Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors

Abstract

The present invention provides for novel metabolic pathways to reduce or eliminate glycerol production and increase product formation. More specifically, the invention provides for a recombinant microorganism comprising a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source, such as lignocellulose, to a product, such as ethanol, wherein the one or more native and/or heterologous enzymes is activated, upregulated, or downregulated. The invention also provides for a recombinant microorganism comprising one or more heterologous enzymes that function to regulate glycerol synthesis and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated.

1 .- 95 . (canceled) 
     
     
         96 . A recombinant yeast comprising a first heterologous pyruvate formate lyase having at least 90% identity to the amino acid sequence of SEQ ID NO: 26 and/or a second heterologous pyruvate formate lyase having at least 90% identity to the amino acid sequence of SEQ ID NO: 60. 
     
     
         97 . The recombinant yeast of  claim 96  comprising said first heterologous pyruvate formate lyase and said second heterologous pyruvate formate lyase. 
     
     
         98 . The recombinant yeast of  claim 96  further comprising a heterologous polypeptide having acetaldehyde dehydrogenase activity. 
     
     
         99 . The recombinant yeast of  claim 98 , wherein said heterologous polypeptide having acetaldehyde dehydrogenase activity is a bifunctional acetaldehyde/alcohol dehydrogenase. 
     
     
         100 . The recombinant yeast of  claim 99 , wherein said bifunctional acetaldehyde/alcohol dehydrogenase is from  Bifidobacterium adolescentis.    
     
     
         101 . The recombinant yeast of  claim 100 , wherein said bifunctional acetaldehyde/alcohol dehydrogenase comprises the amino acid sequence of SEQ ID NO: 100. 
     
     
         102 . The recombinant yeast of  claim 96 , wherein said recombinant yeast comprises a downregulation in the expression of one or more native enzymes that function to produce glycerol or reduce glycerol synthesis and wherein said recombinant yeast produces less glycerol than a control recombinant microorganism with native enzymes that function to produce glycerol and/or regulate glycerol synthesis. 
     
     
         103 . The recombinant yeast of  claim 102 , wherein said one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis is encoded by a gpd1 polynucleotide, a gpd2 polynucleotide, a gpp1 polynucleotide, a gpp2 polynucleotide, or an fps1 polynucleotide. 
     
     
         104 . The recombinant yeast of  claim 96 , wherein said recombinant yeast further comprises a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis. 
     
     
         105 . The recombinant yeast of  claim 104 , wherein said one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis is encoded by a gpd1 polynucleotide, a gpd2 polynucleotide, a gpp1 polynucleotide, a gpp2 polynucleotide, or an fps1 polynucleotide. 
     
     
         106 . The recombinant yeast of  claim 96 , further comprising a deletion of one or more native enzymes encoded by an fdh1 polynucleotide, an fdh2 polynucleotide, or both an fdh1 polynucleotide and an fdh2 polynucleotide. 
     
     
         107 . The recombinant yeast of  claim 96 , wherein said recombinant yeast is selected from the group consisting of  Saccharomyces cerevisiae, Khiyveromyces lactis, Kluyveromvces marxianus, Pichia pastoris, Yarrowia lipolylica, Hansenula polymorpha, Phaffia rhodozyma, Candida uthis, Arxula adeninivorans, Pichia stipitis, Debaryomvces hansenii, Debaryomvces polymorphus, Schizosaccharomyces pombe, Candida albicans , and  Schwanniomyces occidentahs.    
     
     
         108 . The recombinant yeast of  claim 107 , wherein said recombinant yeast is  Saccharomyces cerevisiae.    
     
     
         109 . The recombinant yeast of  claim 96 , wherein said recombinant yeast produces formate. 
     
     
         110 . The recombinant yeast of any one of  claim 109 , wherein said recombinant yeast produces an amount of formate of:
 a. at least 0.012 g/L in 24 hours;   b. at least 0.022 g/L in 48 hours; or   c. at least 2.5 g/L in 142 hours.   
     
     
         111 . The recombinant yeast of  claim 109 , wherein said recombinant yeast produces a formate yield of:
 a. at least 0.05-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   b. at least 0.1-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   c. at least 0.5-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   d. at least 1.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   e. at least 5.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   f. at least 10.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   g. at least 20.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   h. at least 30.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   i. at least 40.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   j. at least 50.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase;   k. at least 75.0-fold more formate than is produced by a recombinant yeast lacking the first and/or the second heterologous pyruvate formate lyase; or   l. at least 100-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.   
     
     
         112 . The recombinant yeast of  claim 96 , wherein said recombinant yeast further comprises a heterologous saccharolytic enzyme. 
     
     
         113 . The recombinant yeast of  claim 112 , wherein said saccharolytic enzyme is selected from the group consisting of amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes. 
     
     
         114 . The recombinant yeast of  claim 113 , wherein said amylases comprises a glucoamylase. 
     
     
         115 . A method of increasing cytosolic formate during the production of ethanol, the method comprises contacting the recombinant yeast of claim  1  with a biomass under conditions to allow cytosolic formate and ethanol production. 
     
     
         116 . The method of  claim 115 , wherein the contact occurs in anaerobic conditions. 
     
     
         117 . The method of  claim 115 , wherein the biomass comprises corn.