Patent application title: PRODUCTION OF BUTANOL
Inventors:
Elizabeth Jenkinson (Abingdon, Oxford, GB)
Preben Krabben (Didcot, Oxfordshire, GB)
Amanda Harding (Abingdon, Oxfordshire, GB)
Edward Green (Bisham, Marlow, Buckinghamshire, GB)
Assignees:
Green Biologics Ltd.
IPC8 Class: AC12P728FI
USPC Class:
4352527
Class name: Micro-organism, per se (e.g., protozoa, etc.); compositions thereof; proces of propagating, maintaining or preserving micro-organisms or compositions thereof; process of preparing or isolating a composition containing a micro-organism; culture media therefor bacteria or actinomycetales; media therefor clostridium
Publication date: 2015-12-24
Patent application number: 20150368678
Abstract:
The present invention relates to methods of making solvents, such as
butanol, acetone or ethanol. In particular, the invention relates to a
process for producing a solvent, comprising the step of culturing a
solventogenic Clostridium sp. in a culture medium in a culture vessel in
the presence of a polysaccharide, wherein the Clostridium sp. is one
which is capable of producing a CGTase, and wherein the polysaccharide is
one which is a substrate for the CGTase, and harvesting solvent from the
culture medium. Preferably, the Clostridium sp. is Clostridium
saccharoperbutylacetonicum N1-4(HMT) or N1-504. The invention also
relates to butanol, acetone and ethanol made by such a process.Claims:
1. A process for culturing a micro-organism, the process comprising the
step: (i) culturing a Clostridium sp. in a culture medium in a culture
vessel in the presence of a polysaccharide, wherein the Clostridium sp.
is one which produces a CGTase, and wherein the polysaccharide is one
which is a substrate for the CGTase.
2.-19. (canceled)
Description:
[0001] The present invention relates to methods of making solvents, such
as butanol, acetone or ethanol. In particular, the invention relates to a
process for producing a solvent, comprising the step of culturing a
solventogenic Clostridium sp. in a culture medium in a culture vessel in
the presence of a polysaccharide, wherein the Clostridium sp. is one
which is capable of producing a CGTase, and wherein the polysaccharide is
one which is a substrate for the CGTase, and harvesting solvent from the
culture medium. Preferably, the Clostridium sp. is Clostridium
saccharoperbutylacetonicum N1-4(HMT) or N1-504. The invention also
relates to butanol, acetone and ethanol made by such a process.
[0002] Solvent-producing Clostridia were first used during the 1920's and 1930's for the industrial production of acetone, butanol and ethanol. During the 1950's the establishment of more efficient petrochemical techniques to synthesise these solvents lead to the abandonment of such large-scale bacterial fermentations. However, in the present environment, with rising oil prices and increasing pressure for the development of biofuels, the interest in Clostridial fermentations for the production of solvents is being renewed. This has also been helped by advancements in the biological understanding of these solventogenic Clostridia and the development of microarray assays. These areas of research have opened up the possibility of engineering new strains capable of over-producing butanol, further improving the economic use of solventogenic fermentations.
[0003] It has long been known that growth by solventogenic Clostridia is biphasic, with exponential growth being characterised by profuse acid production and solvent production occurring at the onset of the stationary phase (review by Jones and Woods, 1986, Microbiological Reviews, 50:484-524). There remains a need, however, for enhanced methods of producing ABE.
[0004] Thang et al. (Appl. Biochem. Biotechnol. (2010) May; 161(1-8):157-70) describes the production of acetone-butanol-ethanol (ABE) in direct fermentation of cassava by Clostridium saccharoperbutylacetonicum N1-4. This paper refers to the hydrolysis of cassava starch into maltose and glucose by the bacteria's own α-amylase and glucoamylase enzymes. These enzymes are said to continue hydrolysing starch throughout the fermentation. Batch fermentations were performed in stirred-tank fermenters wherein the initial pH was 6.2 and the temperature was maintained at 30° C. Anaerobic conditions were ensured by sparging the fermentation broth with oxygen-free nitrogen and by flushing the fermentation broth at the beginning of the fermentation with nitrogen gas.
[0005] It has now been found, however, that certain other Clostridium saccharoperbutylacetonicum strains do not have obvious gene homologues which encode α-amylase or glucoamylase. In these other strains--which include Clostridium saccharoperbutylacetonicum N1-4(HMT) and N1-504--the hydrolysis of starch-based materials such as cassava actually occurs by means of a novel cyclodextrin glucanotransferase (CGTase) enzyme.
[0006] This new knowledge has allowed the conditions for solvent-producing processes involving certain Clostridium saccharoperbutylacetonicum strains to be optimised in order to enhance the activity of the CGTase and hence to increase the production of acetone, butanol and/or ethanol. In particular, the CGTase has been found to perform optimally at a temperature which is significantly different from that used in Thang et al. (supra). Additionally, it has been found that the CGTase is not affected by the presence of oxygen and that growing cultures of some Clostridium saccharoperbutylacetonicum strains may in fact be inoculated aerobically.
[0007] It should be noted that wherein Clostridium thermohydrosulfuricus was previously classified as a Clostridial species, it has now been reclassified as Thermoanaerobacter thermohydrosulfuricus (Collins, et al (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Bacteriol., 44(4), 812-26). The genus Thermoanaerobacter has now clearly established by sequence analysis and shown that it forms a separate and distinct genus from Clostridium sensu stricto (Cluster I) (Stackebrandt et al. (1999) Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol. Med. Microbiol., 24(3), 253-8).
[0008] In one embodiment, therefore, the invention provides a process for culturing a micro-organism, the process comprising the step:
[0009] (i) culturing a Clostridium sp. in a culture medium in a culture vessel in the presence of a polysaccharide, wherein the Clostridium sp. is one which produces a CGTase, and wherein the polysaccharide is one which is a substrate for the CGTase.
[0010] The invention also provides a process for producing a solvent, the process comprising the step:
[0011] (i) culturing a solventogenic Clostridium sp. in a culture medium in a culture vessel in the presence of a polysaccharide, wherein the Clostridium sp. is one which is produces a CGTase, and wherein the polysaccharide is one which is a substrate for the CGTase, and optionally harvesting solvent from the culture medium. Preferably, the solvent is butanol, acetone or ethanol, most preferably butanol.
[0012] In a further embodiment, the invention provides a process for producing butyric acid or an acid derivative thereof, the process comprising the steps:
[0013] (i) culturing a solventogenic Clostridium sp. in a culture medium in a culture vessel in the presence of a polysaccharide, wherein the Clostridium sp. is one which is capable of producing a CGTase, and wherein the polysaccharide is one which is a substrate for the CGTase, and optionally harvesting butyric acid or an acid derivative thereof from the culture medium. Preferably, the butyric acid or acid derivative thereof is subsequently converted to a solvent, most preferably to butanol. Preferably, the CGTase has at least 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3.
