly His Thr Val Ile Ser Lys Ile Leu Ile 50 55 60 Ala Asn Asn Gly Ile Ala Ala Val Lys Glu Ile Arg Ser Val Arg Lys 65 70 75 80 Trp Ala Tyr Glu Thr Phe Gly Asp Asp Arg Thr Val Gln Phe Val Ala 85 90 95 Met Ala Thr Pro Glu Asp Leu Glu Ala Asn Ala Glu Tyr Ile Arg Met 100 105 110 Ala Asp Gln Tyr Ile Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr 115 120 125 Ala Asn Val Asp Leu Ile Val Asp Ile Ala Glu Arg Ala Asp Val Asp 130 135 140 Ala Val Trp Ala Gly Trp Gly His Ala Ser Glu Asn Pro Leu Leu Pro 145 150 155 160 Glu Lys Leu Ser Gln Ser Lys Arg Lys Val Ile Phe Ile Gly Pro Pro 165 170 175 Gly Asn Ala Met Arg Ser Leu Gly Asp Lys Ile Ser Ser Thr Ile Val 180 185 190 Ala Gln Ser Ala Lys Val Pro Cys Ile Pro Trp Ser Gly Thr Gly Val 195 200 205 Asp Thr Val His Val Asp Glu Lys Thr Gly Leu Val Ser Val Asp Asp 210 215 220 Asp Ile Tyr Gln Lys Gly Cys Cys Thr Ser Pro Glu Asp Gly Leu Gln 225 230 235 240 Lys Ala Lys Arg Ile Gly Phe Pro Val Met Ile Lys Ala Ser Glu Gly 245 250 255 Gly Gly Gly Lys Gly Ile Arg Gln Val Glu Arg Glu Glu Asp Phe Ile 260 265 270 Ala Leu Tyr His Gln Ala Ala Asn Glu Ile Pro Gly Ser Pro Ile Phe 275 280 285 Ile Met Lys Leu Ala Gly Arg Ala Arg His Leu Glu Val Gln Leu Leu 290 295 300 Ala Asp Gln Tyr Gly Thr Asn Ile Ser Leu Phe Gly Arg Asp Cys Ser 305 310 315 320 Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Ala Pro Val Thr Ile 325 330 335 Ala Lys Ala Glu Thr Phe His Glu Met Glu Lys Ala Ala Val Arg Leu 340 345 350 Gly Lys Leu Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr 355 360 365 Ser His Asp Asp Gly Lys Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu 370 375 380 Gln Val Glu His Pro Thr Thr Glu Met Val Ser Gly Val Asn Leu Pro 385 390 395 400 Ala Ala Gln Leu Gln Ile Ala Met Gly Ile Pro Met His Arg Ile Ser 405 410 415 Asp Ile Arg Thr Leu Tyr Gly Met Asn Pro His Ser Ala Ser Glu Ile 420 425 430 Asp Phe Glu Phe Lys Thr Gln Asp Ala Thr Lys Lys Gln Arg Arg Pro 435 440 445 Ile Pro Lys Gly His Cys Thr Ala Cys Arg Ile Thr Ser Glu Asp Pro 450 455 460 Asn Asp Gly Phe Lys Pro Ser Gly Gly Thr Leu His Glu Leu Asn Phe 465 470 475 480 Arg Ser Ser Ser Asn Val Trp Gly Tyr Phe Ser Val Gly Asn Asn Gly 485 490 495 Asn Ile His Ser Phe Ser Asp Ser Gln Phe Gly His Ile Phe Ala Phe 500 505 510 Gly Glu Asn Arg Gln Ala Ser Arg Lys His Met Val Val Ala Leu Lys 515 520 525 Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr Leu Ile 530 535 540 Lys Leu Leu Glu Thr Glu Asp Phe Glu Asp Asn Thr Ile Thr Thr Gly 545 550 555 560 Trp Leu Asp Asp Leu Ile Thr His Lys Met Thr Ala Glu Lys Pro Asp 565 570 575 Pro Thr Leu Ala Val Ile Cys Gly Ala Ala Thr Lys Ala Phe Leu Ala 580 585 590 Ser Glu Glu Ala Arg His Lys Tyr Ile Glu Ser Leu Gln Lys Gly Gln 595 600 605 Val Leu Ser Lys Asp Leu Leu Gln Thr Met Phe Pro Val Asp Phe Ile 610 615 620 His Glu Gly Lys Arg Tyr Lys Phe Thr Val Ala Lys Ser Gly Asn Asp 625 630 635 640 Arg Tyr Thr Leu Phe Ile Asn Gly Ser Lys Cys Asp Ile Ile Leu Arg 645 650 655 Gln Leu Ala Asp Gly Gly Leu Leu Ile Ala Ile Gly Gly Lys Ser His 660 665 670 Thr Ile Tyr Trp Lys Glu Glu Val Ala Ala Thr Arg Leu Ser Val Asp 675 680 685 Ser Met Thr Thr Leu Leu Glu Val Glu Asn Asp Pro Thr Gln Leu Arg 690 695 700 Thr Pro Ser Pro Gly Lys Leu Val Lys Phe Leu Val Glu Asn Gly Glu 705 710 715 720 His Ile Ile Lys Gly Gln Pro Tyr Ala Glu Ile Glu Val Met Lys Met 725 730 735 Gln Met Pro Leu Val Ser Gln Glu Asn Gly Ile Val Gln Leu Leu Lys 740 745 750 Gln Pro Gly Ser Thr Ile Val Ala Gly Asp Ile Met Ala Ile Met Thr 755 760 765 Leu Asp Asp Pro Ser Lys Val Lys His Ala Leu Pro Phe Glu Gly Met 770 775 780 Leu Pro Asp Phe Gly Ser Pro Val Ile Glu Gly Thr Lys Pro Ala Tyr 785 790 795 800 Lys Phe Lys Ser Leu Val Ser Thr Leu Glu Asn Ile Leu Lys Gly Tyr 805 810 815 Asp Asn Gln Val Ile Met Asn Ala Ser Leu Gln Gln Leu Ile Glu Val 820 825 830 Leu Arg Asn Pro Lys Leu Pro Tyr Ser Glu Trp Lys Leu His Ile Ser 835 840 845 Ala Leu His Ser Arg Leu Pro Ala Lys Leu Asp Glu Gln Met Glu Glu 850 855 860 Leu Val Ala Arg Ser Leu Arg Arg Gly Ala Val Phe Pro Ala Arg Gln 865 870 875 880 Leu Ser Lys Leu Ile Asp Met Ala Val Lys Asn Pro Glu Tyr Asn Pro 885 890 895 Asp Lys Leu Leu Gly Ala Val Val Glu Pro Leu Ala Asp Ile Ala His 900 905 910 Lys Tyr Ser Asn Gly Leu Glu Ala His Glu His Ser Ile Phe Val His 915 920 925 Phe Leu Glu Glu Tyr Tyr Glu Val Glu Lys Leu Phe Asn Gly Pro Asn 930 935 940 Val Arg Glu Glu Asn Ile Ile Leu Lys Leu Arg Asp Glu Asn Pro Lys 945 950 955 960 Asp Leu Asp Lys Val Ala Leu Thr Val Leu Ser His Ser Lys Val Ser 965 970 975 Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Lys His Tyr Gln Pro Leu 980 985 990 Cys Lys Leu Ser Ser Lys Val Ser Ala Ile Phe Ser Thr Pro Leu Gln 995 1000 1005 His Ile Val Glu Leu Glu Ser Lys Ala Thr Ala Lys Val Ala Leu 1010 1015 1020 Gln Ala Arg Glu Ile Leu Ile Gln Gly Ala Leu Pro Ser Val Lys 1025 1030 1035 Glu Arg Thr Glu Gln Ile Glu His Ile Leu Lys Ser Ser Val Val 1040 1045

