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

Patent metadata

Field	Value
Publication number	US-9719098-B2
Application number	US-201514624163-A
Country	US
Kind code	B2
Filing date	Feb 17, 2015
Priority date	Apr 5, 2011
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

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.

First claim

What is claimed is:

1. A recombinant yeast 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 gpd1, gpd2, gpp1, gpp2 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 fsp1 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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 yeast 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.

19. The recombinant yeast of claim 1 , wherein said yeast is a member of a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces , and Yarrowia.

20. The recombinant yeast of claim 1 , wherein said yeast is a member of a species selected from the group consisting of S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lac tis, K. marxianus , and K. fragilis.

21. The recombinant yeast of claim 1 , wherein said yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorphs, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymmphus, Schizosaccharomyces pombe, Candida albicans , and Schwanniomyces occidentalis.

22. The recombinant yeast of claim 1 , wherein said yeast is Saccharomyces cerevisiae.

23. The recombinant yeast of claim 1 , wherein said pyruvate formate lyase is from one or more of a Bijidobacteria , an Escherichia , a Thennoanaerobacter , a Clostridia , a Streptococcus , a Lactobacillus , a Chlamydom

Assignees

• Lallemand Hungary Liquidity Man Llc

Inventors

• Argyros Aaron
• Sillers William Ryan
• Barrett Trisha
• Caiazza Nicky
• Shaw Iv Arthur J

Classifications

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12P7/40

  containing a carboxyl group {including Peroxycarboxylic acids} · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12Y102/0101

  Acetaldehyde dehydrogenase (acetylating) (1.2.1.10) · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title