RECOMBINANT ENDOXYLANASES AND RELATED COMPOSITIONS AND METHODS OF USE

Abstract

Disclosed are recombinant endoxylanases that are expressed and exported in high levels in bacteria, in particular Bacillus subtilis. The recombinant endoxylanases are based on an endoxylanase selected from Trichoderma reesei or Bacillus pumilus modified with a signal peptide from B. subtilis alpha amylase (AmyE), B. subtilis levanase (SacC), or B. subtilis YwmC. Accordingly, also disclosed are plasmids for transforming bacteria to express and export such recombinant endoxylanases and the resulting recombinant bacterial strains. The disclosure also encompasses compositions for simultaneously degrading and assimilate cellobiose and xylan comprising a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass and a xylose assimilator and methods of producing value-added products, for example succinate, from agricultural biomass using such compositions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/115,566, filed Nov. 18, 2020, the contents of which are incorporated herein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 29,418 byte ASCII (text) file named "SeqList" created on Nov. 17, 2021.

FIELD OF THE INVENTION

[0003] The invention relates to recombinant Bacillus subtilis that can simultaneously degrade and assimilate cellobiose and xylan.

BACKGROUND OF THE INVENTION

[0004] Advent of the 21st century has seen surge in demand of energy, the depletion of fossil fuel reserves and detrimental effect of fossil fuels on environment. Therefore, alternative energy sources have been extensively explored to reduce our dependence on non-renewable fuel sources. Agricultural field waste (also referred to herein as "agricultural biomass") has been considered to be the most abundant energy source and extensive efforts are invested worldwide for valorizing it. Agricultural biomass is mainly made of the biopolymers cellulose, hemicellulose, and lignin. Agricultural biomass is rich renewable source and different methods have been used like pyrolysis, gasification, hydrolysis, etc. to explore its potential as energy and chemical source. For example, hemicellulosic and cellulosic agricultural waste is a promising renewable feedstock for increasing future demand of renewable biofuels and value-added products.

[0005] Hydrolysis of biomass can be done mainly by a chemical or an enzymatic route. The enzymatic route is more environmentally friendly and efficient than the chemical route due to the conversion efficiency of enzymes. However, commercial enzymes are expensive to use. The expense can be a big discouraging factor as the goal of processing agricultural wastes is to find a low-cost alternative. Accordingly, alternative processing method of agricultural waste using the enzymatic route is needed to take advantage of the efficiency of the enzymes while keeping costs low.

SUMMARY OF THE INVENTION

[0006] The disclosure also relates to plasmids for making recombinant bacteria that express and export an enzyme for xylan degradation (an endoxylanase). In some aspects, the plasmid is used for transforming Corynebacterium glutamicum, Bacillus subtilis, or Bacillus coagulans so that they express and export an endoxylanase selected from Trichoderma reesei or Bacillus pumilus or a variant thereof. The endoxylanase or variant thereof from T. reesei or B. pumilus are modified with a signal peptide selected from B. subtilis alpha amylase signal peptide (AmyE), B. subtilis levanase signal peptide (SacC), and B. subtilis YwmC signal peptide, or a variant thereof. In some embodiments, the plasmid comprises a sequence encoding a signal peptide selected from the group consisting of: AmyE, SacC, YwmC, and a variant thereof; and a sequence encoding an endoxylanase selected from Trichoderma reesei or Bacillus pumilus or a variant thereof; wherein the sequence encoding the signal peptide is upstream of the sequence encoding the endoxylanase thereby producing a recombinant endoxylanase modified with the signal peptide. The amino acid sequence of the variant AmyE encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:1. The amino acid sequence of the variant SacC encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:2. The amino acid sequence of the variant YwmC encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:3.

[0007] In certain embodiments, the amino acid sequence of the endoxylanase or variant thereof encoded by the sequence encoding the endoxylanase has at least 90% sequence identity to SEQ ID NO:4. In such embodiments, the amino acid sequence of the variant signal peptide encoded by the sequence encoding the signal peptide has at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In particular embodiments, the amino acid sequence of the recombinant endoxylanase is set forth in SEQ ID NO:6. For example, the amino acid sequence of the plasmid is set forth in SEQ ID NO:7.

[0008] In other embodiments, the sequence encoding the endoxylanase or variant thereof has at least 90% sequence identity to SEQ ID NO:5. In such embodiments, the sequence encoding the signal peptide has at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In particular embodiments, the sequence encoding the signal peptide is set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In certain embodiments, the sequence encoding the endoxylanase is set forth in SEQ ID NO:5.

[0009] The disclosure also relates to compositions and methods of consolidated bioprocessing of plant biomass. The compositions comprise a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass and a xylose assimilator. The recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass is selected from the group consisting of: C. glutamicum, B. subtilis, and B. coagulans. In some aspects, the recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass has been transformed with a plasmid described herein. The xylose assimilator is a bacterium selected from the group consisting of: Escherichia coli, B. coagulans, Lactobacillus pentosus, Lactobacillus brevis, Leuconostoc lactis, a different strain of B. coagulans than the xylose producer, and a different strain of B. subtilis than the xylose producer. In some embodiments, the composition further comprises a media comprising a trace metal solution and M9 media, wherein the trace metal solution comprises sulfate salts of copper, irone, zinc, and magnesium and the M9 media comprises KH.sub.2PO.sub.4, Na.sub.2HPO.sub.4, NaCl, NH.sub.4Cl, glucose, tryptophan, and citrate.

[0010] In some aspects, the compositions comprise recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass is B. subtilis. Thus, in certain embodiments, the composition comprises a recombinant B. subtilis engineered to produce xylose from hydrolyzing the agricultural biomass and E. coli as the xylose assimilator. In some aspects, the xylose assimilator is a succinate producer.

[0011] The methods of producing value-added products from agricultural biomass comprises providing an agricultural biomass, wherein the agricultural biomass comprises xylan; adding a culture of a xylose producer to the agricultural biomass, wherein the xylose producer is a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass; and adding a culture of a xylose assimilator to the agricultural biomass. The recombinant bacteria are selected from the group consisting of: C. glutamicum, B. subtilis, and B. coagulans. In certain implementations, the recombinant bacteria have been transformed with a plasmid described herein. The xylose assimilator is a bacterium selected from the group consisting of: E. coli, B. coagulans, L. pentosus, L. brevis, L. lactis, a different strain of B. coagulans than the xylose producer, and a different strain of B. subtilis than the xylose producer. In certain implementations, the recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass is a recombinant B. subtilis. In some aspects, the corresponding xylose assimilator is E. coli.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 depicts, in accordance with certain embodiments, exemplary recombinant strains of Bacillus subtilis described herein that enable production and export of endoxylanases. Abbreviations in figure: SP1 refers to YwmC, SP2 refers to AmyE, SP3 refers to SacC, Xy(Tr) refers to Xyn2 from Trichoderma reesei, Xy(Bp) refers to XynA from Bacillus pumilus.