[0014] Preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum, more preferably a N1-strain. Even more preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum N1-4(HMT) or N1-504. In some embodiments, the Clostridium sp. is cultured at a temperature of 32-47° C.
[0015] The Clostridium sp. is one which produces or is capable of producing a cyclodextrin glucanotransferase (CGTase). CGTases are also known as cyclodextrin glycosyl transferases and cyclodextrin glucosyltransferases. Whilst CGTases are generally capable of catalysing more than one reaction, the most important activity is the production of cyclic dextrins from substrates such as starch, amylose and other polysaccharides. In this process, the polysaccharide chain is cleaved and the ends are joined by the CGTase in order to produce a cyclic dextrin, i.e. a cyclodextrin. The size of the cyclodextrin (i.e. the number of sugar residues it incorporates) is dependent on the distance apart of the ends.
[0016] The CGTases fall within the general EC classification 2.4.1 (hexosyltransferases). In some embodiments, the CGTase falls within classification EC 2.4.1.248 (cycloisomaltooligosaccharide glucanotransferase). In other embodiments of the invention, the CGTase falls within classification EC 2.4.1.19 (cyclomaltodextrin glucanotransferase).
[0017] In one embodiment, the amino acid sequence of the CGTase:
[0018] (a) comprises the amino acid sequence set forth in SEQ ID NO: 1 or 3;
[0019] (b) comprises an amino acid sequence which has at least 70% sequence identity with SEQ ID NO: 1 or 3;
[0020] (c) is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or 4; or
[0021] (d) is encoded by a nucleotide sequence which has at least 70% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 4.
[0022] Variants or derivatives of the polypeptide of SEQ ID NO: 1 or 3 may also be used. The CGTase may be altered in various ways including substitutions, deletions, truncations, and/or insertions of one or more (e.g. 2-5, 2-10) amino acids, preferably in a manner which does not substantially alter the biological activity of the CGTase. Guidance as to appropriate amino acid changes that do not affect biological activity of the CGTase may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Nat'l. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be also made.
[0023] In particular, substitution of one hydrophobic amino acid such as isoleucine, valine, leucine or methionine for another may be made; or the substitution of one polar amino acid residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, may be made.
[0024] One or more (e.g. 1-5, 1-10) amino acids in the CGTase may be substituted by their corresponding D-amino acids, preferably at the N- and/or C-terminus.
[0025] In particular, the invention provides the use of a variant of the CGTase of SEQ ID NO: 1 or 3, wherein the amino acid sequence of the variant comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 1 or 3, preferably using the blastp method of alignment.
[0026] Percentage amino acid sequence identities and nucleotide sequence identities may be obtained using the BLAST methods of alignment (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; and http://www.ncbi.nlm.nih.gov/BLAST). Preferably the standard or default alignment parameters are used.
[0027] Standard protein-protein BLAST (blastp) may be used for finding similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes. Preferably the standard or default alignment parameters are used. In some instances, the "low complexity filter" may be taken off.
[0028] BLAST protein searches may also be performed with the BLASTX program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. (See Altschul et al. (1997) supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs may be used.
[0029] The invention particularly relates to uses of CGTases of SEQ ID NO: 1 or 3 or to variants of the CGTases of SEQ ID NO: 1 or 3 as defined herein, wherein the amino acid which corresponds to the amino acid at position 223 is a small amino acid, e.g. glycine, alanine, leucine, serine, threonine or valine, preferably glycine. The size of the amino acid residue at position 223 may be involved in determining the size of any cyclodextrin products or ratio of any cyclodextrin products.
[0030] In general, a nucleic acid encoding the CGTase will be stably incorporated into the genome of the Clostridium sp.
[0031] The Clostridium sp. may be a wild-type Clostridium sp. or a mutant (e.g. genetically engineered) Clostridium sp. or a recombinantly-produced Clostridium sp. Preferably, the Clostridium sp. comprises an endogenous CGTase gene (i.e. incorporated into its genome). In other preferred embodiments, the Clostridium sp. does not comprise a vector encoding a heterologous CGTase.
[0032] The Clostridium sp. is one which produces or is capable of producing a CGTase. Preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum, more preferably a N1-strain. Even more preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum N1-4(HMT) or N1-504. In some embodiments, the recombinant bacteria is not Clostridium saccharoperbutylacetonicum N1-4. In other embodiments, the recombinant bacteria is not Clostridium saccharoperbutylacetonicum N1-4(HMT). In other embodiments, the recombinant bacteria is not Clostridium saccharoperbutylacetonicum N1-504.
[0033] Suitable culture vessels and culture media are known in the art. Generally, the culture media will be an aqueous media. The Clostridium sp. is cultured under conditions such that it expresses its endogenous CGTase, thus enabling the digestion of the polysaccharide into a hydrolysate or other product. Suitable conditions are known in the art.
[0034] The process of the invention may be operated in any suitable manner. For example, it may be operated as a batch process, fed-batch process or any form of continuous process or perfusion process. Preferably, the process is allowed to proceed for a time which is sufficient for the CGTase to digest the polysaccharide, either partially or completely.
[0035] In some embodiments of the invention, no additional amylases and/or glucoamylases from non-Clostridial bacteria are present in the culture vessel. For example, Bacillus subtilis amylase is preferably not present in the culture vessel. Preferably, glucoamylase from Rhizopus niveus is not present in the culture vessel.
[0036] Genes encoding α-amylase and glucoamylase are not present in either C. saccharoperbutylacetonicum N1-4 (HMT) or N1-504. In some embodiments of the invention in which these bacteria or derivatives thereof are used, α-amylase and/or glucoamylase may be added to the culture media or the polysaccharide may be pre-treated with α-amylase and/or glucoamylase. In some embodiments, recombinant α-amylase and/or recombinant glucoamylase is produced by the Clostridium sp. (preferably recombinant C. saccharoperbutylacetonicum N1-4 (HMT) or N1-504) in the culture medium.
[0037] The Clostridium sp. is cultured in the culture vessel at a temperature which is optimal for the activity of the CGTase or, in processes for the production of a solvent or butyric acid or acid derivative thereof, which is optimal for the production of the aforementioned products. This temperature range has been found to be 32-47° C. Preferably, the temperature is 32-45° C., more preferably 32-40° C. or 33-40° C., and most preferably 32-36° C. or 33-36° C.
