Carbohydrate-enriched recombinant microorganisms

Patent metadata

Field	Value
Publication number	US-2016333384-A1
Application number	US-201515111733-A
Country	US
Kind code	A1
Filing date	Jan 16, 2015
Priority date	Jan 16, 2014
Publication date	Nov 17, 2016
Grant date	—

Abstract

The present disclosure relates to recombinant microorganisms engineered for enhanced production of a desired carbohydrate, as well as related biomass, and compositions which are useful, inter alia, as animal feed ingredients. The present disclosure also provides related methods.

First claim

What is claimed is:

1 . A recombinant C 1 metabolizing microorganism comprising an exogenous nucleic acid selected from the group consisting of an exogenous nucleic acid that encodes a carbohydrate biosynthesis enzyme and an exogenous nucleic acid that encodes an expression control sequence that is operably linked to a nucleic acid encoding a native carbohydrate biosynthesis enzyme, wherein the recombinant C 1 metabolizing microorganism is capable of converting a natural gas-derived carbon feedstock into a desired carbohydrate.

2 . The recombinant C 1 metabolizing microorganism of claim 1 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme.

3 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 2 , wherein the natural gas-derived feedstock is natural gas.

4 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 3 , wherein the natural gas-derived feedstock is methane.

5 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 4 , wherein the recombinant C 1 metabolizing microorganism is capable of producing the desired carbohydrate at a level that is at least 10% greater than that produced by the native C 1 metabolizing microorganism.

6 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 5 , wherein the C 1 metabolizing microorganism is a methanotroph.

7 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 6 , wherein the desired carbohydrate is selected from the group consisting of a polysaccharide, a disaccharide, and a monosaccharide.

8 . The recombinant C 1 metabolizing microorganism of claim 7 , wherein the desired carbohydrate is a polysaccharide that is a β-glucan.

9 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 8 , wherein the β-glucan is selected from the group consisting of a β-(1,3)-glucan, a β-(1,3)(1,6)-glucan, a β-(1,3)(1,4)-glucan, a β-(1,4)-glucan, and a β-(1,6)-glucan.

10 . The recombinant C 1 metabolizing microorganism of any of claims 8 - 9 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that is a glucan synthase.

11 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 7 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that is a gluconeogenesis enzyme.

12 . The recombinant C 1 metabolizing microorganism of claim 11 , wherein the gluconeogenesis enzyme is selected from the group consisting of a pyruvate carboxylase, a phosphoenolpyruvate carboxykinase, an enolase, a phosphoglycerate mutase, a phosphoglycerate kinase, a glyceraldehyde-3-phosphate dehydrogenase, a Type A aldolase, a fructose 1,6-bisphosphatase, a phosphofructokinase, a phosphoglucose isomerase, a hexokinase, and a glucose-6-phosphate.

13 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 7 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that is a glycogenesis enzyme.

14 . The recombinant C 1 metabolizing microorganism of claim 13 , wherein the glycogenesis enzyme is selected from the group consisting of a glucose-1-phosphate adenyltransferase, a glycogen synthase, and a 1,4-alpha-glucan-branching protein.

15 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 14 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that is endogenous to a bacteria.

16 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 14 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that is endogenous to an organism selected from the group consisting of a yeast, a fungi, and a plant.

17 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 14 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that is endogenous to a microorganism selected from the group consisting of E. coli and C. glutamicum.

18 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 7 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme having an amino acid sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

19 . The recombinant C 1 metabolizing microorganism of any of claims 1 and 3 - 9 , wherein the exogenous nucleic acid encodes an expression control sequence that is operably linked to a nucleic acid encoding a native carbohydrate biosynthesis enzyme.

20 . The recombinant C 1 metabolizing microorganism of claim 19 , wherein the native carbohydrate biosynthesis enzyme is a native gluconeogenesis enzyme.

21 . The recombinant C 1 metabolizing microorganism of claim 19 , wherein the native carbohydrate biosynthesis enzyme is a native glycogenesis enzyme.

22 . The recombinant C 1 metabolizing microorganism of claim 19 , wherein the native carbohydrate biosynthesis enzyme is a native glucan synthase.

23 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 22 , wherein the exogenous nucleic acid is codon optimized for optimal expression from the recombinant C 1 metabolizing microorganism.

24 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 6 , wherein the exogenous nucleic acid encodes a carbohydrate biosynthesis enzyme that comprises a nucleic acid sequence which is at least 85% identical to a nucleic acid reference sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.

25 . The recombinant C 1 metabolizing microorganism of any of claims 1 - 24 , wherein the microorganism exhibits a δ 13 C that is less than −30‰.

26 . The recombinant C 1 metabolizing microorganism of claim 25 , wherein the microorganism exhibits a δ 13 C that is less than −40‰.

27 . A biomass derived from the recombinant C 1 metabolizing microorganism of any of claims 1 - 26 .

28 . The biomass of claim 27 , wherein the biomass exhibits a δ 13 C that is less than −30‰.

29 . The biomass of claim 28 , wherein the biomass exhibits a δ 13 C that is less than −40‰.

30 . A carbohydrate composition comprising carbohydrates extracted from the biomass of any of claims 27 - 29 , wherein the composition exhibits a δ 13 C that is less than −30‰.

31 . The composition of claim 30 , wherein the composition exhibits a δ 13 C that is less than −40‰.

32 . An animal feed comprising the biomass of any of claims 27 - 29 or carbohydrate composition of any of claims 30 - 31 .

33 . The animal feed of claim 32 , further comprising an additive selected from the group consisting of a plant-derived material, an animal-derived material, and a microorganism-derived material.

34 . The animal feed of claim 33 , wherein the additive is microorganism-derived material that is derived from a heterologous microorganism.

35 . The animal feed of claim 33 , wherein the additive is a plant-derived material.

36 . The animal feed of claim 35 , wherein the plant-derived material is derived from corn.

37 . The animal feed of claim 35 , wherein the plant-de

Assignees

• Calysta Inc

Inventors

• Silverman Joshua A
• Giver Lorraine Joan
• Mueller Jana
• Saville Renee M
• Regitsky Drew D

Classifications

• C12Y204/01034

  1,3-Beta-glucan synthase (2.4.1.34) · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C08B37/0024

  beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof · CPC title

• C12N15/74 Primary

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• A23K50/00

  Feeding-stuffs specially adapted for particular animals · CPC title