[0013] FIG. 2 depicts, in accordance with certain embodiments, an exemplary standard curve for convert the absorbance measurements into reducing sugar concentrations for a DNA assay.

[0014] FIGS. 3A and 3B depict, in accordance with certain embodiments, the sugar reducing capabilities of the SSL26 strain in the presence of varying concentrations of xylan.

[0015] FIG. 4 depicts, in accordance with certain embodiments, the results of a xylan depolymerization study using SSL26.

[0016] FIG. 5 depicts, in accordance with certain embodiments, a scheme of in situ breakdown of xylan using the systems or according to the methods described herein.

[0017] FIGS. 6A and 6B depict, in accordance with certain embodiments, the kinetics of the xylan (FIG. 6A) and xylose (FIG. 6B) reactions from the SSL26 strain.

[0018] FIG. 7 depicts, in accordance with certain embodiments, a comparison of the xylose production of the SSL26 strain versus other strains, or bacteria described in the specified prior art. The prior art references all reported xylose production from medium containing 1% xylan. The prior art references are Tsai et al., Applied and Environmental Microbiology, 2010, 76, 7514-7520 (described in the figure as Chen et al., 2010); Zheng et al., Microbial Cell Factories, 2012, 11, 1-11 (Zhao et al., 2012); Liu et al., Bioresource Technology, 2019, 292, 121965-121965 (Yuan., 2019); and Jiang et al., Biotechnology for Biofuels, 2018, 11, 89 (Xin et al., 2018).

[0019] FIG. 8 depicts, in accordance with certain embodiments, a scheme of a coculture process for producing succinate.

[0020] FIG. 9 depicts, in accordance with certain embodiments, succinate production using the coculture systems or according to the methods described herein. "E" represents the amount of Escherichia coli reported as OD.sub.600. B represents the amount of the recombinant B. subtilis reported as OD.sub.600.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed aspects and applications of the invention are described below in the drawings and detailed description of the invention. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0022] In the following description, and for the purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the various aspects of the invention. It will be understood, however, by those skilled in the relevant arts, that the present invention may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices, and technologies to which the disclosed inventions may be applied. The full scope of the inventions is not limited to the examples that are described below.

[0023] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a step" includes reference to one or more of such steps.

[0024] The term "variant" as used herein refers to a variation in the amino acid sequence where the resulting variant amino acid product has at least 80% sequence homology to the original or reference amino acid sequence. In some aspects, the amino acid sequence of the variant amino has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence homology to the original or reference amino acid sequence. In some aspects, the amino acid sequence of the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the original or reference amino acid sequence. In certain embodiments, the amino acid sequence of the variant has less than 8, less than 7, less than 6, or less than 5 insertions, deletions, and/or substitutions when compared the original or reference amino acid sequence.

[0025] Plant biomass is the largest source of carbon on the planet. This huge resource if efficiently utilized can be useful in providing an alternative option to non-renewable sources. Different methods have been used for plant biomass utilization previously and enzymatic degradation is effective due to its high specificity. Signal peptides are short chain polypeptides which play a vital role in enzyme export. Efficient and cost-effective methods are essential for breakdown of cellulosic materials into elementary components. Cost effective enzymatic hydrolysis of the cellulosic and hemicellulosic biomass can play a vital role in making economical biorefineries viable. The disclosure relates to a process of converting agricultural biomass. Instead of using commercial enzymes, the described process aims at engineering bacteria to export enzymes that hydrolyze the biomass during a fermentation process to produce commercially viable products, for example xylose and succinate. Xylan and cellobiose are two chemicals which are formed by polymerization of sugar like glucose, xylose, arabinose, rhamnose, and these sugars can be consumed by bacterial cells as energy source. Accordingly, disclosed herein are plasmids for making recombinant bacteria, such as Bacillus subtilis, that breakdown hemicellulose (for example xylan) into simple sugars. The plasmids described herein enable the expression and export of an endoxylanase from Trichoderma reesei or Bacillus pumilus or a variant thereof. In addition to the transformation of B. subtilis, the plasmid may also be used for transforming Corynebacterium glutamicum or Bacillus coagulans into recombinant bacteria that express and export an endoxylanase from Trichoderma reesei or Bacillus pumilus or a variant thereof. To enable the export of the expressed endoxylanase, the enzyme is modified with a signal peptide from selected from B. subtilis alpha amylase signal peptide (AmyE), B. subtilis levanase signal peptide (SacC), and B. subtilis YwmC signal peptide, or a variant thereof.

[0026] Thus, described herein are plasmids comprising a sequence encoding a signal peptide selected from the group consisting of: AmyE, SacC, YwmC, and a variant thereof; and a sequence encoding an endoxylanase selected from Trichoderma reesei, Bacillus pumilus, or a variant thereof; wherein the sequence encoding the signal peptide is upstream of the sequence encoding the endoxylanase thereby producing a recombinant endoxylanase modified with the signal peptide. The endoxylanase from T. reesei has an amino acid sequence set forth in SEQ ID NO:5, while the endoxylanase from B. pumilus has an amino acid sequence set forth in SEQ ID NO:4. The variant endoxylanase from T. reesei has an amino acid sequence with at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology to SEQ ID NO:5. In some aspects, the variant endoxylanase from T. reesei has an amino acid sequence with at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to SEQ ID NO:5. The variant endoxylanase from B. pumilus has an amino acid sequence with at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology to SEQ ID NO:4. In some aspects, the variant endoxylanase from B. pumilus has an amino acid sequence with at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to SEQ ID NO:4.

[0027] The amino acid sequence of the variant AmyE encoded by the sequence encoding the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to SEQ ID NO:1. In some aspects, the amino acid sequence of the variant AmyE encoded by the sequence encoding the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology to SEQ ID NO:1. The amino acid sequence of the variant SacC encoded by the sequence encoding the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to SEQ ID NO:2. In some aspects, the amino acid sequence of the variant SacC encoded by the sequence encoding the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology to SEQ ID NO:2. The amino acid sequence of the variant YwmC encoded by the sequence encoding the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to SEQ ID NO:3. In some aspects, the amino acid sequence of the variant YwmC encoded by the sequence encoding the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology to SEQ ID NO:3.

[0028] In certain embodiments, the amino acid sequence of the endoxylanase or variant thereof encoded by the sequence encoding the endoxylanase has at least 90% sequence identity to SEQ ID NO:4. In such embodiments, the amino acid sequence of the variant signal peptide encoded by the sequence encoding the signal peptide has at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In particular embodiments, the amino acid sequence of the recombinant endoxylanase is set forth in SEQ ID NO:6. For example, the amino acid sequence of the plasmid is set forth in SEQ ID NO:7. Also described are the recombinant bacteria that express the plasmids described herein, for example, a plasmid having an amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, the recombinant bacteria is C. glutamicum, B. subtilis, or B. coagulans.

