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.

What is claimed: 
     
         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 2 PO 4 , Na 2 HPO 4 , NaCl, NH 4 Cl, 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.