[0038] The initial pH of the culture medium is preferably between pH 5.3 and pH 6.8, more preferably between pH 5.5 and pH 6.2. In some embodiments, it is preferably pH 5.4-5.6 or pH 6.1-6.3, and most preferably about pH 5.5 or about pH 6.2. In other embodiments, the initial pH is 6.4-6.6. In some preferred embodiments, the culture temperature is 34-36° C. and the initial pH is pH 5.4-5.6 or pH 6.1-6.3. During acidogenic growth, the pH may be allowed to drop to about pH 5.3. It may then rise to pH 6.0-6.3 during the reassimilation and solventogenic phase.
[0039] The polysaccharide is one which is a substrate for the CGTase. The polysaccharide may be a lignocellulosic feedstock, i.e. a feedstock comprising hemicellulose, cellulose and lignin; or a cellulosic feedstock.
[0040] In one embodiment, the polysaccharide may be a glucose-containing or glucose-based polysaccharide. In such embodiments, the glucose molecules will in general be linked in the polysaccharide by α(1→4) and/or α(1→6) glycosidic bonds. In some embodiments, the substrate is a starch-based substrate, e.g. starch, amylopectin, amylose or glycogen. Most preferably, the polysaccharide is starch or a starch-based material, e.g. corn, corn starch, corn mash, potato, potato starch, potato mash, potato peeling, potato chips, cassava, cassava starch, cassava chips, sago, sago starch, or `soluble starch` (e.g. as sold by Fisher/Sigma).
[0041] In some embodiments, butyric acid or an acid derivative thereof is harvested from the culture medium. This may be done by any suitable continuous or discontinuous process. Preferably, the harvested butyric acid or an acid derivative thereof is subsequently converted to a solvent, most preferably to butanol.
[0042] In other embodiments, solvent is harvested from the culture medium. This may be done by any suitable continuous or discontinuous process. Solvents may include butanol, ethanol and/or acetone, preferably butanol.
[0043] Isolation of butanol, acetone and/or ethanol from the culture medium may be carried out by any suitable means. Examples of include gas-stripping, pervaporation, distillation and solvent extraction.
[0044] Whilst it has been previously been reported that it has been necessary to sparge the culture media with oxygen-free nitrogen in order to achieve anaerobiosis (Thang et al., supra), it has now been found that the CGTase is not affected by the presence of oxygen and that growing cultures of Clostridium sp. (and C. saccharoperbutylacetonicum N1-4(HMT) and N1-504 in particular) may in fact be inoculated aerobically, with no special precautions necessary to exclude oxygen. Furthermore, the culture vessel may be operated with air in the head-space above the culture medium.
[0045] In some embodiments of the invention, therefore, step (i) comprises inoculating and growing Clostridium sp. (and C. saccharoperbutylacetonicum N1-4(HMT) and N1-504 in particular) in a culture medium in a culture vessel under aerobic conditions or conditions which are not oxygen-free.
[0046] In a further embodiment, the invention relates to a solvent, preferably butanol, acetone or ethanol, which is obtained by a process of the invention. Most preferably, the solvent is butanol.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1 shows the effect of temperature on CGTase activity.
[0048] FIGS. 2-4 show the effect of temperature (30° C., 33° C. and 35° C.) and pH (5.0, 5.3, 5.5, 5.8, 6.2 and 6.8) of CGTase activity.
[0049] FIG. 5 combines photographs of plates from experiments done at 30° C., 33° C. and 35° C., all at pH 6.2, taken from FIGS. 2 to 4 and scaled so that the photo of each plate is the same size, to enable comparison between the three different temperatures. The clearing zone on the 35° C. plate is clearly the largest and most intense.
EXAMPLES
[0050] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
Example 1
Effect of Temperature on CGTase Activity
Method:
[0051] Freeze dried Clostridium saccharoperbutylacetonicum N1-4 (HMT) was grown up in RCM overnight before inoculating into CGM containing either 5% starch or 5% glucose. Cultures were grown at 32° C. for 8 hours before being subcultured a second time into CGM containing 5% starch or 5% glucose. These cultures were grown overnight at 32° C. before recovering the supernatant by centrifugation. This was filter sterilised using a 0.2 μm filter and sodium azide was added to a final concentration of 0.02%. 500 μl of supernatant was concentrated approx. 10-fold using 10 kDa MWCO Vivaspin columns according to manufacturer's instructions.
[0052] Starch agar plates were prepared using 1.5% agar and 1% soluble starch. pH was not adjusted and was approx. 6.5.
[0053] 10 μl of unconcentrated supernatant sample or 2 μl of the concentrated samples were spotted onto the plates and left to soak in. As a control, B. amyloliquefaciens α-amylase was diluted 1000-fold and 2 μl was spotted onto the plate. 5 μl dH2O was used as a negative control. The plates were wrapped in foil and incubated at 33° C., 36° C., 40° C., 45° C., 50° C., 55° C. or 60° C. overnight.
[0054] Areas of starch degradation were visualised by adding iodine solution to the plate for 20-30 sec. Excess iodine was removed before imaging.
Results:
[0055] The results are shown in FIG. 1. No CGTase activity was observed above 50° C. Some activity was seen between 33° C. and 45° C., with optimal activity at 36° C., based on intensity and size of clearing zone. (This was based on a estimated measurement: the clearing zone of concentrated spots in the sample at 36° C. was approx. 1.5× larger than clearing zone at 33° C. and 1.3× larger than clearing zone at 40° C.).
Example 2
Effect of Temperature and pH on CGTase Activity
Method:
[0056] Supernatant was prepared from Clostridium saccharoperbutylacetonicum N1-4 (HMT) cultures grown for 72 hours, 32° C. in CGM supplemented with 5% glucose or 5% soluble starch. The cultures were centrifuged at 4000×g, 10 min, RT and the supernatant was removed and filter sterilised using a 0.2 μm filter. Sodium azide was added to a final concentration of 0.02% before storage at 4° C. Supernatants were concentrated 10-fold using Vivaspin columns according to the manufacturer's instructions.
[0057] Experiments using growing cultures were all inoculated aerobically and subsequently maintained in an anaerobic environment.
[0058] Starch agar plates were prepared using 1.5% agar and 1% soluble starch. The pH of the agar was adjusted to pH 5.0, 5.3, 5.5, 5.8 or 6.2 with 10% HCl prior to autoclaving. The pH of the unadjusted starch agar was pH 6.8.