[0029] The recombinant bacteria described herein can breakdown the biopolymers found in plant biomass in a one-pot process. Accordingly, a one-pot fermentation system to process agricultural waste product involving a polymer degradation process are also described, and the disclosure also relates to compositions and methods of consolidated bioprocessing of plant biomass. The end products of the one-pot fermentation system to process agricultural biomass produces can be a wide range of useful products, like succinate, ethanol, lactate, etc. The composition and methods relate to bacterial digestion of agricultural biomass, for example, the breakdown of hemicellulose into simple sugars by bacteria, for example, the breakdown of xylan into xylose and then further into value-added products, such as succinate, ethanol, or lactate. Accordingly, the described system and method for processing agricultural biomass can also help reduce the production cost value-added products from agricultural biomass.

[0030] The compositions comprise a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass and a xylose assimilator. The recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass is selected from the group consisting of: C. glutamicum, B. subtilis, and B. coagulans. In some aspects, the recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass has been transformed with at least one plasmid described herein. The xylose assimilator is a bacterium selected from the group consisting of: Escherichia coli, B. coagulans, Lactobacillus pentosus, Lactobacillus brevis, Leuconostoc lactis, a different strain of B. coagulans than the xylose producer, and a different strain of B. subtilis than the xylose producer. In some aspects, the compositions comprise recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass is B. subtilis. Thus, in certain embodiments, the composition comprises a recombinant B. subtilis engineered to produce xylose from hydrolyzing the agricultural biomass and E. coli as the xylose assimilator. In some aspects, the xylose assimilator is a succinate producer.

[0031] Hydrolysis reaction mainly involves lysis of the long biopolymeric chains in reaction mixture predominantly consisting of water. The polymer degradation comprises a first step involving xylan degradation and a second step involving cellobiose degradation. Both steps involve using different signal peptides. For xylan degradation, the best results were displayed by the recombinant endoxylanase YwMC-XynA with an amino acid sequence set forth in SEQ ID NO:6. The highest yields obtained were 6.7 g/L of xylose from 1% xylan.

[0032] In some embodiments, the composition further comprises a media designed for in situ breakdown of the polymers. This media comprises: a trace metal solution and M9 media. In some aspects, the media further comprises CaCl.sub.2). In some aspects, the media pH is adjusted to 6, for example with sodium phosphate and citric acid. In a particular embodiment, the one-pot fermentation system comprises the following in 5 ml of the media:

[0033] M9 media (10.times.) 0.5 ml

[0034] Trace metal solution (1000.times.) 5 ul

[0035] CaCl2 0.5 ul

[0036] Chloramphenicol 5 ug/ml

[0037] Seed culture OD.sub.600 of 1.7 50 ul.

[0038] The trace metal solution comprises sulfate salts of copper, irone, zinc, and magnesium. In some embodiments, the trace metal solution (1000.times.) has the following components:

[0039] CuSO.sub.4 60 mM

[0040] FeSO.sub.4 60 mM

[0041] ZnSO.sub.4 60 mM

[0042] MgSO.sub.4 2000 mM

[0043] The M9 media comprises KH.sub.2PO.sub.4, Na.sub.2HPO.sub.4, NaCl, NH.sub.4C1, glucose, tryptophan, and citrate. In some embodiments, M9 media (10.times.) has the following components:

[0044] KH.sub.2PO.sub.4--0.6 g in 100 ml

[0045] Na.sub.2HPO.sub.4--3.21 g in 100 ml

[0046] NaCl--0.5 g in 100 ml

[0047] NH.sub.4Cl--1 g in 100 ml

[0048] Glucose--20 g in 100 ml

[0049] Tryptophan--50 g in 100 ml

[0050] Citrate--1.79 g in 100 ml

[0051] The methods of producing value-added products from agricultural biomass comprises providing an agricultural biomass, wherein the agricultural biomass comprises xylan; adding a culture of a xylose producer to the agricultural biomass, wherein the xylose producer is a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass; and adding a culture of a xylose assimilator to the agricultural biomass. The recombinant bacteria are selected from the group consisting of: C. glutamicum, B. subtilis, and B. coagulans. In certain implementations, the recombinant bacteria have been transformed with a plasmid described herein. The xylose assimilator is a bacterium selected from the group consisting of: E. coli, B. coagulans, L. pentosus, L. brevis, L. lactis, a different strain of B. coagulans than the xylose producer, and a different strain of B. subtilis than the xylose producer. In certain implementations, the recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass is a recombinant B. subtilis. In some aspects, the corresponding xylose assimilator is E. coli.

[0052] In some aspects, the cultures of the xylose producer or the xylose assimilator comprise the above-described media designed for in situ breakdown of the polymers.

[0053] The disclosed process has the highest pentose sugar yields compared to pre-existing processes. It is also cost effective and environment friendly compared to other hydrolysis methods.

Illustrative, Non-Limiting Examples in Accordance with Certain Embodiments

[0054] The disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

1) Screening of Recombinant Strains Containing Signal Peptides and Enzymes Showing Highest Hydrolysis Activity

[0055] A DNS assay was used for evaluating endo-1,4-O-xylanase activity in the supernatant of the recombinant B. subtilis culture. The recombinant B. subtilis strain tested were genetically engineered to express an endoxylanase modified with a signal peptide from the alpha amylase of B. subtilis (AmyE, SEQ ID NO:1), from the levanase of B. subtilis (SacC, SEQ ID NO:2), or the YwmC protein of B. subtilis (SEQ ID NO:3). FIG. 1 depicts four exemplary strains.

[0056] Engineered B. subtilis strains were grown in 5 mL of 2.times.YT media until the early logarithmic growth phase (OD600=0.8) at 37.degree. C. and 250 rpm and induced with 1 mM isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) at the exponential phase for expression and secretion of the target enzyme. The strains were grown further for 24 hours to accumulate endo-1,4-.beta.-xylanase in the extracellular media and supernatant samples were collected to be used for enzyme reactions. Birchwood xylan (1% w/v) in citrate phosphate buffer (20 .mu.L) (pH adjusted as required) was mixed with supernatants (20 .mu.L) containing the secreted enzyme at a 1:1 volume ratio in a flat bottom 96-well plate to a total volume of 40 .mu.L and was incubated at 50.degree. C. for 5, 10, 15, 30, and 60 mins to understand the kinetics of xylan depolymerization. The amount of reducing sugar produced through the action of endo-1,4-.beta.-xylanase on xylan was estimated using 3, 5 dinitrosalicyclic acid (DNS) assay. The enzyme reaction was ended by adding 160 .mu.L of DNS and by heating at 105.degree. C. for 20 mins. Color change at the end of the DNS reaction was measured at an absorbance of 570 nm. A standard curve was used to convert the absorbance measurements into reducing sugar concentrations (FIG. 2).

[0057] The SSL26 strain, which is genetically engineered to express the enodxylanase from B. Pumilus modified with the signal peptide of the YwmC protein of B. subtilis, resulted in the highest hydrolysis activity (FIGS. 3A and 3B).