1050 Lys Val Ala Tyr Gly Ser Ser Asn Pro Lys Arg Ser Glu Pro Asp 1055 1060 1065 Leu Asn Ile Leu Lys Asp Leu Ile Asp Ser Asn Tyr Val Val Phe 1070 1075 1080 Asp Val Leu Leu Gln Phe Leu Thr His Gln Asp Pro Val Val Thr 1085 1090 1095 Ala Ala Ala Ala Gln Val Tyr Ile Arg Arg Ala Tyr Arg Ala Tyr 1100 1105 1110 Thr Ile Gly Asp Ile Arg Val His Glu Gly Val Thr Val Pro Ile 1115 1120 1125 Val Glu Trp Lys Phe Gln Leu Pro Ser Ala Ala Phe Ser Thr Phe 1130 1135 1140 Pro Thr Val Lys Ser Lys Met Gly Met Asn Arg Ala Val Ala Val 1145 1150 1155 Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln Ser Ser Pro Leu Arg 1160 1165 1170 Glu Gly Ile Leu Met Ala Val Asp His Leu Asp Asp Val Asp Glu 1175 1180 1185 Ile Leu Ser Gln Ser Leu Glu Val Ile Pro Arg His Gln Ser Ser 1190 1195 1200 Ser Asn Gly Pro Ala Pro Asp Arg Ser Gly Ser Ser Ala Ser Leu 1205 1210 1215 Ser Asn Val Ala Asn Val Cys Val Ala Ser Thr Glu Gly Phe Glu 1220 1225 1230 Ser Glu Glu Glu Ile Leu Val Arg Leu Arg Glu Ile Leu Asp Leu 1235 1240 1245 Asn Lys Gln Glu Leu Ile Asn Ala Ser Ile Arg Arg Ile Thr Phe 1250 1255 1260 Met Phe Gly Phe Lys Asp Gly Ser Tyr Pro Lys Tyr Tyr Thr Phe 1265 1270 1275 Asn Gly Pro Asn Tyr Asn Glu Asn Glu Thr Ile Arg His Ile Glu 1280 1285 1290 Pro Ala Leu Ala Phe Gln Leu Glu Leu Gly Arg Leu Ser Asn Phe 1295 1300 1305 Asn Ile Lys Pro Ile Phe Thr Asp Asn Arg Asn Ile His Val Tyr 1310 1315 1320 Glu Ala Val Ser Lys Thr Ser Pro Leu Asp Lys Arg Phe Phe Thr 1325 1330 1335 Arg Gly Ile Ile Arg Thr Gly His Ile Arg Asp Asp Ile Ser Ile 1340 1345 1350 Gln Glu Tyr Leu Thr Ser Glu Ala Asn Arg Leu Met Ser Asp Ile 1355 1360 1365 Leu Asp Asn Leu Glu Val Thr Asp Thr Ser Asn Ser Asp Leu Asn 1370 1375 1380 His Ile Phe Ile Asn Phe Ile Ala Val Phe Asp Ile Ser Pro Glu 1385 1390 1395 Asp Val Glu Ala Ala Phe Gly Gly Phe Leu Glu Arg Phe Gly Lys 1400 1405 1410 Arg Leu Leu Arg Leu Arg Val Ser Ser Ala Glu Ile Arg Ile Ile 1415 1420 1425 Ile Lys Asp Pro Gln Thr Gly Ala Pro Val Pro Leu Arg Ala Leu 1430 1435 1440 Ile Asn Asn Val Ser Gly Tyr Val Ile Lys Thr Glu Met Tyr Thr 1445 1450 1455 Glu Val Lys Asn Ala Lys Gly Glu Trp Val Phe Lys Ser Leu Gly 1460 1465 1470 Lys Pro Gly Ser Met His Leu Arg Pro Ile Ala Thr Pro Tyr Pro 1475 1480 1485 Val Lys Glu Trp Leu Gln Pro Lys Arg Tyr Lys Ala His Leu Met 1490 1495 1500 Gly Thr Thr Tyr Val Tyr Asp Phe Pro Glu Leu Phe Arg Gln Ala 1505 1510 1515 Ser Ser Ser Gln Trp Lys Asn Phe Ser Ala Asp Val Lys Leu Thr 1520 1525 1530 Asp Asp Phe Phe Ile Ser Asn Glu Leu Ile Glu Asp Glu Asn Gly 1535 1540 1545 Glu Leu Thr Glu Val Glu Arg Glu Pro Gly Ala Asn Ala Ile Gly 1550 1555 1560 Met Val Ala Phe Lys Ile Thr Val Lys Thr Pro Glu Tyr Pro Arg 1565 1570 1575 Gly Arg Gln Phe Val Val Val Ala Asn Asp Ile Thr Phe Lys Ile 1580 1585 1590 Gly Ser Phe Gly Pro Gln Glu Asp Glu Phe Phe Asn Lys Val Thr 1595 1600 1605 Glu Tyr Ala Arg Lys Arg Gly Ile Pro Arg Ile Tyr Leu Ala Ala 1610 1615 1620 Asn Ser Gly Ala Arg Ile Gly Met Ala Glu Glu Ile Val Pro Leu 1625 1630 1635 Phe Gln Val Ala Trp Asn Asp Ala Ala Asn Pro Asp Lys Gly Phe 1640 1645 1650 Gln Tyr Leu Tyr Leu Thr Ser Glu Gly Met Glu Thr Leu Lys Lys 1655 1660 1665 Phe Asp Lys Glu Asn Ser Val Leu Thr Glu Arg Thr Val Ile Asn 1670 1675 1680 Gly Glu Glu Arg Phe Val Ile Lys Thr Ile Ile Gly Ser Glu Asp 1685 1690 1695 Gly Leu Gly Val Glu Cys Leu Arg Gly Ser Gly Leu Ile Ala Gly 1700 1705 1710 Ala Thr Ser Arg Ala Tyr His Asp Ile Phe Thr Ile Thr Leu Val 1715 1720 1725 Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly 1730 1735 1740 Gln Arg Ala Ile Gln Val Glu Gly Gln Pro Ile Ile Leu Thr Gly 1745 1750 1755 Ala Pro Ala Ile Asn Lys Met Leu Gly Arg Glu Val Tyr Thr Ser 1760 1765 1770 Asn Leu Gln Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn Gly Val 1775 1780 1785 Ser His Leu Thr Ala Val Asp Asp Leu Ala Gly Val Glu Lys Ile 1790 1795 1800 Val Glu Trp Met Ser Tyr Val Pro Ala Lys Arg Asn Met Pro Val 1805 1810 1815 Pro Ile Leu Glu Thr Lys Asp Thr Trp Asp Arg Pro Val Asp Phe 1820 1825 1830 Thr Pro Thr Asn Asp Glu Thr Tyr Asp Val Arg Trp Met Ile Glu 1835 1840 1845 Gly Arg Glu Thr Glu Ser Gly Phe Glu Tyr Gly Leu Phe Asp Lys 1850 1855 1860 Gly Ser Phe Phe Glu Thr Leu Ser Gly Trp Ala Lys Gly Val Val 1865 1870 1875 Val Gly Arg Ala Arg Leu Gly Gly Ile Pro Leu Gly Val Ile Gly 1880 1885 1890 Val Glu Thr Arg Thr Val Glu Asn Leu Ile Pro Ala Asp Pro Ala 1895 1900 1905 Asn Pro Asn Ser Ala Glu Thr Leu Ile Gln Glu Pro Gly Gln Val 1910 1915 1920 Trp His Pro Asn Ser Ala Phe Lys Thr Ala Gln Ala Ile Asn Asp 1925 1930 1935 Phe Asn Asn Gly Glu Gln Leu Pro Met Met Ile Leu Ala Asn Trp 1940 1945 1950 Arg Gly Phe Ser Gly Gly Gln Arg Asp Met Phe Asn Glu Val Leu 1955 1960 1965 Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu Val Asp Tyr Lys Gln 1970 1975 1980 Pro Ile Ile Ile Tyr Ile Pro Pro Thr Gly Glu Leu Arg Gly Gly 1985 1990 1995 Ser Trp Val Val Val Asp Pro Thr Ile Asn Ala Asp Gln Met Glu 2000 2005 2010 Met Tyr Ala Asp Val Asn Ala Arg Ala Gly Val Leu Glu Pro Gln 2015 2020 2025 Gly Met Val Gly Ile Lys Phe Arg Arg Glu Lys Leu Leu Asp Thr 2030 2035 2040 Met Asn Arg Leu Asp Asp Lys Tyr Arg Glu Leu Arg Ser Gln Leu 2045 2050 2055 Ser Asn Lys Ser Leu Ala Pro Glu Val His Gln Gln Ile Ser Lys 2060 2065 2070 Gln Leu Ala Asp Arg Glu Arg Glu Leu Leu Pro Ile Tyr Gly Gln 2075 2080 2085 Ile Ser Leu Gln Phe Ala Asp Leu His Asp Arg Ser Ser Arg Met 2090 2095 2100 Val Ala Lys Gly Val Ile Ser Lys Glu Leu Glu Trp Thr Glu Ala 2105 2110 2115 Arg Arg Phe Phe Phe Trp Arg Leu Arg Arg Arg Leu Asn Glu Glu 2120 2125 2130 Tyr Leu Ile Lys Arg Leu Ser His Gln Val Gly Glu Ala Ser Arg 2135 2140 2145 Leu Glu Lys Ile Ala Arg Ile Arg Ser Trp Tyr Pro Ala Ser Val 2150 2155 2160 Asp His Glu Asp Asp Arg Gln Val Ala Thr Trp Ile Glu Glu Asn 2165 2170 2175 Tyr Lys Thr Leu Asp Asp Lys Leu Lys Gly Leu Lys Leu Glu Ser 2180 2185 2190 Phe Ala Gln