[0059] Supernatants were spotted onto starch agar in 5 μl volumes. Alpha amylase (from Bacillus amyloliquefaciens, Sigma) was diluted 100-fold in dH2O and 2 μl was plated as a positive control for starch degradation. A 5 μl volume of dH2O was plated as a negative control. The plates were wrapped in foil before being incubated for 18 h at 30° C., 33° C. or 35° C. Areas of starch degradation were visualised by adding iodine solution to the plate for 20-30 sec. Excess iodine was removed before imaging.
Results
[0060] The results are shown in FIGS. 2-4. At 30° C. optimal enzyme activity was between pH 5.8-6.8, with very little difference in size or intensity of clearing zone under these conditions. The enzyme had very little activity below pH 5.5 when incubated at 30° C.
[0061] At 33° C. optimal enzyme activity is between pH 5.5-6.8. As seen at 30° C., there is only slight differences in size or intensity of clearing zones under these conditions. Enzyme has very little activity below pH 5.3 when incubated at 33° C.
[0062] At 35° C. enzyme activity was observed between pH 5.5-6.8. Optimal activity (based on size and intensity of clearing zones) was seen at pH 5.5 and pH 6.2. The enzyme has very little activity below pH 5.3 when incubated at 35° C.
[0063] FIG. 5 shows plates from experiments done at 30° C., 33° C. and 35° C., all at pH 6.2. The plates have been scaled to make the plate sizes comparable between the three different temperatures. The clearing zone on the 35° C. plate is clearly the largest and the most intense.
[0064] The above results show that the CGTase enzyme has most activity at about 35° C.; the optimal pHs were pH 5.5 and pH 6.2. Furthermore, CGTase activity appears to be lower at 30° C. across the pH range compared to the 33° C. and 35° C. plates. No CGTase enzyme activity was observed below pH 5.3.
[0065] The enzyme plate assays were all carried out aerobically. Therefore the enzyme is not affected by the presence of oxygen.
TABLE-US-00001 SEQUENCES SEQ ID NO: 1 Clostridium saccharoperbutylacetonicum strain N1-4(HMT) MFRRKFNKVILSILVATIVSSTNMFMSGSKAQAAIGNLSENDTIYQIMVDRFYDGDKTNN ATGDAFRNTENLEDDFRYMHGGDWQGVIDKLDYIKGMGYSAIWISPVAEPQMWSRADGTG KVWPTAYHGYNVKDPNKANPYFGTKEKLKELVDKAHEKGIKVIIDIVPNHVGDYMLGKQA YYDIKGFEPAAPFNNPNWYHHNGDIDWSREHSDPQMLDDHDLGGLDDLNQDNSDAKAAMN NAIKSWFDYTGADAARVDAAKCMKPSYINELQKYIGVNTFGENFDMNVDFVKKWVGSDAE WGMLDFPLYQAINNDFASGQSFDDMSSSGTCSIKNILAQDNKYNGYANHMVTFIDNHDRN RFLTVANGNVKKLQNALVFMFTVRGVPTVFQGTEQNKGNANGASINGIADTWNRWSMVKK DYNGNVITDYFNENTDTYKLINKLNSFRQKYEALREGTQREMWSSPHLYAFSRRMDSGEN VGQEVVNVFNNSDGDQSATIPIRAESTIKVGDKFVNLFDVNDSITVQQGGVTGKQISVNL GENSGKIYVVNNETPNPDQKNVQYKVSYKNTNAQKVTLHYGTNGWKNIQDVNMTKNSNGE FEATITVNNNDILNYCIHIISPTDYWDNNGGQNWNVKVTKAEDYINDGVKSNLKSVNTTT SAAIDSGIDSTVNR The predicted N-terminal signal sequence is highlighted (predicted using signalP). SEQ ID NO: 2 Clostridium saccharoperbutylacetonicum strain N1-4(HMT) ATGTTTAGAAGAAAATTTAACAAGGTAATATTATCTATCTTAGTTGCAACAATTGTTTCA AGCACTAACATGTTTATGAGTGGAAGCAAGGCACAAGCGGCAATTGGAAATCTAAGTGAA AACGATACTATTTATCAAATTATGGTAGACAGATTTTATGATGGAGATAAAACAAATAAT GCTACAGGAGATGCATTTCGTAATACAGAAAATCTTGAAGATGATTTTAGATATATGCAC GGCGGAGATTGGCAAGGTGTTATTGATAAGTTAGATTATATTAAGGGCATGGGATACTCA GCCATTTGGATATCACCGGTTGCGGAACCACAAATGTGGTCTAGAGCTGATGGCACAGGA AAAGTATGGCCTACAGCTTATCATGGATATAATGTGAAAGATCCCAATAAGGCAAATCCT TATTTTGGAACAAAAGAAAAGCTAAAGGAGTTAGTAGATAAAGCTCACGAAAAGGGGATT AAAGTAATAATAGATATAGTTCCAAATCATGTTGGGGATTATATGTTAGGAAAACAAGCT TATTATGACATCAAGGGGTTTGAGCCGGCAGCACCTTTTAATAATCCAAATTGGTATCAT CATAATGGCGATATTGATTGGTCAAGAGAACACTCTGATCCCCAAATGTTAGATGATCAT GATTTGGGCGGTTTAGATGATTTAAATCAAGATAATTCTGATGCTAAGGCAGCTATGAAT AATGCTATTAAGTCATGGTTTGATTATACTGGAGCTGATGCAGCAAGGGTTGACGCAGCA AAATGTATGAAACCATCTTATATTAACGAGTTACAAAAGTATATAGGAGTTAATACTTTT GGAGAAAATTTTGATATGAATGTAGATTTTGTGAAGAAGTGGGTTGGATCCGATGCAGAA TGGGGAATGCTAGATTTTCCATTATATCAAGCAATAAATAATGATTTTGCATCAGGACAA