2) In Situ Hemicellulose Depolymerization

[0058] Xylan depolymerization studies were conducted in 3M media with starting pH of 6.0 as it was found to be optimal for the endo-1,4-.beta.-xylanase activity (FIG. 4). Engineered strains were grown in 3M media with 1.30% xylan at 37.degree. C. and 250 rpm for 4 hours. The strains were induced with 0, 0.1, 0.2, 1 and, 2 mM IPTG to express and secrete the xylanase enzyme. Following induction, xylan depolymerization was carried out at 37.degree. C. for 4 days. The fermentation batches were sampled at 24, 48, 72, and 96 hrs after IPTG induction for OD.sub.600 measurement as well as for the quantification of, substrate and product concentrations using HPLC (FIG. 5).

[0059] As shown in FIG. 6B, higher expression of the endoxylanase (induced by greater concentrations of IPTG) does not necessarily result in the highest production of xylose. As such, too high expression of the enzyme can negatively affect the export of the enzyme by the bacteria. The optimal concentration of IPTG to induce expression of the endoxylanase in the best strain of B. subtilis described herein is 0.2 mM.

[0060] As shown in FIG. 7, xylose production from medium containing 1% xylan of the SSL26 strain when compared to other strains, or bacteria described in the prior art (Tsai et al., Applied and Environmental Microbiology, 2010, 76, 7514-7520; Zheng et al., Microbial Cell Factories, 2012, 11, 1-11; Liu et al., Bioresource Technology, 2019, 292, 121965-121965; and Jiang et al., Biotechnology for Biofuels, 2018, 11, 89) is at least about two times greater.

3) Coculture Cultivation for the Consolidated Bioprocessing of Xylan to Succinate

[0061] Consolidated bioprocessing (CBP) refers to the combination of multiple biological events required to convert a starting material into a desired end product in a one-pot reaction. For CBP of xylan to succinate, the multiple biological events include the production of xylose from xylan and the metabolism of xylose into succinate (FIG. 9). The first successful CBP of xylan to succinate using a coculture of a B. subtilis xylose producer and a succinate producer that is a different species of bacteria is described in this example (FIG. 8).

[0062] The seed culture of the B. subtilis strain SSL26 was grown overnight in glass culture tubes in 2.times.YT media, shaking at 250 rpm at 37.degree. C. The next day, 50 .mu.L of seed culture was added to 5 mL of 3M media containing xylan (1% or 5%) at 37.degree. C. under aerobic conditions. As the cells reached the exponential growth phase (OD.sub.600 of 0.8), induction was initiated by adding 0.2 mM IPTG. Protein expression and in situ xylan depolymerization were carried out under aerobic conditions for 24 hours after induction. In parallel, an E. coli strain X2S engineered for converting xylose to succinic acid was cultured overnight in glass culture tubes in LB media at 37.degree. C., 250 rpm. The overnight culture of the X2S E. coli strain was centrifuged at 5000 rpm for 5 minutes and washed with M9 minimal media to avoid the carryover of nutrient traces to the next coculture stage. Coculture studies were conducted at different inoculation volume ratios of X2S to SSL26 (0.5:1; 1:1; 1.5:1; 2:1) and cultured in luer-lock tubes to produce succinic acid from xylan. The coculture was supplemented with 0.1 M KHCO.sub.3 as a bicarbonate source and the CBP of xylan to succinate was carried out under microaerobic conditions for 96 hours.

[0063] As shown in FIG. 9, the volume ratio of 0.5:1 of X2S to SSL26 resulted in the greatest succinate production. The simple sugars produced in this coculture can be used in fermentation processes for production of fuels and products.