Asp Leu Ala Lys Lys Ile Arg Ser Asp His Asp Asn 2195 2200 2205 Ala Ile Asp Gly Leu Ser Glu Val Ile Lys Met Leu Ser Thr Asp 2210 2215 2220 Asp Lys Glu Lys Leu Leu Lys Thr Leu Lys 2225 2230 171428DNAArtificial sequenceoptimized gapN sequence 17ttgacaaaac aatacaaaaa ctacgttaat ggtgaatgga aactaagtga gaatgaaatc 60aagatatacg aacctgcctc aggcgcagaa ctgggttctg ttcctgcaat gtccactgag 120gaagtggatt acgtgtatgc ttcagccaaa aaggctcagc ctgcatggag atccctaagt 180tacattgaaa gagctgccta tttgcataaa gtcgcagaca tattgatgag ggataaagag 240aagattggcg ctgtgctttc taaggaagtc gctaagggat acaaatctgc agtatctgag 300gtagttagaa cagcagagat tatcaattac gctgccgagg aaggtcttag aatggaggga 360gaggtacttg aaggaggatc atttgaagca gcatccaaaa agaagatcgc tgtagtaagg 420agagaaccag taggccttgt tctagccatc agtcctttca actatccagt caacttagct 480ggctccaaaa tcgctcctgc cttaatcgct ggtaatgtca ttgctttcaa gccacctact 540caagggtcta tttcaggttt gttgttggcc gaggcttttg ctgaagcagg tctgccagct 600ggtgttttca atacaattac aggtagagga tctgaaattg gagactacat tgtcgaacat 660caagctgtca actttatcaa tttcacaggt tcaacaggaa ttggcgagag aatagggaaa 720atggcaggta tgcgtccaat catgttagaa ctaggcggga aagactctgc aatcgtgttg 780gaagatgctg atttggaact taccgccaaa aacatcattg ccggtgcatt cggttattct 840ggacaaagat gtactgcagt taaacgtgtt ttagtaatgg aatcagtggc agatgaactt 900gtggaaaaga tcagggaaaa agtccttgca ctgactatcg gtaatccaga agatgatgct 960gacatcaccc cacttattga cactaagtca gctgattatg ttgaaggttt gatcaatgat 1020gcaaatgata agggtgccgc tgccttaaca gagatcaaaa gagaaggtaa cttaatctgc 1080ccaatcctgt ttgataaggt tactactgat atgagattgg cttgggagga accatttggt 1140cctgttttgc ctatcataag agttacctct gttgaggaag ctatagagat atctaacaaa 1200tcagaatacg gcttacaagc ctctatcttt actaatgatt tcccaagagc atttggaata 1260gctgaacaac tagaagtagg tacagttcac attaacaaca aaacccagag aggcacagac 1320aatttcccat ttctaggggc caaaaagtca ggggctggaa ttcaaggcgt gaaatactcc 1380attgaagcta tgactacagt gaaaagtgtt gtctttgaca taaagtga 14281830DNAArtificial sequenceprimer 18aaacaagcgg ccgcactagt ttgacaaaac 301934DNAArtificial sequenceprimer 19ttaattaaga gctcagatct ttatttgata tcaa 342030DNAArtificial sequenceprimer 20cgggatcccg ctcgagatga agagattagg 302134DNAArtificial sequenceprimer 21ggggtacccc aagcttgggt tactttctag tacg 342253DNAArtificial sequenceprimer 22gttgggattc cattgttgat aaaggcgcac acaccatagc ttcaaaatgt ttc 532332DNAArtificial sequenceprimer 23gtgcaatgta gatcttcgag cgtcccaaaa cc 322452DNAArtificial sequenceprimer 24gacgctcgaa gatctacatt gcacaagata aaaatatatc atcatgaaca at 522543DNAArtificial sequenceprimer 25gcctttatca acaatggaat cccaaccgcc gtcccgtcaa gtc 432648DNAArtificial sequenceprimer 26acaacaaata taaaacaagc ggccgcacta tgaagagatt aggtactc 482746DNAArtificial sequenceprimer 27ggcgaagaat tgttaattaa gagctcggta ccccaagctt gggtta 462851DNAArtificial sequenceprimer 28gttgggattc cattgttgat aaaggcggaa gtaccttcaa agaatggggt c 512937DNAArtificial sequenceprimer 29cttgtgcaat gtagagcgac ctcatgctat acctgag 373047DNAArtificial sequenceprimer 30atgaggtcgc tctacattgc acaagataaa aatatatcat catgaac 47318721DNAArtificial sequenceplasmid pSP-B1 31tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 1860ttcgctatta cgccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 1920atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt 1980tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta 2040tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta 2100tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc 2160gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca 2220gatcttatcg tcgtcatcct tgtaatccat cgatactagt gcggccgctt gttttatatt 2280tgttgtaaaa agtagataat tacttccttg atgatctgta aaaaagagaa aaagaaagca 2340tctaagaact tgaaaaacta cgaattagaa aagaccaaat atgtatttct tgcattgacc 2400aatttatgca agtttatata tatgtaaatg taagtttcac gaggttctac taaactaaac 2460cacccccttg gttagaagaa aagagtgtgt gagaacaggc tgttgttgtc acacgattcg 2520gacaattctg tttgaaagag agagagtaac agtacgatcg aacgaacttt gctctggaga 2580tcacagtggg catcatagca tgtggtacta aaccctttcc cgccattcca gaaccttcga 2640ttgcttgtta caaaacctgt gagccgtcgc taggaccttg ttgtgtgacg aaattggaag 2700ctgcaatcaa taggaagaca ggaagtcgag cgtgtctggg ttttttcagt tttgttcttt 2760ttgcaaacaa atcacgagcg acggtaattt ctttctcgat aagaggccac gtgctttatg 2820agggtaacat caattcaaga aggagggaaa cacttccttt ttctggccct gataatagta 2880tgagggtgaa gccaaaataa aggattcgcg cccaaatcgg catctttaaa tgcaggtatg 2940cgatagttcc tcactctttc cttactcacg agtaattctt gcaaatgcct attatgcaga 3000tgttataata tctgtgcgtc ttgagttgaa gtcaggaatc taaaataaaa attaaggtta 3060ataaaaagag gaaagaaaaa aaaattaatc gatttacaga aacttgcaca ctaaaaatac 3120acaactaaaa gcaattacag tatgggaagt catcgacgtt atctctacta tagtatatta 3180tcatttctat tattatcctg ctcagtggta cttgcaaaac aagataagac cccattcttt 3240gaaggtactt ccaggccggc cgcacacacc atagcttcaa aatgtttcta ctcctttttt 3300actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac 3360agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg 3420tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa 3480atttttatca cgtttctttt tcttgaaaat tttttttttt gatttttttc tctttcgatg 3540acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3600tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3660taatctaagt tttaattaca aggatcccgc tcgagatgcg tccattacat ccaattgatt 3720tcatcttcct atctttagaa aagagacagc aaccaatgca tgtaggcggg ctttttctct 3780tccaaattcc tgacaatgct ccagacacat tcatccaaga cttggttaat gatatcagaa 3840tttccaagtc aatacctgtt ccacctttca ataacaaact caacggcttg ttctgggacg 3900aggatgaaga gttcgatctg gatcatcact ttcgacatat cgcattacca caccctggta 3960gaatcaggga gttactcatc tacatttccc aagagcattc aacattactt gatagagcca 4020agccattgtg gacatgcaac ataatcgaag gtatagaagg caatagattt gccatgtact 4080tcaagataca tcacgctatg gttgatggag tcgctggaat gagactaatc gagaagagcc 4140tttctcacga tgttacagaa aagtcaattg taccaccttg gtgtgtagaa ggtaaacgag 4200ctaagcgttt acgtgaacca aaaaccggaa agatcaaaaa