TCTTTTGATGACATGTCATCATCAGGTACTTGCTCTATTAAAAATATTTTAGCACAAGAC AATAAATATAATGGTTATGCAAATCATATGGTGACTTTTATAGATAATCATGATCGTAAT AGATTTTTAACAGTAGCAAATGGTAATGTAAAAAAACTTCAAAATGCACTTGTTTTCATG TTTACTGTAAGAGGGGTACCAACAGTATTTCAAGGTACAGAACAAAACAAAGGTAATGCA AATGGAGCAAGTATAAATGGTATTGCAGATACATGGAATCGTTGGTCAATGGTTAAAAAG GATTACAATGGAAATGTAATTACAGATTATTTTAATGAGAATACAGATACTTATAAACTA ATTAACAAATTGAATTCATTTAGGCAAAAATATGAAGCCTTAAGAGAAGGTACTCAAAGA GAAATGTGGTCTTCACCACATTTATATGCATTCTCAAGAAGGATGGATTCAGGAGAAAAT GTTGGACAAGAAGTTGTAAATGTATTTAATAATTCAGATGGAGATCAAAGTGCGACCATT CCAATTAGAGCTGAAAGTACTATAAAAGTTGGAGATAAATTTGTAAATCTTTTTGATGTA AATGATTCGATCACAGTTCAACAAGGAGGTGTTACAGGAAAACAAATATCAGTGAATTTA GGAGAAAATAGTGGGAAGATTTATGTTGTTAATAATGAAACACCAAATCCAGATCAAAAG AACGTACAATATAAAGTTTCATATAAGAATACTAATGCACAAAAAGTAACACTTCATTAT GGAACTAATGGATGGAAAAACATTCAAGATGTAAATATGACTAAGAATTCCAATGGAGAA TTTGAAGCAACTATTACAGTAAATAATAATGATATTCTAAATTACTGTATTCATATTATT TCACCAACAGACTATTGGGATAATAATGGTGGACAGAATTGGAATGTAAAAGTGACTAAG GCAGAAGATTATATAAATGATGGTGTAAAGAGTAATTTGAAGAGCGTTAATACAACTACA TCAGCAGCTATAGACTCTGGGATTGATAGTACTGTAAATCGTTAA SEQ ID NO: 3 Clostridium saccharoperbutylacetonicum strain N1-504 MFRRKFNKVILSILVATIVSSTNMFMSGSKAQAAIGNLSENDTIYQIMVDRFYDGDKTNN ATGDAFRNTENLEDDFRYMHGGDWQ GVIDKLDYIKGMGYSAIWISPVAEPQMWSRADGTGKVWPTAYHGYNVKDPNKANPYFGTK EKLKELVDKAHEKGIKVIIDIVPNHVGDYMLGKQAYYDIKGFEPAAPFNNPNWYHHNGDI DWSREHSDPQMLDDHDLGGLDDLNQDNSDAKAAMNNAIKSWFDYTGADAARVDAAKCMKP SYINELQKYIGVNTFGENFDMNVDFVKKWVGSDAEWGMLDFPLYQAINNDFASGQSFDDM SSSGTCSIKNILAQDNKYNGYANHMVTFIDNHDRNRFLTVANGNVKKLQNALVFMFTVRG VPTVFQGTEQNKGNGNGAILNGIADTWNRWSMVKKDYNGNIITDYFNENTDTYKLISKLN SFRQKYEALREGTQREMWSSPHLYAFSRRMDSGENVGQEVVNVFNNSDGDQSATIPIRAE STIKVGDKLVNLFDVNDSITVQQGGVTGKQISVNLGENSGKIYVVNNETPNPDQKNVQYK VSYKNTNAQKVTLHYGTNGWKNIQDVNMTKNSNGEFEATITVNNNDILNYCIHIISPTDY WDNNGGQNWNVKVTKAEDYINDGVKSNLKSVNTTTSAAIESGIDSTVNR The predicted N-terminal signal sequence is highlighted (predicted using signalP). SEQ ID NO: 4 Clostridium saccharoperbutylacetonicum strain N1-504 atgtttagaagaaaatttaacaaggtaatattatctattttagttgcaacaattgtttca agcactaacatgttt ATGAGTGGAAGCAAGGCACAAGCGGCAATTGGAAATTTAAGTGAAAACGATACTATTTAT CAAATTATGGTAGACAGATTTTATGATGGAGATAAAACAAATAATGCTACAGGAGATGCA TTTCGTAATACAGAAAATCTTGAAGATGATTTTAGATATATGCACGGCGGAGATTGGCAA GGTGTTATTGATAAGTTAGATTATATTAAGGGCATGGGATACTCAGCCATTTGGATATCA CCGGTTGCGGAACCACAAATGTGGTCTAGAGCTGATGGCACAGGAAAAGTATGGCCTACA GCTTACCATGGATATAATGTGAAAGATCCCAATAAGGCAAATCCTTATTTTGGAACAAAA GAAAAGCTAAAGGAGTTAGTAGATAAAGCTCACGAAAAGGGGATTAAAGTAATAATAGAT ATAGTTCCAAATCATGTTGGGGATTATATGTTAGGAAAACAAGCTTATTATGACATCAAG GGGTTTGAGCCGGCAGCACCTTTTAATAATCCAAATTGGTATCATCATAATGGCGATATT GATTGGTCAAGAGAACACTCTGATCCCCAAATGTTAGATGATCATGATTTGGGCGGTTTA GATGATTTAAATCAAGATAATTCTGATGCTAAGGCAGCTATGAATAATGCTATTAAGTCA TGGTTTGATTATACTGGAGCTGATGCAGCAAGGGTTGACGCAGCAAAATGTATGAAACCA TCTTATATTAACGAGTTACAAAAGTATATAGGAGTTAATACTTTTGGAGAAAATTTTGAT ATGAATGTAGATTTTGTGAAGAAGTGGGTTGGATCCGATGCAGAATGGGGAATGCTAGAT TTTCCATTATATCAAGCAATAAATAATGATTTTGCATCAGGACAATCTTTTGATGACATG TCATCATCAGGTACTTGCTCTATTAAAAATATTTTAGCACAAGACAATAAATATAATGGT TATGCAAATCATATGGTGACTTTTATAGATAATCATGATCGTAATAGATTTTTAACAGTA GCAAATGGTAATGTTAAAAAACTTCAAAATGCACTTGTTTTCATGTTTACTGTAAGAGGG GTACCAACAGTATTTCAAGGTACAGAACAAAACAAAGGTAATGGAAATGGAGCAATTCTA AATGGTATTGCAGATACATGGAATCGTTGGTCAATGGTTAAAAAGGACTATAATGGAAAT ATAATTACAGATTATTTTAATGAGAATACAGATACTTATAAACTAATTAGCAAATTGAAT TCATTTAGGCAAAAATATGAAGCCTTAAGAGAAGGTACTCAAAGAGAAATGTGGTCTTCA CCACATTTATATGCATTCTCAAGAAGGATGGATTCAGGAGAAAATGTTGGACAAGAAGTT GTAAATGTATTTAATAATTCAGATGGAGATCAAAGTGCGACCATTCCAATTAGAGCTGAA AGTACTATAAAAGTTGGAGATAAACTTGTAAATCTTTTTGATGTAAATGATTCGATCACA GTTCAACAAGGAGGTGTTACAGGAAAACAAATATCAGTGAATTTAGGAGAAAATAGTGGG AAGATTTATGTTGTTAATAATGAAACACCAAATCCAGATCAAAAGAACGTACAATATAAA GTTTCATATAAGAATACTAATGCACAAAAAGTAACACTTCATTATGGAACTAATGGATGG AAAAACATTCAAGATGTAAATATGACTAAGAATTCCAATGGAGAATTTGAAGCAACTATT ACAGTAAATAATAATGATATTCTAAATTACTGTATTCATATTATTTCACCAACAGACTAT TGGGATAATAATGGTGGACAGAATTGGAATGTAAAAGTGACTAAGGCAGAAGATTATATA AATGATGGTGTAAAGAGTAATTTGAAGAGCGTTAATACAACTACATCAGCAGCGATAGAA TCTGGTATTGATAGTACTGTAAATCGTTAA