Sequence CWU 1

1

7135PRTBacillus subtilis 1Met Phe Ala Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly1 5 10 15Phe Leu Leu Leu Phe His Leu Val Leu Ala Gly Pro Ala Ala Ala Ser 20 25 30Ala Ala Ala 35226PRTBacillus subtilis 2Met Lys Lys Arg Leu Ile Gln Val Met Ile Met Phe Thr Leu Leu Leu1 5 10 15Thr Met Ala Phe Ser Ala Asp Ala Ala Ala 20 25325PRTBacillus subtilis 3Met Lys Lys Arg Phe Ser Leu Ile Met Met Thr Gly Leu Leu Phe Gly1 5 10 15Leu Thr Ser Pro Ala Phe Ala Ala Ala 20 254228PRTBacillus pumilus 4Met Asn Leu Lys Arg Leu Arg Leu Leu Phe Val Met Cys Ile Gly Phe1 5 10 15Val Leu Thr Leu Thr Ala Val Pro Ala His Ala Glu Thr Ile Tyr Asp 20 25 30Asn Arg Ile Gly Thr His Gly Gly Tyr Asp Phe Glu Leu Trp Lys Asp 35 40 45Tyr Gly Asn Thr Ser Met Thr Leu Asn Asn Gly Gly Ala Phe Ser Ala 50 55 60Gln Trp Asn Asn Ile Gly Asn Ala Leu Phe Arg Lys Gly Lys Lys Phe65 70 75 80Asp Ser Thr Lys Thr His His Gln Leu Gly Asn Ile Ser Ile Asn Tyr 85 90 95Asn Ala Ala Phe Asn Pro Gly Gly Asn Ser Tyr Leu Cys Val Tyr Gly 100 105 110Trp Thr Gln Ser Pro Leu Ala Glu Tyr Tyr Ile Val Glu Ser Trp Gly 115 120 125Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Ser Phe Tyr Ala Asp Gly 130 135 140Gly Thr Tyr Asp Ile Tyr Glu Thr Leu Arg Val Asn Gln Pro Ser Ile145 150 155 160Ile Gly Asp Ala Thr Phe Lys Gln Tyr Trp Ser Val Arg Gln Thr Lys 165 170 175Arg Thr Ser Gly Thr Val Ser Val Ser Gln His Phe Arg Lys Trp Glu 180 185 190Ser Leu Gly Met Pro Met Gly Lys Met Tyr Glu Thr Ala Leu Thr Val 195 200 205Glu Gly Tyr Arg Ser Asn Gly Ser Ala Asn Val Phe Thr Asn Gln Leu 210 215 220Met Ile Gly Gln2255223PRTTrichoderma resei 5Met Val Ser Phe Thr Ser Leu Leu Ala Gly Val Ala Ala Ile Ser Gly1 5 10 15Val Leu Ala Ala Pro Ala Ala Glu Val Glu Ser Val Ala Val Glu Lys 20 25 30Arg Gln Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr 35 40 45Ser Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro 50 55 60Gly Gly Gln Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly65 70 75 80Gly Lys Gly Trp Gln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser 85 90 95Gly Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp 100 105 110Ser Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr 115 120 125Tyr Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp 130 135 140Gly Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser145 150 155 160Ile Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn 165 170 175His Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp 180 185 190Ala Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala 195 200 205Val Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser 210 215 2206240PRTArtificial SequenceYwMC-XynA 6Asp Pro Arg Lys Asp Phe His Ser Gln Asp Cys Phe Leu Asp Leu His1 5 10 15Leu Leu Leu Gln Arg Pro Val Ile Lys Asp Gly Tyr Cys Leu Cys Val 20 25 30Leu Asp Leu Cys His Arg Leu Cys Gln Leu Met Arg Lys Arg Phe Met 35 40 45Ile Ile Glu Ser Ala His Thr Ala Asp Thr Ile Leu Asn Tyr Gly Lys 50 55 60Ile Thr Glu Ile Arg Gln His Ser Ile Met Ala Ala His Leu Ala His65 70 75 80Asn Gly Thr Ile Ser Glu Met Leu Cys Phe Glu Lys Gly Arg Ser Leu 85 90 95Ile Pro Leu Lys Leu Ile Ile Asn Leu Ala Thr Ser Pro Ser Ile Thr 100 105 110Thr Gln Pro Leu Thr Gln Ala Gly Ile Pro Thr Tyr Val Ser Thr Ala 115 120 125Gly His Asn Leu His Leu Asn Thr Thr Leu Leu Ser His Gly Ala Leu 130 135 140Ile Ala Gln Gln Glu Arg Ile Lys Asp His Phe Met Pro Met Ala Ala145 150 155 160His Met Thr Phe Met Lys Arg Tyr Val Ser Ile Ser Leu Leu Leu Leu 165 170 175Glu Thr Leu Pro Leu Ser Asn Ile Gly Val Tyr Val Lys Gln Asn Val 180 185 190Arg Ala Glu Leu Tyr Pro Ser Val Ser Ile Ser Glu Asn Gly Lys Ala 195 200 205Val Cys Gln Trp Gly Lys Cys Met Lys Gln His Leu Lys Ala Thr Glu 210 215 220Ala Thr Glu Val Arg Met Cys Leu Pro Thr Ser Leu Asp Asn Asn Leu225 230 235 24072499PRTArtificial SequenceYwMC-XynA Plasmid 7Leu Ser Tyr Trp Tyr Asp Trp Phe Ala Gln Lys Lys Leu Leu Phe Arg1 5 10 15Thr Tyr Cys Ile Val Leu Glu Asn Arg Leu Lys Val Gln Ser Ala Leu 20 25 30Ser His Ile Ile Lys Ala Ser His Ala Tyr Leu Thr Ile Pro Glu Ser 35 40 45Ser Thr Ile Leu His Asp Asn His His Lys Gln Asn Asp Val Pro Val 50 55 60Lys Ile Ala Val Asn Ile Leu Asn Tyr Leu Tyr Ile Phe Leu Leu Trp65 70 75 80Val Glu Gly Asn Tyr Tyr Tyr Tyr Tyr Leu Ser Thr Gln Met Lys Ser 85 90 95Met Glu Lys Glu Lys Lys His Phe Gln Val Val Phe Trp Glu Thr Ile 100 105 110Ser Pro Asn His Tyr Ile Ser Leu His Gln Lys Gly Ile Asn His Lys 115 120 125Thr Leu Ser His Ser Leu Gln Glu Ser Lys Tyr Gln Arg Met Phe Ile 130 135 140His His Gln Lys Leu Tyr Lys Val Ala Leu Thr Tyr Pro Asn Asn Leu145 150 155 160Thr Leu Arg Arg Tyr Cys Asn Gln Phe Lys Leu Tyr Leu Ser Leu Ser 165 170 175Pro Leu Ser Leu Arg Lys Met Gln Gly Lys Ile Tyr Ile Leu Leu Val 180 185 190Leu Cys Phe Gly Ile Lys His Tyr Gln Phe Leu Trp Leu Tyr Lys Ser 195 200 205Phe Val Gly Ser Asn Asn Asp Ile Ser Leu Phe Ser Ser Asn Cys Leu 210 215 220Asn Gln Phe Tyr Ser Ser Phe Asp Met Pro Pro Lys Phe Leu Ser Lys225 230 235 240Val Asn Leu Gly Gly Leu Leu Val Cys Phe Leu His Asn Gln Ser Phe 245 250 255Phe Lys Ser Gln Tyr Tyr Cys Asn Ile Asn Ile Tyr Phe Lys Asn Ile 260 265 270Pro Leu Tyr Pro Ile Phe Val Cys Thr Asn Gly Cys Phe Ser Arg Ile 275 280 285Lys Thr Thr Leu Lys Asn Val Val Phe Cys Val Phe Leu Lys Asp Leu 290 295 300Ser Val Ala Lys Asn Pro Phe Leu Ser Tyr Leu Asp Asn Lys Gly Asn305 310 315 320Tyr Cys Arg Ser Ser Ile Pro Thr Ala Ser Pro Val Thr Met Ala Cys 325 330 335Cys Arg His Ser Pro Phe Arg Leu Arg Asn Cys Trp Glu Gly Arg Ser 340 345 350Val Arg Ala Ser Ser Leu Leu Arg Gln Leu Ala Lys Gly Gly Cys Ala 355 360 365Ala Arg Arg Leu Ser Trp Val Thr Pro Gly Phe Ser Gln Ser Arg Arg 370 375 380Cys Lys Thr Thr Ala Ser Glu Phe Glu Leu Arg Pro Leu Thr Leu Ile385 390 395 400Ala Leu Arg Ser Leu Pro Ala Phe Gln Ser Gly Asn Leu Ser Cys Gln 405 410 415Leu His Ile Gly Gln Arg Ala Gly Arg Gly Gly Leu Arg Ile Gly Arg 420 425 430Gln Gly Gly Phe Ser Phe His Gln Asp Gly Gln Gln Leu Ile Ala Leu 435 