Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors
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. A recombinant microorganism comprising:
(a) a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis, wherein said one or more enzymes is encoded by a glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-phosphate dehydrogenase (gpd2) glycerol 3-phosphatase 1′, glycerol 3-phosphatase 2 or fps1 polynucleotide; and
(b) 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 one of said metabolic pathways comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase, wherein one of said metabolic pathways comprises conversion of acetyl-CoA to ethanol by an acetaldehyde dehydrogenase, alcohol dehydrogenase, or a bifunctional acetaldehyde/alcohol dehydrogenase, and wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated.
2. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
3. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpp1 polynucleotide and a gpp2 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
4. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to regulate glycerol synthesis is encoded by an fps1 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
5. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to regulate glycerol synthesis is encoded by an fps1 polynucleotide and said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
6. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide.
7. The recombinant microorganism of claim 6 , further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
8. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and said one or more native enzymes that function to regulate glycerol synthesis is encoded by an fps1 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide.
9. The recombinant microorganism of claim 8 , further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
10. The recombinant microorganism of claim 2 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
11. The recombinant microorganism of claim 3 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
12. The recombinant microorganism of claim 4 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
13. The recombinant microorganism of claim 5 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
14. The recombinant microorganism of claim 6 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
15. The recombinant microorganism of claim 7 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
16. The recombinant microorganism of claim 8 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
17. The recombinant microorganism of claim 9 , wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.
18. The recombinant microorganism of claim 1 , wherein said one or more native enzymes that function to produce glycerol is encoded by a gpd2 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.