Sequence CWU
1
1
41674PRTClostridium saccharoperbutylacetonicum 1Met Phe Arg Arg Lys Phe
Asn Lys Val Ile Leu Ser Ile Leu Val Ala 1 5
10 15 Thr Ile Val Ser Ser Thr Asn Met Phe Met Ser
Gly Ser Lys Ala Gln 20 25
30 Ala Ala Ile Gly Asn Leu Ser Glu Asn Asp Thr Ile Tyr Gln Ile
Met 35 40 45 Val
Asp Arg Phe Tyr Asp Gly Asp Lys Thr Asn Asn Ala Thr Gly Asp 50
55 60 Ala Phe Arg Asn Thr Glu
Asn Leu Glu Asp Asp Phe Arg Tyr Met His 65 70
75 80 Gly Gly Asp Trp Gln Gly Val Ile Asp Lys Leu
Asp Tyr Ile Lys Gly 85 90
95 Met Gly Tyr Ser Ala Ile Trp Ile Ser Pro Val Ala Glu Pro Gln Met
100 105 110 Trp Ser
Arg Ala Asp Gly Thr Gly Lys Val Trp Pro Thr Ala Tyr His 115
120 125 Gly Tyr Asn Val Lys Asp Pro
Asn Lys Ala Asn Pro Tyr Phe Gly Thr 130 135
140 Lys Glu Lys Leu Lys Glu Leu Val Asp Lys Ala His
Glu Lys Gly Ile 145 150 155
160 Lys Val Ile Ile Asp Ile Val Pro Asn His Val Gly Asp Tyr Met Leu
165 170 175 Gly Lys Gln
Ala Tyr Tyr Asp Ile Lys Gly Phe Glu Pro Ala Ala Pro 180
185 190 Phe Asn Asn Pro Asn Trp Tyr His
His Asn Gly Asp Ile Asp Trp Ser 195 200
205 Arg Glu His Ser Asp Pro Gln Met Leu Asp Asp His Asp
Leu Gly Gly 210 215 220
Leu Asp Asp Leu Asn Gln Asp Asn Ser Asp Ala Lys Ala Ala Met Asn 225
230 235 240 Asn Ala Ile Lys
Ser Trp Phe Asp Tyr Thr Gly Ala Asp Ala Ala Arg 245
250 255 Val Asp Ala Ala Lys Cys Met Lys Pro
Ser Tyr Ile Asn Glu Leu Gln 260 265
270 Lys Tyr Ile Gly Val Asn Thr Phe Gly Glu Asn Phe Asp Met
Asn Val 275 280 285
Asp Phe Val Lys Lys Trp Val Gly Ser Asp Ala Glu Trp Gly Met Leu 290
295 300 Asp Phe Pro Leu Tyr
Gln Ala Ile Asn Asn Asp Phe Ala Ser Gly Gln 305 310
315 320 Ser Phe Asp Asp Met Ser Ser Ser Gly Thr
Cys Ser Ile Lys Asn Ile 325 330
335 Leu Ala Gln Asp Asn Lys Tyr Asn Gly Tyr Ala Asn His Met Val
Thr 340 345 350 Phe
Ile Asp Asn His Asp Arg Asn Arg Phe Leu Thr Val Ala Asn Gly 355
360 365 Asn Val Lys Lys Leu Gln
Asn Ala Leu Val Phe Met Phe Thr Val Arg 370 375
380 Gly Val Pro Thr Val Phe Gln Gly Thr Glu Gln
Asn Lys Gly Asn Ala 385 390 395
400 Asn Gly Ala Ser Ile Asn Gly Ile Ala Asp Thr Trp Asn Arg Trp Ser
405 410 415 Met Val
Lys Lys Asp Tyr Asn Gly Asn Val Ile Thr Asp Tyr Phe Asn 420
425 430 Glu Asn Thr Asp Thr Tyr Lys
Leu Ile Asn Lys Leu Asn Ser Phe Arg 435 440
445 Gln Lys Tyr Glu Ala Leu Arg Glu Gly Thr Gln Arg
Glu Met Trp Ser 450 455 460
Ser Pro His Leu Tyr Ala Phe Ser Arg Arg Met Asp Ser Gly Glu Asn 465
470 475 480 Val Gly Gln
Glu Val Val Asn Val Phe Asn Asn Ser Asp Gly Asp Gln 485
490 495 Ser Ala Thr Ile Pro Ile Arg Ala
Glu Ser Thr Ile Lys Val Gly Asp 500 505
510 Lys Phe Val Asn Leu Phe Asp Val Asn Asp Ser Ile Thr
Val Gln Gln 515 520 525
Gly Gly Val Thr Gly Lys Gln Ile Ser Val Asn Leu Gly Glu Asn Ser 530
535 540 Gly Lys Ile Tyr
Val Val Asn Asn Glu Thr Pro Asn Pro Asp Gln Lys 545 550
555 560 Asn Val Gln Tyr Lys Val Ser Tyr Lys
Asn Thr Asn Ala Gln Lys Val 565 570
575 Thr Leu His Tyr Gly Thr Asn Gly Trp Lys Asn Ile Gln Asp
Val Asn 580 585 590
Met Thr Lys Asn Ser Asn Gly Glu Phe Glu Ala Thr Ile Thr Val Asn
595 600 605 Asn Asn Asp Ile
Leu Asn Tyr Cys Ile His Ile Ile Ser Pro Thr Asp 610
615 620 Tyr Trp Asp Asn Asn Gly Gly Gln
Asn Trp Asn Val Lys Val Thr Lys 625 630
635 640 Ala Glu Asp Tyr Ile Asn Asp Gly Val Lys Ser Asn
Leu Lys Ser Val 645 650
655 Asn Thr Thr Thr Ser Ala Ala Ile Asp Ser Gly Ile Asp Ser Thr Val
660 665 670 Asn Arg
22025DNAClostridium saccharoperbutylacetonicum 2atgtttagaa gaaaatttaa
caaggtaata ttatctatct tagttgcaac aattgtttca 60agcactaaca tgtttatgag
tggaagcaag gcacaagcgg caattggaaa tctaagtgaa 120aacgatacta tttatcaaat
tatggtagac agattttatg atggagataa aacaaataat 180gctacaggag atgcatttcg
taatacagaa aatcttgaag atgattttag atatatgcac 240ggcggagatt ggcaaggtgt
tattgataag ttagattata ttaagggcat gggatactca 300gccatttgga tatcaccggt