440 445His Arg Leu Ala Leu Arg Glu Leu Gln Gln Ala Val His Ala Gly Leu 450 455 460Pro Gln Gln Ala Lys Ile Leu Phe Asp Gly Gly Arg Arg Asp Ile Thr465 470 475 480Ala Val Phe Gly Ile Val Val Ser His Tyr Arg Asp Ile Arg Thr Asn 485 490 495Ala Gln Pro Gly Leu Gly Asn Gly Ala His Cys Ala Gln Arg His Leu 500 505 510Ile Val Gly Asn Gln His Arg Ser Gly Asn Asp Ala Leu Ile Gln His 515 520 525Leu His Gly Leu Leu Lys Thr Gly His Gly Thr Pro Val Ala Phe Pro 530 535 540Phe Arg Tyr Arg Leu Asn Leu Ile Ala Ser Glu Ile Phe Met Pro Ala545 550 555 560Ser Gln Thr Gln Thr Arg Arg Asp Arg Thr Trp Ala Arg Gln Arg Asp 565 570 575Leu Leu Val Thr Gln Cys Asp Gln Met Leu His Ala Gln Ser Arg Thr 580 585 590Val Phe Met Gly Glu Asn Asn Thr Val Asp Gly Cys Leu Val Arg Asp 595 600 605Ile Lys Lys Arg Arg Asn Ile Ser Ala Gly Ser Phe His Ser Asn Gly 610 615 620Ile Leu Val Ile Gln Arg Ile Val Asn Asp Gln Pro Thr Asp Ala Leu625 630 635 640Arg Glu Lys Ile Val His Arg Arg Phe Thr Gly Phe Asp Ala Ala Ser 645 650 655Phe Tyr His Arg His His His Ala Gly Thr Gln Leu Ile Gly Ala Arg 660 665 670Phe Asn Arg Arg Asp Asn Leu Arg Arg Arg Val Gln Gly Gln Thr Gly 675 680 685Gly Gly Asn Ala Asn Gln Gln Arg Leu Phe Ala Arg Gln Leu Leu Cys 690 695 700His Ala Val Gly Asn Val Ile Gln Leu Arg His Arg Arg Phe His Phe705 710 715 720Phe Pro Arg Phe Arg Arg Asn Val Ala Gly Leu Val His His Ala Gly 725 730 735Asn Gly Leu Ile Arg Asp Thr Gly Ile Leu Cys Asp Ile Val Arg Tyr 740 745 750Trp Phe His Gln Asn Arg Leu Pro Pro Phe Glu Tyr Leu Ile Asp Arg 755 760 765Asn Gln Met Lys His Ser Phe His Tyr Pro Tyr Ser Val Met Ala Thr 770 775 780Ile Thr Lys Gln Leu Val Arg Thr Ile Phe Gln Pro Thr Gln Thr Ser785 790 795 800Asn Leu Thr Asn Val Val Phe Glu Ser Ile Thr Tyr Val Arg Phe Lys 805 810 815Cys Asn Arg Phe Phe Gly Arg Lys Pro Arg Phe His Arg Asn Leu Gly 820 825 830Thr Lys Gly Gly Lys Asp His Lys Ile Phe Lys Ile Ser His Trp Lys 835 840 845Gly Asp Met Leu Leu Glu Leu Arg Asn Cys Glu Arg Ile Thr Ile Pro 850 855 860Ile Arg Arg Lys Asp Pro Cys Leu Glu Ser Thr Ser Leu Ile Thr Ile865 870 875 880Thr Ile Thr Ile Thr Asn Val Pro Gly Ala Ala Arg Leu Met Ser Gly 885 890 895Leu Phe Ser Arg His Ala Ser Met Glu Ile Phe Val Cys Asn Lys Val 900 905 910Tyr Thr Leu Pro Gly Thr Asn Gly Asn Ile Arg Gly Tyr Arg Leu Ile 915 920 925Arg Asn Ser Pro Cys Thr Asn Lys Ile Arg Trp Ile Ser Ser Val Tyr 930 935 940Phe Gly Arg Ser Ser Glu Arg Ile Trp Arg Leu Ala Phe His Asn Ile945 950 955 960Lys Gly Lys Asn Lys Ala Ile Cys Ser Arg Asn His Gly Arg Arg Tyr 965 970 975Ser Arg Ser Ser Lys Thr Ile Gln Pro Tyr Asn Gly Ile Asp Arg Lys 980 985 990Gly Gly Arg Leu Met Val Arg Lys Leu Gly Asp Leu Pro Arg Lys Pro 995 1000 1005Gln Gly Asp Arg Ser Ser Leu Lys Asn Pro Tyr Met Asp Leu Thr 1010 1015 1020Asp Ser Glu Ser Lys Glu Thr Thr Glu Val Lys Gln Thr Glu Pro 1025 1030 1035Lys Arg Lys Lys Ala Leu Leu Lys Thr Met Lys Val Asp Val Ser 1040 1045 1050Ile His Asn Lys Ile Lys Ser Leu His Glu Ile Leu Ala Ala Ser 1055 1060 1065Glu Gly Asn Ser Tyr Tyr Leu Glu Asp Thr Ile Glu Arg Ala Ile 1070 1075 1080Asp Lys Met Val Glu Thr Leu Pro Glu Ser Gln Lys Thr Phe Tyr 1085 1090 1095Glu Tyr Glu Leu Lys Lys Arg Thr Asn Lys Gly Asp Arg Leu Gln 1100 1105 1110Thr Ser Leu Phe Phe Lys Lys Tyr Glu His Ile Tyr Ile Arg Glu 1115 1120 1125Leu Arg Lys Thr Trp Glu Lys Tyr Phe Lys Ser Ser Lys Asn Met 1130 1135 1140Ile Arg Leu Phe Gln Asn Met Lys Asn Ser Val Cys Phe Lys Asn 1145 1150 1155Lys Gln Lys Lys Ser Thr Arg Asn Leu Asn Leu Thr Asn Ser Gly 1160 1165 1170Gln Thr Glu Lys Leu Asn Leu Arg Arg Gly Lys Gly Gly Phe Ile 1175 1180 1185Leu Val Phe Asn Tyr Leu His Phe Asn Ile Leu Leu Asn Pro Ile 1190 1195 1200Gln Val Lys Ile Leu Phe Tyr Thr Val Pro Leu Gly Asp Arg Gly 1205 1210 1215Gly Thr Leu Val Lys Thr Lys Glu Ile Gln Met Asn Cys Ile Ile 1220 1225 1230Asp Ser Lys Pro Leu Glu Ile Lys Ser Pro Gly Arg Glu Ser Lys 1235 1240 1245Lys Gly Lys Lys Lys Phe Ile Met Leu Leu Lys Arg Lys Arg Arg 1250 1255 1260Tyr Gly Gln Lys Ser Lys Lys Thr Phe Leu Thr Asn Leu Val Arg 1265 1270 1275Ile Tyr Leu Thr Lys Val Ile Ile Gln Ser Ile Ile Thr Ser Leu 1280 1285 1290Ile Phe Gly Ala Ile Phe Val Leu Lys Glu Leu Arg Ser Met Leu 1295 1300 1305Thr Leu Val Met His Thr Ala Ala Lys Thr Phe Ala Phe Leu Val 1310 1315 1320Tyr Lys Gln Ser Leu Lys Lys Trp Thr Arg Leu Leu Leu Gln Leu 1325 1330 1335Glu Ala Thr Asn Cys Leu Lys Gly Thr Val Leu Phe Gly Arg Thr 1340 1345 1350Ser Val Ile Lys Pro Arg Ile Thr Gln Arg Asn Pro Arg Phe Leu 1355 1360 1365Arg Leu Asp Val Arg Phe Leu Cys Phe Gln Lys Asn Phe Met Glu 1370 1375 1380Thr Leu Ile Leu Lys Phe Gln Met Thr Arg Lys His Met Arg Arg 1385 1390 1395Leu Lys Arg Lys Lys Arg Val Phe Gln Arg Phe Lys Lys Ser Thr 1400 1405 1410Met Asn Leu Leu Lys Lys Trp Met Ser Gln Lys Gln Leu Ile Phe 1415 1420 1425Gln Arg Pro Tyr Asn Met Thr Gln Cys Met Lys Ile Tyr Ser Val 1430 1435 1440Lys Glu Lys Phe Glu Lys Lys Ser Lys Asn Lys Tyr Leu Ile Leu 1445 1450 1455Gln His Leu Leu Arg Val Tyr Gln Gln Leu Lys Arg Lys Lys Ser 1460 1465 1470Thr Val Leu Lys Ala Lys Cys Lys Ile Val Ser Leu Ser Leu Leu 1475 1480 1485Leu Ile Pro Gly Leu Lys Thr Leu Arg Ser Lys Leu Lys Ile Lys 1490 1495 1500Ile Val Tyr Tyr Leu Tyr Arg Val Asn Leu His Leu Asn Gly Leu 1505 1510 1515Arg Lys Asp Ile Lys Gln Leu Lys Gln Ser Leu Lys Lys Leu Asp 1520 1525 1530Met Phe Ser Lys Lys Ser Asn Glu Lys Cys Asn Lys Leu Leu Lys 1535 1540 1545Tyr Phe Ser Ser Phe Phe Tyr Leu Glu Ile Val Lys Lys Ile Ser 1550 1555 1560Gly Arg Tyr Gln Tyr Leu Met Ser Thr Asp Leu Asn Leu Phe Arg 1565 1570 1575Leu Glu Leu Ile Ile Asn Thr Thr Asn Asn Leu Met Arg Asp Lys 1580 1585 1590Glu Asp Thr Lys Ile Leu Ile Ser Ile Pro Ile Lys Phe Gln Gly 1595 1600 1605Gly Asp Asn Leu Ile Arg Gly Leu Ser Thr Arg Lys Asp Pro Asn 1610 1615 1620Lys Ile Phe Thr Arg Val Ile Thr Leu Ile Asn Phe Leu Met Gly 1625 1630 1635Glu Gly Leu Lys Phe Asn Asp Lys Glu Asn Asn Leu Leu Arg Lys 1640 1645 1650Ala Phe Lys Arg Lys Glu Leu Cys Asp Ala Lys Leu Phe Thr Phe