gattatgtct ggtatcaaat 4260ctcaacttca agccacgcct actgtcattc aagagttgtc tcaaacagtg ttcaaagaca 4320ttggtagaaa cccagatcac gtgtcctcat ttcaagcgcc atgttctatt ctgaaccaaa 4380gagtatccag tagtagaaga tttgcagcac agtcttttga tcttgatagg ttcagaaaca 4440ttgcaaagtc tctgaacgtc accataaacg acgtggttct agctgtttgc tctggggcac 4500tgagagctta tctaatgtca cataacagct tgccatcaaa accattgatt gcgatggttc 4560ctgcctctat acgtaatgat gattcagatg tcagtaacag aataacaatg atccttgcca 4620acctagctac tcataaggat gatcctttgc agagattaga aatcattaga agatcagtgc 4680aaaactcaaa gcagagattc aaaaggatga ccagtgatca aatcttgaat tactctgcag 4740tggtatacgg tccagctggt ctgaatatca tatcaggaat gatgccaaaa agacaagcct 4800ttaacttagt tatctccaat gtacctggtc cacgagaacc tctctactgg aacggagcta 4860agttggatgc actttaccca gcctctatcg ttttagatgg tcaggctttg aacattacaa 4920tgactagtta tctagacaag ctagaagttg ggttgattgc gtgtagaaat gccctaccta 4980gaatgcagaa tttgctgact cacttagaag aggagattca actctttgaa ggcgtcatcg 5040caaaacaaga ggatatcaaa actgcaaact aacccaagct tggtaccgcg gctagctaag 5100atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 5160ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 5220gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 5280acgctcgaag atcctccgga tcgtttcgcc ggcgtttatc cagctgcatt aatgaatcgg 5340ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 5400ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5460acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5520aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5580tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5640aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5700gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 5760acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5820accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5880ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5940gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 6000gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 6060ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 6120gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 6180cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 6240cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 6300gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 6360tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 6420gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6480agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6540tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6600agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6660gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6720catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6780ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 6840atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 6900tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 6960cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 7020cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 7080atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 7140aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 7200ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 7260aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aacgaagcat 7320ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc gctaattttt caaacaaaga 7380atctgagctg catttttaca gaacagaaat gcaacgcgaa agcgctattt taccaacgaa 7440gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac 7500aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgagagcgc tattttacca 7560acaaagaatc tatacttctt ttttgttcta caaaaatgca tcccgagagc gctatttttc 7620taacaaagca tcttagatta ctttttttct cctttgtgcg ctctataatg cagtctcttg 7680ataacttttt gcactgtagg tccgttaagg ttagaagaag gctactttgg tgtctatttt 7740ctcttccata aaaaaagcct gactccactt cccgcgttta ctgattacta gcgaagctgc 7800gggtgcattt tttcaagata aaggcatccc cgattatatt ctataccgat gtggattgcg 7860catactttgt gaacagaaag tgatagcgtt gatgattctt cattggtcag aaaattatga 7920acggtttctt ctattttgtc tctatatact acgtatagga aatgtttaca ttttcgtatt 7980gttttcgatt cactctatga atagttctta ctacaatttt tttgtctaaa gagtaatact 8040agagataaac ataaaaaatg tagaggtcga gtttagatgc aagttcaagg agcgaaaggt 8100ggatgggtag gttatatagg gatatagcac agagatatat agcaaagaga tacttttgag 8160caatgtttgt ggaagcggta ttcgcaatat tttagtagct cgttacagtc cggtgcgttt 8220ttggtttttt gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc tctgaagttc 8280ctatactttc tagagaatag gaacttcgga ataggaactt caaagcgttt ccgaaaacga 8340gcgcttccga aaatgcaacg cgagctgcgc acatacagct cactgttcac gtcgcaccta 8400tatctgcgtg ttgcctgtat atatatatac atgagaagaa cggcatagtg cgtgtttatg 8460cttaaatgcg tacttatatg cgtctattta tgtaggatga aaggtagtct agtacctcct 8520gtgatattat cccattccat gcggggtatc gtatgcttcc ttcagcacta ccctttagct 8580gttctatatg ctgccactcc tcaattggat tagtctcatc cttcaatgct atcatttcct 8640ttgatattgg atcatactaa gaaaccatta ttatcatgac attaacctat aaaaataggc 8700gtatcacgag gccctttcgt c 8721328768DNAArtificial sequenceplasmid pSP-B2 32tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 1860ttcgctatta cgccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 1920atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt 1980tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta 2040tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta 2100tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc 2160gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca 2220gatcttatcg tcgtcatcct tgtaatccat cgatactagt gcggccgctt gttttatatt 2280tgttgtaaaa agtagataat tacttccttg atgatctgta aaaaagagaa aaagaaagca 2340tctaagaact tgaaaaacta cgaattagaa aagaccaaat atgtatttct tgcattgacc 2400aatttatgca agtttatata tatgtaaatg taagtttcac gaggttctac taaactaaac 2460cacccccttg gttagaagaa aagagtgtgt gagaacaggc tgttgttgtc acacgattcg 2520gacaattctg tttgaaagag agagagtaac agtacgatcg aacgaacttt gctctggaga 2580tcacagtggg catcatagca tgtggtacta aaccctttcc cgccattcca gaaccttcga 2640ttgcttgtta caaaacctgt gagccgtcgc taggaccttg ttgtgtgacg aaattggaag 2700ctgcaatcaa taggaagaca ggaagtcgag cgtgtctggg ttttttcagt tttgttcttt 2760ttgcaaacaa atcacgagcg acggtaattt ctttctcgat aagaggccac gtgctttatg 2820agggtaacat caattcaaga aggagggaaa cacttccttt ttctggccct gataatagta 2880tgagggtgaa gccaaaataa aggattcgcg cccaaatcgg catctttaaa tgcaggtatg 2940cgatagttcc tcactctttc cttactcacg agtaattctt gcaaatgcct attatgcaga 3000tgttataata tctgtgcgtc ttgagttgaa gtcaggaatc taaaataaaa attaaggtta 3060ataaaaagag gaaagaaaaa aaaattaatc gatttacaga aacttgcaca ctaaaaatac 3120acaactaaaa gcaattacag tatgggaagt catcgacgtt atctctacta tagtatatta 3180tcatttctat tattatcctg ctcagtggta cttgcaaaac aagataagac cccattcttt 3240gaaggtactt ccaggccggc cgcacacacc atagcttcaa aatgtttcta ctcctttttt 3300actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac 3360agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg 3420tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa 3480atttttatca cgtttctttt tcttgaaaat tttttttttt gatttttttc tctttcgatg 3540acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3600tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3660taatctaagt tttaattaca aggatcccgc tcgagatgaa gagattaggt actctagacg 3720ctagttggct tgcagtcgaa tccgaagata cgccaatgca cgtgggcact ctccaaatct 3780tctcattacc agaaggtgct ccagagacat ttctacgtga tatggttaca aggatgaaag 3840aggcaggaga tgttgcccca ccatggggtt acaagctcgc atggtccggt ttccttggca 3900gggttattgc tcctgcctgg aaggtagaca aagatatcga tttggattat catgtccgac 3960atagtgcatt gccaagacca ggtggtgaaa gagagctagg gatacttgtt tctagattac 4020actccaaccc tttagatttc tctagaccac tatgggaatg ccatgtcatt gaaggtcttg 4080aaaacaacag atttgcactg tatactaaga tgcatcactc tatgattgat gggatatctg 4140gagtaagatt gatgcaaaga gtattgacca ctgacccaga gagatgtaac atgcctcctc 4200catggacagt tagacctcac cagagaagag gagctaaaac agataaagag gcttctgtgc 4260ctgctgcggt ttctcaagca atggacgcct tgaagctcca agcggatatg gcccctagac 4320tatggcaagc tggcaatcgt ctagtacatt ctgtcagaca ccctgaggat ggcttaacag 4380ctccattcac cggtccagtg tctgtcctta accatagagt tacagcgcag agaagattcg 4440ctactcaaca ctaccaacta gatagattga aaaacttagc gcatgccagt ggtggttcac 4500tgaatgatat agtgctttac ttatgtggta ctgccttgag aaggtttttg gctgagcaga 4560ataacttgcc tgacacacct ttaacggcag gaattccagt gaatatcaga ccagctgatg 4620acgaaggcac cggaacacaa atctcattca tgattgctag tttggctact gacgaagctg 4680atcctctcaa tagattacaa cagatcaaaa cctcaacacg aagggcgaag gagcatctcc 4740aaaagttgcc taagtcagca ctaacacaat acacaatgct gctgatgtca ccttacatct 4800tacaattgat gagcggattg ggaggtagaa tgaggccagt tttcaatgtt actataagca 4860atgtccctgg gcctgagggg acattgtatt acgaaggagc tagattggaa gccatgtacc 4920cagtttccct tatcgcccac ggtggtgcct tgaacatcac atgcctgtct tacgctggct 4980cccttaactt tgggtttacc ggttgtcgtg atactttacc atcaatgcaa aagttagcag 5040tctatactgg tgaagcattg gatgaactcg aatctctaat tctgccacca aagaagcgtg 5100cccgtactag aaagtaaccc aagcttgggg taccgcggct agctaagatc cgctctaacc 5160gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 5220ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 5280tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc 5340ctccggatcg tttcgccggc gtttatccag ctgcattaat gaatcggcca acgcgcgggg 5400agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 5460gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5520gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 5580cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 5640aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 5700tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 5760ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 5820ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 5880cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 5940ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 6000gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 6060atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6120aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 6180aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 6240gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 6300cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 6360gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 6420tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 6480ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 6540ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 6600atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 6660cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 6720tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 6780aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 6840tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 6900ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 6960agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 7020gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 7080agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 7140accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 7200gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 7260cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 7320ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt 7380tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat 7440ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc 7500atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc 7560tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat 7620acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct 7680tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca 7740ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa 7800aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt 7860caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa 7920cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta 7980ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac 8040tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata 8100aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt 8160atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga 8220agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa 8280agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag 8340agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa 8400tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg 8460cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac 8520ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc 8580attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg 8640ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc 8700atactaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 8760ctttcgtc 8768338766DNAArtificial sequenceplasmid pSP-B3 33tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620aagcactaaa tcggaaccct

aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 1860ttcgctatta cgccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 1920atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt 1980tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta 2040tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta 2100tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc 2160gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca 2220gatcttatcg tcgtcatcct tgtaatccat cgatactagt gcggccgctt gttttatatt 2280tgttgtaaaa agtagataat tacttccttg atgatctgta aaaaagagaa aaagaaagca 2340tctaagaact tgaaaaacta cgaattagaa aagaccaaat atgtatttct tgcattgacc 2400aatttatgca agtttatata tatgtaaatg taagtttcac gaggttctac taaactaaac 2460cacccccttg gttagaagaa aagagtgtgt gagaacaggc tgttgttgtc acacgattcg 2520gacaattctg tttgaaagag agagagtaac agtacgatcg aacgaacttt gctctggaga 2580tcacagtggg catcatagca tgtggtacta aaccctttcc cgccattcca gaaccttcga 2640ttgcttgtta caaaacctgt gagccgtcgc taggaccttg ttgtgtgacg aaattggaag 2700ctgcaatcaa taggaagaca ggaagtcgag cgtgtctggg ttttttcagt tttgttcttt 2760ttgcaaacaa atcacgagcg acggtaattt ctttctcgat aagaggccac gtgctttatg 2820agggtaacat caattcaaga aggagggaaa cacttccttt ttctggccct gataatagta 2880tgagggtgaa gccaaaataa aggattcgcg cccaaatcgg catctttaaa tgcaggtatg 2940cgatagttcc tcactctttc cttactcacg agtaattctt gcaaatgcct attatgcaga 3000tgttataata tctgtgcgtc ttgagttgaa gtcaggaatc taaaataaaa attaaggtta 3060ataaaaagag gaaagaaaaa aaaattaatc gatttacaga aacttgcaca ctaaaaatac 3120acaactaaaa gcaattacag tatgggaagt catcgacgtt atctctacta tagtatatta 3180tcatttctat tattatcctg ctcagtggta cttgcaaaac aagataagac cccattcttt 3240gaaggtactt ccaggccggc cgcacacacc atagcttcaa aatgtttcta ctcctttttt 3300actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac 3360agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg 3420tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa 3480atttttatca cgtttctttt tcttgaaaat tttttttttt gatttttttc tctttcgatg 3540acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3600tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3660taatctaagt tttaattaca aggatcccgc tcgagttgac cgacgtgatt accacaaacc 3720aaagatacat gactcagacc gatttcatgt cttggagaat ggaggaagat ccaatactga 3780gaagcacgat cgttgcagtg gccctgttgg acagaaggcc tgatcaaagt agatttgttg 3840atatgatgag aagagctgtc gatctagttc cattgtttcg tagaaccgcc attgaagatc 3900cactcggctt ggctcctcca agatgggccg atgatagaga ttttgaccta tcatggcatc 3960taagacgata tactttagcg gaacctagga cttgggacgg cgtcctagat ttcgcacgta 4020ctgcagagat gacagctttt gataaacgta gacctttgtg ggagttcaca atcttagatg 4080gtcttaatga tggtagatca gcgttggtta tgaaggttca ccattcactc acggatggtg 4140tctctggtat gcaaattgcc agagaaatcg tggactttac tagagaaggt acgccacgac 4200caggacgtac agatagagct acagctgttc ctcatggagg ctcttctaga cctccttcta 4260gacttagttg gtatagagat acagctgcag acgtaacaca ccgagctgcg aacatcttgg 4320gtagaaattc tgttaggcta gttagagcgc cacgtgctac atggagagaa gccactgcgt 4380tagctggttc cactttaaga ttaaccagac cagttgtttc cacattgtca ccagtgatga 4440ctaagagatc aacaagacga cattgtgctg tcatcgacgt ccctgtagaa gctctcgcac 4500aggctgcagc agccgcagct gggtctatca atgacgcttt ccttgctgca gtcctgttgg 4560gtatggcaaa gtaccataga cttcatggtg ccgaaatcag agaattacgt atgactttac 4620caatatcttt aaggacagaa acagatccat taggtgggaa tagaatttcc ctagccagat 4680tcgctttgcc tactgatatt gatgatccag ctgagttgat gaggagggta cacgctactg 4740tagatgcatg gagaagagaa ccagcaatac cattttcccc tatgattgct ggtgccgtaa 4800acttacttcc tgcctcaact ttagggaaca tgttgaaaca cgttgacttt gtagcatcta 4860acgtcgctgg ctcaccagtt cctctattca tagccggatc agagatccta cattactacg 4920cgttctcacc aactcttgga tctgcattca atgttacgct gatgagttac accactcaat 4980gctgtgtcgg gataaacgct gatacagacg ctgtacctga tcttgccaca ctgaccgaaa 5040gtttggcaga tggattcaga gccgttttgg gcttatgtgc taagactaca gacacaagag 5100tggtggtggc tagctaaccc aagcttggta ccgcggctag ctaagatccg ctctaaccga 5160aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt 5220agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta 5280cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaagatcct 5340ccggatcgtt tcgccggcgt ttatccagct gcattaatga atcggccaac gcgcggggag 5400aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 5460cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 5520atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 5580taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 5640aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5700tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5760gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 5820cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 5880cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 5940atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6000tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 6060ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6120acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 6180aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 6240aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 6300tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 6360cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 6420catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 6480ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 6540aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 6600ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 6660caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 6720attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 6780agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 6840actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6900ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6960ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 7020gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 7080atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 7140cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 7200gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 7260gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 7320ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg cttcattttg 7380tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt 7440ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat 7500ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg 7560catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac 7620ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta 7680gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact 7740gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa 7800agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca 7860agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca 7920gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt 7980ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc 8040tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa 8100aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat 8160atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag 8220cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag 8280tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag 8340aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg 8400caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc 8460tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt 8520atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat 8580tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc 8640actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat 8700actaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 8760ttcgtc 8766348346DNAArtificial sequenceplasmid pSP-B4 34tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 1860ttcgctatta cgccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 1920atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt 1980tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta 2040tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta 2100tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc 2160gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca 2220gatcttatcg tcgtcatcct tgtaatccat cgatactagt gcggccgctt gttttatatt 2280tgttgtaaaa agtagataat tacttccttg atgatctgta aaaaagagaa aaagaaagca 2340tctaagaact tgaaaaacta cgaattagaa aagaccaaat atgtatttct tgcattgacc 2400aatttatgca agtttatata tatgtaaatg taagtttcac gaggttctac taaactaaac 2460cacccccttg gttagaagaa aagagtgtgt gagaacaggc tgttgttgtc acacgattcg 2520gacaattctg tttgaaagag agagagtaac agtacgatcg aacgaacttt gctctggaga 2580tcacagtggg catcatagca tgtggtacta aaccctttcc cgccattcca gaaccttcga 2640ttgcttgtta caaaacctgt gagccgtcgc taggaccttg ttgtgtgacg aaattggaag 2700ctgcaatcaa taggaagaca ggaagtcgag cgtgtctggg ttttttcagt tttgttcttt 2760ttgcaaacaa atcacgagcg acggtaattt ctttctcgat aagaggccac gtgctttatg 2820agggtaacat caattcaaga aggagggaaa cacttccttt ttctggccct gataatagta 2880tgagggtgaa gccaaaataa aggattcgcg cccaaatcgg catctttaaa tgcaggtatg 2940cgatagttcc tcactctttc cttactcacg agtaattctt gcaaatgcct attatgcaga 3000tgttataata tctgtgcgtc ttgagttgaa gtcaggaatc taaaataaaa attaaggtta 3060ataaaaagag gaaagaaaaa aaaattaatc gatttacaga aacttgcaca ctaaaaatac 3120acaactaaaa gcaattacag tatgggaagt catcgacgtt atctctacta tagtatatta 3180tcatttctat tattatcctg ctcagtggta cttgcaaaac aagataagac cccattcttt 3240gaaggtactt ccaggccggc cgcacacacc atagcttcaa aatgtttcta ctcctttttt 3300actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac 3360agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg 3420tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa 3480atttttatca cgtttctttt tcttgaaaat tttttttttt gatttttttc tctttcgatg 3540acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3600tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3660taatctaagt tttaattaca aggatcccgc tcgagatgtt ctggccaacc aaaaaggatt 3720tgaaaactgc aatggaagta tttgctctct tccaatgggc cctttccgct ttggtaattg 3780tcactactgt gatcatagtc aatttgtatc ttgttgtgtt tacatcatat tggccagtga 3840cggtcttaat gttgacatgg ttagcattcg attggaaaac accagaaaga ggtggcagga 3900gattcacatg tgtccgtaag tggagattgt ggaagcacta ctctgattac ttccctttga 3960aaatggttaa gactaaggac atatcaccag atagaaacta catcttagta tgtcatccac 4020atggtcttat ggcacattca tgtttcggac atttcgccac agatacaact ggattcagta 4080agacttttcc tggtatcact ccttacatgc taacattagg cgcctttttc tgggttccat 4140tccttagaga ctatgttatg tccactggct catgctctgt gtccagaagc tcaatggact 4200tcctcctaac acaaaaagga actggaaaca tgttggttgt agttgtaggt ggtttagctg 4260agtgtcgtta ctctacgcca ggctctacaa ccctgttttt gaaaaagaga cagggtttcg 4320tgagaactgc gttgaagcat ggtgtttctc tgatcccagc ttacgctttc ggggaaactg 4380atctctacga tcaacacata ttcacaccag gtggttttgt caatagattt cagaaatggt 4440ttcaaaagat ggtacacatc tacccatgcg ctttctatgg cagagggctc accaaaaact 4500catgggggct actaccttat tcacagcctg ttaccacagt ggttggagaa cctttacctc 4560tgccaaagat tgaaaaccct tccgaagaga ttgttgcgaa gtaccataca ctgtacatcg 4620atgcacttag gaagctattc gaccaacaca aaactaagtt tggtattagt gaaacccaag 4680agttggtcat tgtttaaccc aagcttggta ccgcggctag ctaagatccg ctctaaccga 4740aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt 4800agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta 4860cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaagatcct 4920ccggatcgtt tcgccggcgt ttatccagct gcattaatga atcggccaac gcgcggggag 4980aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 5040cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 5100atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 5160taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 5220aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5280tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5340gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 5400cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 5460cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 5520atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 5580tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 5640ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 5700acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 5760aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 5820aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5880tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5940cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 6000catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 6060ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 6120aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 6180ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 6240caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 6300attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 6360agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 6420actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6480ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6540ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 6600gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 6660atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 6720cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 6780gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6840gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 6900ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg cttcattttg 6960tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt 7020ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat 7080ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg 7140catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac 7200ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta 7260gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact 7320gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa 7380agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca 7440agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca 7500gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt 7560ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc 7620tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa 7680aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat 7740atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag 7800cggtattcgc aatattttag tagctcgtta cagtccggtg

