Engineered microorganisms with enhanced fermentation activity

Abstract

Provided herein are genetically modified microorganisms that have enhanced fermentation activity, and methods for making and using such microorganisms.

1 - 35 . (canceled) 
     
     
         36 . A method for producing ethanol, comprising contacting an engineered yeast with a feedstock that contains one or more hexose sugars under conditions in which the yeast synthesizes ethanol, which yeast comprises:
 (i) alterations that add or increase a phosphogluconate dehydratase activity, a 2-keto-3-deoxygluconate-6-phosphate aldolase activity, and a 6-phosphogluconolactonase activity;   (ii) alterations that reduce or substantially remove a phosphofructokinase 1 (PFK1) activity, phosphofructokinase 2 (PFK2) activity, or PFK1 and PFK2 activity; and   (iii) a nucleotide sequence identification tag having a nucleotide sequence chosen from the following six (6) nucleotide sequences:   
       
         
           
                 
               
                   (SEQ ID NO: 448) 
                 
                 
               
                   GCGTCCATACCGGACCGTCCATCCGTCCCGGCGGGCTATCGTTAGTCCC 
                 
                     
                 
                   CGCGAGCGGATTCCGAGGTGTCGATGACGCGCTCGGTCCCCGCATCTCG 
                 
                     
                 
                   GGGTGGCCCGCACCTAGCTTAAGCGGACTACGAAGCGCGGGGCGAGCGG 
                 
                     
                 
                   CGACGATCGCGTACTCACACTCGGACCTCGCGGGTCGGCTCGGAGCCCT 
                 
                     
                 
                   GGTCA 
                 
                     
                 
                 
               
                   (SEQ ID NO: 449) 
                 
                 
               
                   AGCGGTCAGTGCACGGGACGCGATCGGGCACCCTCGACGCAGCGATGGG 
                 
                     
                 
                   CCGTGCGTCGTGTAGTCCGATAGTGCCGGCGTCGCTCGGTAAGCCCCTT 
                 
                     
                 
                   ATACCTGCGCGCTGGCGAGAGATGGGTTCGCGAGTCTAGCGCGATCGCT 
                 
                     
                 
                   CTAGAGGGTCCAGGAGTACCTACACGGCGCGAGGCGCGGACATCCTAGG 
                 
                     
                 
                   GCGCA 
                 
                     
                 
                 
               
                   (SEQ ID NO: 450) 
                 
                 
               
                   CCCCTGCGTTTGCCGAGCGACGAGTCCTACACCCTGTCCGCGCCCGAGC 
                 
                     
                 
                   AGGGTCGTCCCCGCGAACCGACGGATGCGCGGCCCGAATCGCCTAGACC 
                 
                     
                 
                   CCTACGGGGCGGCTCGCTCGGCCCCGCCTGACCGGTCGATCCCACGAGA 
                 
                     
                 
                   CCCCGCCCTATAGGGAGAGCACCGACCCGCCTCCTCGGGCCTTACGGCG 
                 
                     
                 
                   TGCGA 
                 
                     
                 
                 
               
                   (SEQ ID NO: 451) 
                 
                 
               
                   GCCAGTGTAGAGATCCGGGGATCCCCAGCGCCTGGAGCTAGGCCCACGG 
                 
                     
                 
                   CGTCTGACCGGGTGTACCGGGCCCCCTAGGACGGGTGCGCCCGTAGTCC 
                 
                     
                 
                   GTCTGCGAGGGGGCCGTCCGGTCGGGGGCATCCGGCGCTCCGCGGGGAG 
                 
                     
                 
                   GCGCTACGTGCCCGACCGGGGGAGTCGAGTCTCTATGCTCGCGACCGCG 
                 
                     
                 
                   TGCGA 
                 
                     
                 
                 
               
                   (SEQ ID NO: 452) 
                 
                 
               
                   AAGCGCGCACTACGTCAGGCATAGCGTACTGGGCTTGCGGAGCCACGCG 
                 
                     
                 
                   GGCGCGGAGCGGGCCGGTTGAGTGCGGGATAGACGGACCGTACGCATGC 
                 
                     
                 
                   CTCAAGTCGACGGTACGGGGGGCAGGGTAGCTGGGATCCGAGGCGGGTA 
                 
                     
                 