1655 1660 1665Ser Leu Thr Leu Ser Asn Ser Ser Ile Ser Asn Cys Cys Cys Ile 1670 1675 1680Ile Lys Leu Ile Leu Phe Cys Thr Thr Phe Ser Gly Ile Asn Thr 1685 1690 1695Ser Glu Ala Cys Phe Ile Asn Ser Gly Ser Leu Lys Ser Met Arg 1700 1705 1710Ser Ile Tyr Gly Ile Ala Ser Thr Gln Val Ser Leu Ser Ala Ser 1715 1720 1725Ser Glu Cys Phe Ser Tyr Thr Ser Met Gly Ile Ser Lys Phe Ser 1730 1735 1740Ser Cys Ser Lys Val Ala Lys Ser Leu Trp Asn Phe Leu Ser Phe 1745 1750 1755Ser Ser Thr Phe Leu Ser Lys Thr Asn Thr Leu Thr Ser Glu Ser 1760 1765 1770Met Ala Asp Val Phe Pro Val Ile Ile Ser Ile Pro Asn Leu Leu 1775 1780 1785Asp Arg Asn Ser Gly Arg Lys Ser Phe Gly Glu Arg Ile Phe Leu 1790 1795 1800Cys Ser Asn Ile Ser Asp Thr Ala Pro Ser Lys Ser Val Gly Glu 1805 1810 1815Gly Ile Leu Pro Ile Ser Ser His Phe Val Glu Lys Ser Tyr Ser 1820 1825 1830Ser Ile Tyr Leu Ser Tyr Tyr Arg Thr Val Glu Leu Phe Asn Gln 1835 1840 1845Gly Ser Val Leu Phe Phe Ile Ile Leu Lys Leu Cys Ser Leu Gln 1850 1855 1860Leu Asp Leu Phe Phe Gln Ile Ser Arg Val Thr Ser Leu Ala Asp 1865 1870 1875Pro Ala Arg Gly Pro Ala Val Arg Trp His Phe Ser Gly Lys Cys 1880 1885 1890Ala Arg Asn Pro Tyr Leu Phe Ile Phe Leu Asn Thr Phe Lys Tyr 1895 1900 1905Val Ser Ala His Glu Thr Ile Thr Leu Ile Asn Ala Ser Ile Ile 1910 1915 1920Leu Lys Lys Glu Glu Tyr Glu Tyr Ser Thr Phe Pro Cys Arg Pro 1925 1930 1935Tyr Ser Leu Phe Cys Gly Ile Leu Pro Ser Cys Phe Cys Ser Pro 1940 1945 1950Arg Asn Ala Gly Glu Ser Lys Arg Cys Arg Ser Val Gly Cys Thr 1955 1960 1965Ser Gly Leu His Arg Thr Gly Ser Gln Gln Arg Asp Pro Glu Phe 1970 1975 1980Ser Pro Arg Arg Thr Phe Ser Asn Asp Glu His Phe Ser Ser Ala 1985 1990 1995Met Trp Arg Gly Ile Ile Pro Tyr Arg Arg Ala Arg Ala Thr Arg 2000 2005 2010Ser Pro His Thr Leu Phe Ser Glu Leu Gly Val Leu Thr Ser His 2015 2020 2025Arg Lys Ala Ser Tyr Gly Trp His Asp Ser Lys Arg Ile Met Gln 2030 2035 2040Cys Cys His Asn His Glu His Cys Gly Gln Leu Thr Ser Asp Asn 2045 2050 2055Asp Arg Arg Thr Glu Gly Ala Asn Arg Phe Phe Ala Gln His Gly 2060 2065 2070Gly Ser Cys Asn Ser Pro Ser Leu Gly Thr Gly Ala Glu Ser His 2075 2080 2085Thr Lys Arg Arg Ala His His Asp Ala Cys Ser Asn Gly Asn Asn 2090 2095 2100Val Ala Gln Thr Ile Asn Trp Arg Thr Thr Tyr Ser Ser Phe Pro 2105 2110 2115Ala Thr Ile Asn Arg Leu Asp Gly Gly Gly Ser Cys Arg Thr Thr 2120 2125 2130Ser Ala Leu Gly Pro Ser Gly Trp Leu Val Tyr Cys Ile Trp Ser 2135 2140 2145Arg Ala Trp Val Ser Arg Tyr His Cys Ser Thr Gly Ala Arg Trp 2150 2155 2160Ala Leu Pro Tyr Arg Ser Tyr Leu His Asp Gly Glu Ser Gly Asn 2165 2170 2175Tyr Gly Thr Lys Thr Asp Arg Asp Arg Cys Leu Thr Asp Ala Leu 2180 2185 2190Val Thr Val Arg Pro Ser Leu Leu Ile Tyr Thr Leu Asp Phe Lys 2195 2200 2205Thr Ser Phe Leu Ile Lys Asp Leu Gly Glu Asp Pro Phe Ser His 2210 2215 2220Asp Gln Asn Pro Leu Thr Val Phe Val Pro Leu Ser Val Arg Pro 2225 2230 2235Arg Arg Lys Asp Gln Arg Ile Phe Leu Arg Ser Phe Phe Ser Ala 2240 2245 2250Arg Asn Leu Leu Leu Ala Asn Lys Lys Thr Thr Ala Thr Ser Gly 2255 2260 2265Gly Leu Phe Ala Gly Ser Arg Ala Thr Asn Ser Phe Ser Glu Gly 2270 2275 2280Asn Trp Leu Gln Gln Ser Ala Asp Thr Lys Tyr Cys Pro Ser Ser 2285 2290 2295Val Ala Val Val Arg Pro Pro Leu Gln Glu Leu Cys Ser Thr Ala 2300 2305 2310Tyr Ile Pro Arg Ser Ala Asn Pro Val Thr Ser Gly Cys Cys Gln 2315 2320 2325Trp Arg Val Val Ser Tyr Arg Val Gly Leu Lys Thr Ile Val Thr 2330 2335 2340Gly Gly Ala Ala Val Gly Leu Asn Gly Gly Phe Val His Thr Ala 2345 2350 2355Gln Leu Gly Ala Asn Asp Leu His Arg Thr Glu Ile Pro Thr Ala 2360 2365 2370Ala Met Arg Lys Arg His Ala Ser Arg Arg Glu Lys Gly Gly Gln 2375 2380 2385Val Ser Gly Lys Arg Gln Gly Arg Asn Arg Arg Ala His Glu Gly 2390 2395 2400Ala Ser Arg Gly Lys Arg Leu Val Ser Leu Ser Cys Arg Val Ser 2405 2410 2415Pro Pro Leu Thr Ala Ser Ile Phe Val Met Leu Val Arg Gly Ala 2420 2425 2430Glu Pro Met Glu Lys Arg Gln Gln Arg Gly Leu Phe Thr Val Pro 2435 2440 2445Gly Leu Leu Leu Ala Phe Cys Ser His Val Leu Ser Cys Val Ile 2450 2455 2460Pro Phe Cys Gly Pro Tyr Tyr Arg Leu Val Ser Tyr Arg Ser Pro 2465 2470 2475Gln Pro Asn Asp Arg Ala Gln Arg Val Ser Glu Arg Gly Ser Gly 2480 2485 2490Arg Ala Pro Asn Thr His 2495