cgtttttggt tttttgaaag 7860tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag 7920aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg 7980caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc 8040tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt 8100atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat 8160tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc 8220actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat 8280actaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 8340ttcgtc 8346358772DNAArtificial sequenceplasmid pSP-B5 35tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 1860ttcgctatta cgccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 1920atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt 1980tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta 2040tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta 2100tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc 2160gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca 2220gatcttatcg tcgtcatcct tgtaatccat cgatactagt gcggccgctt gttttatatt 2280tgttgtaaaa agtagataat tacttccttg atgatctgta aaaaagagaa aaagaaagca 2340tctaagaact tgaaaaacta cgaattagaa aagaccaaat atgtatttct tgcattgacc 2400aatttatgca agtttatata tatgtaaatg taagtttcac gaggttctac taaactaaac 2460cacccccttg gttagaagaa aagagtgtgt gagaacaggc tgttgttgtc acacgattcg 2520gacaattctg tttgaaagag agagagtaac agtacgatcg aacgaacttt gctctggaga 2580tcacagtggg catcatagca tgtggtacta aaccctttcc cgccattcca gaaccttcga 2640ttgcttgtta caaaacctgt gagccgtcgc taggaccttg ttgtgtgacg aaattggaag 2700ctgcaatcaa taggaagaca ggaagtcgag cgtgtctggg ttttttcagt tttgttcttt 2760ttgcaaacaa atcacgagcg acggtaattt ctttctcgat aagaggccac gtgctttatg 2820agggtaacat caattcaaga aggagggaaa cacttccttt ttctggccct gataatagta 2880tgagggtgaa gccaaaataa aggattcgcg cccaaatcgg catctttaaa tgcaggtatg 2940cgatagttcc tcactctttc cttactcacg agtaattctt gcaaatgcct attatgcaga 3000tgttataata tctgtgcgtc ttgagttgaa gtcaggaatc taaaataaaa attaaggtta 3060ataaaaagag gaaagaaaaa aaaattaatc gatttacaga aacttgcaca ctaaaaatac 3120acaactaaaa gcaattacag tatgggaagt catcgacgtt atctctacta tagtatatta 3180tcatttctat tattatcctg ctcagtggta cttgcaaaac aagataagac cccattcttt 3240gaaggtactt ccaggccggc cgcacacacc atagcttcaa aatgtttcta ctcctttttt 3300actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac 3360agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg 3420tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa 3480atttttatca cgtttctttt tcttgaaaat tttttttttt gatttttttc tctttcgatg 3540acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3600tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3660taatctaagt tttaattaca aggatcccgc tcgagatgag attactgacc gctgtcgatc 3720aactctttct attgttggag agtagaaagc acccaatgca cgttggtgga ctgttcctat 3780tcgagcttcc agagaatgct gacattagtt tcgttcacca gcttgttaag caaatgcaag 3840attccgacgt accaccaaca ttcccattca atcaggttct ggaacacatg atgttttgga 3900aggaggacaa aaactttgac gtagaacatc atctacacca tgtggcttta ccaaaacctg 3960ccagagttag agaattactc atgtacgttt ccagggaaca tgggaggttg ctcgatagag 4020caatgccact atgggagtgc catgtgatcg aaggtattca accagagact gaaggttctc 4080cagagagatt cgcattgtat ttcaagattc atcattcctt agtcgatggt atcgccgcta 4140tgaggttggt gaaaaagtca ttatcacagt caccaaacga accagttacc cttccaatct 4200ggtctttgat ggctcaccat agaaaccaaa tcgatgccat cttcccaaag gaaagatcag 4260ccttgcgtat cttaaaggaa caagtttcta caatcaagcc tgtgtttact gaactcttga 4320ataacttcaa aaactacaat gacgatagtt acgtcagcac ttttgacgct cctagatcaa 4380tccttaaccg tagaatttct gcctcaagac gtattgcagc gcagtcatac gatatcaaaa 4440gattcaatga catagcggag agaatcaaca tttccaaaaa cgatgtggtt ttggcagtat 4500gttccggtgc tattagaaga taccttatct ctatggatgc tttaccatca aaacctctga 4560tagcattcgt tcctatgtct ttgcgaactg atgatagtat agctggaaac caattgagtt 4620ttgtactagc gaatctgggc acacatttgg atgatccatt atctagaatc aagctcattc 4680atcgtagcat gaacaactct aagagaagat tcagaaggat gaaccaagca caagttatca 4740attactccat agtatcttac gcatgggaag gcattaactt ggccactgat cttttcccta 4800aaaagcaagc ctttaactta atcatctcta acgtcccagg ctcagaaaaa cctttgtatt 4860ggaatggtgc aagattagaa tcactatatc ctgcttcaat cgtgtttaac ggacaagcta 4920tgaatatcac gcttgcatct tacttggaca agatggaatt cggtataact gcttgttcta 4980aagctctacc tcatgtccaa gatatgttga tgcttattga ggaagagcta caactgctgg 5040aatctgttag caaggaacta gaattcaatg ggattacagt aaaagataag tcagagaaaa 5100agctgaaaaa gttggcccct taacccaagc ttggtaccgc ggctagctaa gatccgctct 5160aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt 5220tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg 5280cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 5340gatcctccgg atcgtttcgc cggcgtttat ccagctgcat taatgaatcg gccaacgcgc 5400ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 5460ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 5520cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 5580gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 5640tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 5700ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 5760atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 5820gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 5880tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 5940cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 6000cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 6060tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6120cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6180cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6240gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 6300gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 6360gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 6420ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 6480atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 6540agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 6600ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 6660tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 6720ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 6780caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 6840gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 6900atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 6960accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7020aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7080gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7140tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7200aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7260ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 7320aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc 7380attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct 7440gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg 7500cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 7560gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat 7620ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc 7680atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt 7740tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat 7800aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt 7860ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg 7920tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct 7980tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat 8040tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa 8100cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta 8160ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg 8220tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt 8280tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt 8340ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg 8400aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt 8460gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc 8520gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta 8580tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat 8640gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg 8700gatcatacta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 8760ggccctttcg tc 8772