tgcggaacca caaatgtggt ctagagctga tggcacagga 360aaagtatggc ctacagctta
tcatggatat aatgtgaaag atcccaataa ggcaaatcct 420tattttggaa caaaagaaaa
gctaaaggag ttagtagata aagctcacga aaaggggatt 480aaagtaataa tagatatagt
tccaaatcat gttggggatt atatgttagg aaaacaagct 540tattatgaca tcaaggggtt
tgagccggca gcacctttta ataatccaaa ttggtatcat 600cataatggcg atattgattg
gtcaagagaa cactctgatc cccaaatgtt agatgatcat 660gatttgggcg gtttagatga
tttaaatcaa gataattctg atgctaaggc agctatgaat 720aatgctatta agtcatggtt
tgattatact ggagctgatg cagcaagggt tgacgcagca 780aaatgtatga aaccatctta
tattaacgag ttacaaaagt atataggagt taatactttt 840ggagaaaatt ttgatatgaa
tgtagatttt gtgaagaagt gggttggatc cgatgcagaa 900tggggaatgc tagattttcc
attatatcaa gcaataaata atgattttgc atcaggacaa 960tcttttgatg acatgtcatc
atcaggtact tgctctatta aaaatatttt agcacaagac 1020aataaatata atggttatgc
aaatcatatg gtgactttta tagataatca tgatcgtaat 1080agatttttaa cagtagcaaa
tggtaatgta aaaaaacttc aaaatgcact tgttttcatg 1140tttactgtaa gaggggtacc
aacagtattt caaggtacag aacaaaacaa aggtaatgca 1200aatggagcaa gtataaatgg
tattgcagat acatggaatc gttggtcaat ggttaaaaag 1260gattacaatg gaaatgtaat
tacagattat tttaatgaga atacagatac ttataaacta 1320attaacaaat tgaattcatt
taggcaaaaa tatgaagcct taagagaagg tactcaaaga 1380gaaatgtggt cttcaccaca
tttatatgca ttctcaagaa ggatggattc aggagaaaat 1440gttggacaag aagttgtaaa
tgtatttaat aattcagatg gagatcaaag tgcgaccatt 1500ccaattagag ctgaaagtac
tataaaagtt ggagataaat ttgtaaatct ttttgatgta 1560aatgattcga tcacagttca
acaaggaggt gttacaggaa aacaaatatc agtgaattta 1620ggagaaaata gtgggaagat
ttatgttgtt aataatgaaa caccaaatcc agatcaaaag 1680aacgtacaat ataaagtttc
atataagaat actaatgcac aaaaagtaac acttcattat 1740ggaactaatg gatggaaaaa
cattcaagat gtaaatatga ctaagaattc caatggagaa 1800tttgaagcaa ctattacagt
aaataataat gatattctaa attactgtat tcatattatt 1860tcaccaacag actattggga
taataatggt ggacagaatt ggaatgtaaa agtgactaag 1920gcagaagatt atataaatga
tggtgtaaag agtaatttga agagcgttaa tacaactaca 1980tcagcagcta tagactctgg
gattgatagt actgtaaatc gttaa 20253674PRTClostridium
saccharoperbutylacetonicum 3Met Phe Arg Arg Lys Phe Asn Lys Val Ile Leu
Ser Ile Leu Val Ala 1 5 10
15 Thr Ile Val Ser Ser Thr Asn Met Phe Met Ser Gly Ser Lys Ala Gln
20 25 30 Ala Ala
Ile Gly Asn Leu Ser Glu Asn Asp Thr Ile Tyr Gln Ile Met 35
40 45 Val Asp Arg Phe Tyr Asp Gly
Asp Lys Thr Asn Asn Ala Thr Gly Asp 50 55
60 Ala Phe Arg Asn Thr Glu Asn Leu Glu Asp Asp Phe
Arg Tyr Met His 65 70 75
80 Gly Gly Asp Trp Gln Gly Val Ile Asp Lys Leu Asp Tyr Ile Lys Gly
85 90 95 Met Gly Tyr
Ser Ala Ile Trp Ile Ser Pro Val Ala Glu Pro Gln Met 100
105 110 Trp Ser Arg Ala Asp Gly Thr Gly
Lys Val Trp Pro Thr Ala Tyr His 115 120
125 Gly Tyr Asn Val Lys Asp Pro Asn Lys Ala Asn Pro Tyr
Phe Gly Thr 130 135 140
Lys Glu Lys Leu Lys Glu Leu Val Asp Lys Ala His Glu Lys Gly Ile 145
150 155 160 Lys Val Ile Ile
Asp Ile Val Pro Asn His Val Gly Asp Tyr Met Leu 165
170 175 Gly Lys Gln Ala Tyr Tyr Asp Ile Lys
Gly Phe Glu Pro Ala Ala Pro 180 185
190 Phe Asn Asn Pro Asn Trp Tyr His His Asn Gly Asp Ile Asp
Trp Ser 195 200 205
Arg Glu His Ser Asp Pro Gln Met Leu Asp Asp His Asp Leu Gly Gly 210
215 220 Leu Asp Asp Leu Asn
Gln Asp Asn Ser Asp Ala Lys Ala Ala Met Asn 225 230
235 240 Asn Ala Ile Lys Ser Trp Phe Asp Tyr Thr
Gly Ala Asp Ala Ala Arg 245 250
255 Val Asp Ala Ala Lys Cys Met Lys Pro Ser Tyr Ile Asn Glu Leu
Gln 260 265 270 Lys
Tyr Ile Gly Val Asn Thr Phe Gly Glu Asn Phe Asp Met Asn Val 275
280 285 Asp Phe Val Lys Lys Trp
Val Gly Ser Asp Ala Glu Trp Gly Met Leu 290 295
300 Asp Phe Pro Leu Tyr Gln Ala Ile Asn Asn Asp
Phe Ala Ser Gly Gln 305 310 315
320 Ser Phe Asp Asp Met Ser Ser Ser Gly Thr Cys Ser Ile Lys Asn Ile
325 330 335 Leu Ala
Gln Asp Asn Lys Tyr Asn Gly Tyr Ala Asn His Met Val Thr 340
345 350 Phe Ile Asp Asn His Asp Arg
Asn Arg Phe Leu Thr Val Ala Asn Gly 355 360
365 Asn Val Lys Lys Leu Gln Asn Ala Leu Val Phe Met