Claims

1. A plasmid comprising: a sequence encoding a signal peptide selected from the group consisting of: B. subtilis alpha amylase signal peptide (AmyE), B. subtilis levanase signal peptide (SacC), B. subtilis YwmC signal peptide, and a variant thereof; and a sequence encoding an endoxylanase selected from Trichoderma reesei or Bacillus pumilus or a variant thereof; wherein: the sequence encoding the signal peptide is upstream of the sequence encoding the endoxylanase thereby producing a recombinant endoxylanase modified with the signal peptide; the amino acid sequence of the variant AmyE encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:1; the amino acid sequence of the variant SacC encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:2; and the amino acid sequence of the variant YwmC encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:3.

2. The plasmid of claim 1, wherein the amino acid sequence of the endoxylanase or variant thereof encoded by the sequence encoding the endoxylanase has at least 90% sequence identity to SEQ ID NO:4.

3. The plasmid of claim 2, wherein the amino acid sequence of the variant signal peptide encoded by the sequence encoding the signal peptide has at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

4. The plasmid of claim 2, wherein the amino acid sequence of the recombinant endoxylanase is set forth in SEQ ID NO:6.

5. The plasmid of claim 4, wherein the amino acid sequence of the plasmid is set forth in SEQ ID NO:7.

6. The plasmid of claim 1, wherein the sequence encoding the endoxylanase or variant thereof has at least 90% sequence identity to SEQ ID NO:5.

7. The plasmid of claim 6, wherein the sequence encoding the signal peptide has at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

8. The plasmid of claim 6, wherein the sequence encoding the signal peptide is set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

9. The plasmid of claim 8, wherein the sequence encoding the endoxylanase is set forth in SEQ ID NO:5.

10. A method of producing value-added products from agricultural biomass, the method comprising: providing an agricultural biomass, wherein the agricultural biomass comprises cellobiose and xylan; adding a culture of a xylose producer to the agricultural biomass, wherein the xylose producer is a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass, wherein the recombinant bacteria is selected from the group consisting of: Corynebacterium glutamicum, Bacillus subtilis, and Bacillus coagulans; and adding a culture of a xylose assimilator to the agricultural biomass, wherein the xylose assimilator is a bacterium selected from the group consisting of: Escherichia coli, B. coagulans, Lactobacillus pentosus, Lactobacillus brevis, Leuconostoc lactis, a different strain of B. coagulans than the xylose producer, and a different strain of B. subtilis than the xylose producer.

11. The method of claim 10, wherein the xylose producer is a recombinant B. subtilis.

12. The method of claim 11, wherein the recombinant B. subtilis has been transformed with the plasmid of claim 1.

13. The method of claim 11, wherein the recombinant B. subtilis has been transformed with the plasmid of claim 5.

14. The method of claim 11, wherein the xylose assimilator is a succinate producer.

15. The method of claim 14, wherein is succinate producer is E. coli.

16. A composition for simultaneously degrading and assimilate cellobiose and xylan comprising: a recombinant bacteria engineered to produce xylose from hydrolyzing the agricultural biomass, wherein the recombinant bacteria is selected from the group consisting of: Corynebacterium glutamicum, Bacillus subtilis, and Bacillus coagulans; and a xylose assimilator, wherein the xylose assimilator is a bacterium selected from the group consisting of: Escherichia coli, B. coagulans, Lactobacillus pentosus, Lactobacillus brevis, Leuconostoc lactis, a different strain of B. coagulans than the xylose producer, and a different strain of B. subtilis than the xylose producer.

17. The composition of claim 16, further comprising a media comprising a trace metal solution and M9 media, wherein the trace metal solution comprises sulfate salts of copper, irone, zinc, and magnesium and the M9 media comprises KH.sub.2PO.sub.4, Na.sub.2HPO.sub.4, NaCl, NH.sub.4Cl, glucose, tryptophan, and citrate.

18. The composition of claim 16, wherein the recombinant bacteria is transformed with a plasmid comprising: a sequence encoding a signal peptide selected from the group consisting of: B. subtilis alpha amylase signal peptide (AmyE), B. subtilis levanase signal peptide (SacC), B. subtilis YwmC signal peptide, and a variant thereof; and a sequence encoding an endoxylanase selected from Trichoderma reesei or Bacillus pumilus or a variant thereof; wherein: the sequence encoding the signal peptide is upstream of the sequence encoding the endoxylanase thereby producing a recombinant endoxylanase modified with the signal peptide; the amino acid sequence of the variant AmyE encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:1; the amino acid sequence of the variant SacC encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:2; and the amino acid sequence of the variant YwmC encoded by the sequence encoding the signal peptide has at least 80% identity to SEQ ID NO:3.

19. The composition of claim 18, wherein the recombinant bacteria is B. subtilis.

20. The composition of claim 19, wherein the xylose assimilator is a bacterium that is a succinate producer.