Claims

1. A fungal cell system for producing fatty acyl ethyl esters (FAEE), said system comprising: a) a fungal cell, and b) an expression vector encoding at least one wax synthase, wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester transporter(s) of said fungal cell.

2. A fungal cell system according to claim 1, wherein said fungal cell is a yeast cell.

3. A fungal cell system according to claim 1, wherein said fungal cell is selected from the group of fungal cells consisting of Saccharomyces, Saccharomyces cerevisae Hansenula polymorpha, Kluyveromyces, Pichia, Candida albicans, Aspergilli, Rhodotorula rubra, Torulopsis, Trichosporon cutaneum, Trichoderma reesei, Apiofrichum curvafum, Yarrowia lipolytica, and Cryptococcus curvatus.

4. A fungal cell system according to any one of claims 1-3, wherein said modification is genetic and is effectuated by the introduction of one or more exogenous expression vector(s) into said fungal cell.

5. A fungal cell system according to claim 4, wherein said one or more exogenous expression vector(s) is a plasmid.

6. A fungal cell system according to any one of claims 1-5, wherein said genetic modification provides for an increased supply of fatty acyls to the metabolism of said fungal cell.

7. A fungal cell system according to any one of claims 1-6, wherein said fungal cell is genetically modified to stimulate overproduction of fatty acids.

8. A fungal cell system according to any one of claims 1-7, wherein the modification is performed to any one or more of the following genes, or its expression products: ACB1 (ACBP, acyl-CoA-binding protein), ACC1 (Acetyl-CoA carboxylase), FAS1, FAS2 (Fatty acid synthase), gapN (NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase), ACS1 (Acetyl-CoA synthetase), DGA1 (Acyl-CoA:diacylglycerol acyltransferas), LRO1 (Lecithin: cholesterol acyltransferase), ARE1, ARE2 (Acyl-CoA:sterol acyltransferase), PDX1 (Peroxisomal acyl-CoA oxidase).

9. A fungal cell system according to claim 8, wherein the modification is performed by a knockout/deletion of one or more of the genes DGA1, LRO1, ARE1, ARE2, and PDX1.

10. A fungal cell system according to claim 8, wherein the modification is performed by overexpressing one or more gene product(s) by the introduction of one or more expression vector(s) encoding said one or more gene product(s), said one or more gene product(s) being selected from the group consisting of: acyl-CoA-binding protein, Acetyl-CoA carboxylase (ACC1), NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase, Fatty acid synthases (FAS1, FAS2) and Acetyl-CoA synthetase (ACS1).

11. A fungal cell system according to claim 10, wherein said modification provides for an overexpression of ACC1 in combination with an increased expression of FAS1 and FAS2.

12. A fungal cell system according to any one of claim 10 or 11, wherein the modification of ACC1 is performed by the introduction of an expression vector and an increased expression of FAS1/FAS2 is performed by replacing the promoter thereof.

13. A fungal cell system according to claim 12, wherein Ser659Ala and Ser1157Ala of said ACC1 gene is replaced (SEQ ID NO:16).

14. A fungal cell system according to any one of the preceding claims, wherein said wax synthase encoded by said expression vector is heterologous.

15. A fungal cell system according to any one of the preceding claims, wherein said wax synthase is obtained from the one or more of the species Mycobacterium, Rhodococcus, Acinetobacter, Mus Musculus and/or Marinobacter.

16. A fungal cell system according to claim 15, wherein said at least one wax synthase is selected from the group consisting of Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter articus 273-4.

17. A fungal cell system according to claim 16, wherein the gene expressing said wax synthase is codon optimized and comprises a sequence encoded by any one of SEQ ID NO:1, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and/or SEQ ID NO 7.

18. A fungal cell system according to claim 16 or 17, wherein said wax synthase is encoded by one or more of the following expression vectors pSP-B1, pSP-B2, pSP-B3, pSP-B4 and/or pSP-B5.

19. A fungal cell system according to any one of the preceding claims, wherein said expression vector encoding said one or more wax synthase(s) is an episomal plasmid (single copy plasmids) or a high-copy plasmid.

20. A fungal cell system according to any one of the preceding claims, wherein said expression vector encoding said wax synthase provides for chromosomal integration into the chromosome of said fungal cell.

21. A fungal cell system according to any one of the preceding claims, wherein carbohydrates are supplied as an external substrate to said fungal cell system for the production of FAEE.

22. A fungal cell system according to claim 21, wherein said carbohydrates are selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose.

23. A fungal cell system according to any one of the preceding claims, wherein additionally either or both of genes Eht1p and Eeb1p of said fungal cell, are overexpressed by said fungal cell.

24. Use of an fatty acyl ester, such as a fatty acyl ethyl ester (FAEE), produced by a fungal cell system according to any one of the preceding claims as a component in a biofuel, such as biodiesel, a lubricant, cosmetic, linoleum, printing ink, and/or a solid wax ester used for candles and/or polishes.

25. A composition comprising a fungal cell system according to any one of the preceding claims, said composition further comprising at least one additional component selected from the group consisting of: buffers; stabilizers; protease-inhibiting agents; hydrolytic enzymes, saccharolytic enzymes; cell membrane- and/or cell wall-preserving compounds, nutritional media appropriate to the cell; and the like.

26. A method for producing fatty acyl ethyl esters (FAEE), said method comprising: a) providing a fungal cell system according to any one of claims 1-23 and, b) adding one or more sources of carbohydrates as an external substrate to said fungal cell system in a culture broth; c) and wherein said FAEE are thereafter retrieved by extraction from said culture broth.

27. A method according to claim 26, wherein said carbohydrates are selected from the group consisting of: glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose.

28. A composition comprising: a) a fungal cell which metabolism is modified thereby possessing an increased flux towards fatty acid biosynthesis; and b) one or more expression vectors encoding one or more wax synthase(s)

29. Use of a composition according to claim 28 for producing biofuel esters, such as biodiesel, lubricants, cosmetics, linoleum and printing inks, and/or the solid waxes used for candles and polishes.

30. A yeast cell having an increased metabolic flux towards fatty acid ester biosynthesis, said yeast cell expressing at least one wax synthase selected from the group consisting of Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 and Psychrobacter articus 273-4) in combination with over-expressing the protein ACBP (acyl-CoA-binding protein)

31. A yeast cell according to claim 30, wherein said yeast cell is Saccharomyces cerevisae.

32. A fungal cell system for overproducing fatty acids, comprising a fungal cell having an increased metabolic flux towards fatty acid biosynthesis, characterized by at least over-expressing ACC1 (Acetyl-CoA carboxylase).

33. A fungal cell system for overproducing fatty acids, comprising a) a fungal cell having an increased metabolic flux towards fatty acid biosynthesis, characterized by at least over-expressing ACC1 (Acetyl-CoA carboxylase), and wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester and/or fatty acids transporter(s) of said fungal cell.

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