Phe Thr Val Arg 370 375 380
Gly Val Pro Thr Val Phe Gln Gly Thr Glu Gln Asn Lys Gly Asn Gly 385
390 395 400 Asn Gly Ala
Ile Leu Asn Gly Ile Ala Asp Thr Trp Asn Arg Trp Ser 405
410 415 Met Val Lys Lys Asp Tyr Asn Gly
Asn Ile Ile Thr Asp Tyr Phe Asn 420 425
430 Glu Asn Thr Asp Thr Tyr Lys Leu Ile Ser Lys Leu Asn
Ser Phe Arg 435 440 445
Gln Lys Tyr Glu Ala Leu Arg Glu Gly Thr Gln Arg Glu Met Trp Ser 450
455 460 Ser Pro His Leu
Tyr Ala Phe Ser Arg Arg Met Asp Ser Gly Glu Asn 465 470
475 480 Val Gly Gln Glu Val Val Asn Val Phe
Asn Asn Ser Asp Gly Asp Gln 485 490
495 Ser Ala Thr Ile Pro Ile Arg Ala Glu Ser Thr Ile Lys Val
Gly Asp 500 505 510
Lys Leu Val Asn Leu Phe Asp Val Asn Asp Ser Ile Thr Val Gln Gln
515 520 525 Gly Gly Val Thr
Gly Lys Gln Ile Ser Val Asn Leu Gly Glu Asn Ser 530
535 540 Gly Lys Ile Tyr Val Val Asn Asn
Glu Thr Pro Asn Pro Asp Gln Lys 545 550
555 560 Asn Val Gln Tyr Lys Val Ser Tyr Lys Asn Thr Asn
Ala Gln Lys Val 565 570
575 Thr Leu His Tyr Gly Thr Asn Gly Trp Lys Asn Ile Gln Asp Val Asn
580 585 590 Met Thr Lys
Asn Ser Asn Gly Glu Phe Glu Ala Thr Ile Thr Val Asn 595
600 605 Asn Asn Asp Ile Leu Asn Tyr Cys
Ile His Ile Ile Ser Pro Thr Asp 610 615
620 Tyr Trp Asp Asn Asn Gly Gly Gln Asn Trp Asn Val Lys
Val Thr Lys 625 630 635
640 Ala Glu Asp Tyr Ile Asn Asp Gly Val Lys Ser Asn Leu Lys Ser Val
645 650 655 Asn Thr Thr Thr
Ser Ala Ala Ile Glu Ser Gly Ile Asp Ser Thr Val 660
665 670 Asn Arg 42025DNAClostridium
saccharoperbutylacetonicum 4atgtttagaa gaaaatttaa caaggtaata ttatctattt
tagttgcaac aattgtttca 60agcactaaca tgtttatgag tggaagcaag gcacaagcgg
caattggaaa tttaagtgaa 120aacgatacta tttatcaaat tatggtagac agattttatg
atggagataa aacaaataat 180gctacaggag atgcatttcg taatacagaa aatcttgaag
atgattttag atatatgcac 240ggcggagatt ggcaaggtgt tattgataag ttagattata
ttaagggcat gggatactca 300gccatttgga tatcaccggt tgcggaacca caaatgtggt
ctagagctga tggcacagga 360aaagtatggc ctacagctta ccatggatat aatgtgaaag
atcccaataa ggcaaatcct 420tattttggaa caaaagaaaa gctaaaggag ttagtagata
aagctcacga aaaggggatt 480aaagtaataa tagatatagt tccaaatcat gttggggatt
atatgttagg aaaacaagct 540tattatgaca tcaaggggtt tgagccggca gcacctttta
ataatccaaa ttggtatcat 600cataatggcg atattgattg gtcaagagaa cactctgatc
cccaaatgtt agatgatcat 660gatttgggcg gtttagatga tttaaatcaa gataattctg
atgctaaggc agctatgaat 720aatgctatta agtcatggtt tgattatact ggagctgatg
cagcaagggt tgacgcagca 780aaatgtatga aaccatctta tattaacgag ttacaaaagt
atataggagt taatactttt 840ggagaaaatt ttgatatgaa tgtagatttt gtgaagaagt
gggttggatc cgatgcagaa 900tggggaatgc tagattttcc attatatcaa gcaataaata
atgattttgc atcaggacaa 960tcttttgatg acatgtcatc atcaggtact tgctctatta
aaaatatttt agcacaagac 1020aataaatata atggttatgc aaatcatatg gtgactttta
tagataatca tgatcgtaat 1080agatttttaa cagtagcaaa tggtaatgtt aaaaaacttc
aaaatgcact tgttttcatg 1140tttactgtaa gaggggtacc aacagtattt caaggtacag
aacaaaacaa aggtaatgga 1200aatggagcaa ttctaaatgg tattgcagat acatggaatc
gttggtcaat ggttaaaaag 1260gactataatg gaaatataat tacagattat tttaatgaga
atacagatac ttataaacta 1320attagcaaat tgaattcatt taggcaaaaa tatgaagcct
taagagaagg tactcaaaga 1380gaaatgtggt cttcaccaca tttatatgca ttctcaagaa
ggatggattc aggagaaaat 1440gttggacaag aagttgtaaa tgtatttaat aattcagatg
gagatcaaag tgcgaccatt 1500ccaattagag ctgaaagtac tataaaagtt ggagataaac
ttgtaaatct ttttgatgta 1560aatgattcga tcacagttca acaaggaggt gttacaggaa
aacaaatatc agtgaattta 1620ggagaaaata gtgggaagat ttatgttgtt aataatgaaa
caccaaatcc agatcaaaag 1680aacgtacaat ataaagtttc atataagaat actaatgcac
aaaaagtaac acttcattat 1740ggaactaatg gatggaaaaa cattcaagat gtaaatatga
ctaagaattc caatggagaa 1800tttgaagcaa ctattacagt aaataataat gatattctaa
attactgtat tcatattatt 1860tcaccaacag actattggga taataatggt ggacagaatt
ggaatgtaaa agtgactaag 1920gcagaagatt atataaatga tggtgtaaag agtaatttga
agagcgttaa tacaactaca 1980tcagcagcga tagaatctgg tattgatagt actgtaaatc
gttaa 2025
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