                   GGCGTCGGCCGCGACTGTGCCCGTACGACGGGAGAACCCCCCGCGCGAG 
                 
                     
                 
                   TTGGA 
                 
                     
                 
                 
               
                   (SEQ ID NO: 453) 
                 
                 
               
                   ACGTCGGCAGGCCCGCTCGGTTCCGAGCACCGGATCGACGCTACACGAG 
                 
                     
                 
                   GCCCGACACTAGGCGCGTACTCCGGGGGGGTCCGCCTCCGTCCCGTGAG 
                 
                     
                 
                   TATCGCGGGCGGGAACAGGGCGGGCTGCCGGGGCCGACCGGTGTGGGGC 
                 
                     
                 
                   GTGACTCCGACCGACTCGGGCGAGGGCCGCCTAGTCGCGAAGGACGCGC 
                 
                     
                 
                   GACCA 
                 
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         37 . The method of  claim 36 , wherein the yeast is a  Saccharomyces  spp. yeast. 
     
     
         38 . The method of  claim 37 , wherein the yeast is a  Saccharomyces cerevisiae  yeast. 
     
     
         39 . The method of  claim 36 , wherein the engineered yeast synthesizes ethanol to about 85% to about 99% of theoretical yield. 
     
     
         40 . The method of  claim 36 , comprising recovering ethanol synthesized by the engineered yeast. 
     
     
         41 . The method of  claim 36 , wherein the conditions are fermentation conditions. 
     
     
         42 . The method of  claim 36 , wherein the yeast comprises heterologous polynucleotides that encode independently a phosphogluconate dehydratase enzyme and a 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme. 
     
     
         43 . The method of  claim 42 , wherein the polynucleotides encoding the phosphogluconate dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase enzyme independently are from an  Escherichia  spp. microbe or  Psuedomonas  spp. microbe. 
     
     
         44 . The method of  claim 43 , wherein the  Escherichia  spp. microbe is an  Escherichia coli  strain. 
     
     
         45 . The method of  claim 43 , wherein the  Pseudomonas  spp. microbe is a  Pseudomonas aeruginosa  strain. 
     
     
         46 . The method of  claim 43 , wherein the polynucleotide that encodes the phosphogluconate dehydratase enzyme is from a  Pseudomonas aeruginosa  strain. 
     
     
         47 . The method of  claim 46 , wherein the polynucleotide that encodes the phosphogluconate dehydratase enzyme encodes an amino acid sequence comprising the polypeptide sequence of SEQ ID NO: 70. 
     
     
         48 . The method of  claim 36 , wherein the yeast comprises a heterologous polynucleotide, or multiple copies of an endogenous polynucleotide, that encodes a 6-phosphogluconolactonase enzyme. 
     
     
         49 . The method of  claim 48 , wherein the polynucleotide that encodes the 6-phosphogluconolactonase enzyme encodes an amino acid sequence comprising the polypeptide sequence of SEQ ID NO: 120. 
     
     
         50 . The method of  claim 36 , wherein the yeast comprises a genetic alteration that increases or adds a glucose-6-phosphate dehydrogenase activity. 
     
     
         51 . The method of  claim 50 , wherein the yeast comprises a heterologous polynucleotide that encodes a glucose-6-phosphate dehydrogenase enzyme, or wherein the yeast comprises multiple copies of an endogenous polynucleotide that encodes a glucose-6-phosphate dehydrogenase enzyme. 
     
     
         52 . The method of  claim 51 , wherein the polynucleotide that encodes the glucose-6-phosphate dehydrogenase enzyme encodes an amino acid sequence comprising the polypeptide sequence of any one of SEQ ID NOS: 119, 123, 127 and 129. 
     
     
         53 . The method of  claim 36 , wherein the yeast comprises an alteration in one or more polynucleotides that inhibits production of one or more enzymes chosen from a phosphoglucoisomerase (PGI) enzyme, 6-phosphogluconate dehydrogenase (decarboxylating) enzyme, transketolase enzyme, transaldolase enzyme, or combination thereof. 
     
     
         54 . The method of  claim 53 , wherein the transketolase enzyme is encoded by a TKL-1 coding sequence comprising SEQ ID NO: 428. 
     
     
         55 . The method of  claim 53 , wherein the transaldolase is encoded by a TAL-1 coding sequence comprising SEQ ID NO: 426.