Carbohydrate-enriched recombinant microorganisms

Patent metadata

Field	Value
Publication number	US-10337043-B2
Application number	US-201515111733-A
Country	US
Kind code	B2
Filing date	Jan 16, 2015
Priority date	Jan 16, 2014
Publication date	Jul 2, 2019
Grant date	Jul 2, 2019

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 methanotrophic bacterium, comprising an exogenous nucleic acid that encodes a glucan synthase,
wherein the recombinant methanotrophic bacterium is capable of producing glucan at a level that is greater than that produced by the parent methanotrophic bacterium; and
wherein the methanotrophic bacterium is selected from the group consisting of Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas , and Methylocella.

2. The recombinant C 1 metabolizing microorganism of claim 1 , wherein the C 1 substrate feedstock comprises natural gas or methane.

3. The recombinant methanotrophic bacterium of claim 1 , wherein the methanotrophic bacterium is an obligate methanotrophic bacterium or a facultative methanotrophic bacterium.

4. The recombinant methanotrophic bacterium of claim 1 , wherein the glucan produced is a β-glucan.

5. The recombinant methanotrophic bacterium of claim 4 , 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.

6. The recombinant methanotrophic bacterium of claim 1 , wherein the encoded exogenous glucan synthase comprises a β-1,3-glucan synthase.

7. The recombinant methanotrophic bacterium of claim 1 , wherein the exogenous nucleic acid encoding the glucan synthase is endogenous to an organism selected from the group consisting of a bacteria, a yeast, a fungi, and a plant.

8. The recombinant methanotrophic bacterium of claim 1 , wherein the exogenous nucleic acid encoding the glucan synthase is endogenous to Escherichia coli or Corynebacterium glutamicum.

9. The recombinant methanotrophic bacterium of claim 1 , wherein the exogenous nucleic acid encodes a glucan synthase that comprises a nucleic acid sequence having at least 85% sequence identity 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, and 25.

10. The recombinant methanotrophic bacterium of claim 1 , wherein the exogenous nucleic acid encodes a glucan synthase having an amino acid sequence that has at least 90% sequence identity 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, and 26.

11. The recombinant methanotrophic bacterium of claim 1 , wherein the exogenous nucleic acid comprises an expression control sequence that is operably linked to the nucleic acid encoding the glucan synthase.

12. The recombinant methanotrophic bacterium of claim 1 , wherein the exogenous nucleic acid is codon optimized for the methanotrophic bacterium.

13. The recombinant methanotrophic bacterium of claim 1 , wherein the methanotrophic bacterium further comprises an exogenous polynucleotide encoding one or more gluconeogenesis enzymes 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.

14. The recombinant methanotrophic bacterium of claim 13 , wherein the further exogenous polynucleotide encoding one or more gluconeogenesis enzymes comprises an expression control sequence that is operably linked to the polynucleotide encoding the one or more gluconeogenesis enzymes, wherein the one or more gluconeogenesis enzymes are heterologous gluconeogenesis enzymes, native gluconeogenesis enzymes, or a combination thereof.

15. The recombinant methanotrophic bacterium of claim 1 , wherein the carbohydrates of the recombinant methanotrophic bacterium exhibits a δ 13 C that is less than −30‰ or is less than −40‰.

16. The recombinant methanotrophic bacterium of claim 1 , wherein the methanotrophic bacterium is selected from the group consisting of Methylococcus capsulatus Bath, Methylomonas sp. 16a, Methylosinus trichosporium OB3b, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus Y, Methylomonas flagellata AJ-3670, Methylacidiphilum infernorum, Methylacidiphdum fumariolicum, Methylomicrobium alcaliphilum , and Methyloacida kamchatkensis.

17. A biomass derived from whole and/or lysed cells of the recombinant methanotrophic bacterium of claim 1 .

18. The biomass of claim 17 , wherein the biomass exhibits a δ 13 C that is less than −30‰ or is less than −40‰.

19. A carbohydrate composition, comprising carbohydrates extracted from a biomass derived from the methanotrophic bacterium of claim 1 , wherein the composition exhibits a δ 13 C that is less than −30‰ or is less than −40‰.

20. An animal feed, comprising the recombinant methanotrophic bacterium of claim 1 , a biomass derived from whole and/or lysed cells of the recombinant methanotrophic bacterium of claim 1 , or a carbohydrate composition comprising carbohydrates extracted from a biomass derived from the recombinant methanotrophic bacterium of claim 1 .

21. The animal feed of claim 20 , further comprising an additive selected from the group consisting of a plant-derived material, an animal-derived material, and a microorganism-derived material.

22. The animal feed of claim 21 , wherein the additive is microorganism-derived material that is derived from a microorganism heterologous to the methanotrophic bacterium.

23. The animal feed of claim 21 , wherein the additive is a plant-derived material that is derived from corn, soybean meal or pea protein.

24. The animal feed of claim 21 , wherein the additive is an animal-derived material that is fish meal.

25. A method of producing a desired carbohydrate, the method comprising culturing the recombinant methanotrophic bacterium of claim 1 in the presence of a C 1 substrate feedstock comprising methane under conditions sufficient to produce the glucan.

26. The method of claim 25 , wherein the C 1 substrate feedstock comprises natural gas.

27. The method of claim 25 , wherein the methanotrophic bacterium is selected from the group consisting of Methylococcus capsulatus Bath, Methylomonas sp. 16a, Methylosinus trichosporium OB3b, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus Y, Methylomonas flagellata AJ-3670, Methylacidiphilum infernorum, Methylacidiphilum fumariolicum, Methylomicrobium alcaliphilum , and Methyloacida kamchatkensis.

Assignees

• Calysta Inc

Inventors

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

Classifications

• C12P19/04

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• A23K50/00

  Feeding-stuffs specially adapted for particular animals · CPC title

• C12Y204/01034

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

• A23K20/163

  Sugars; Polysaccharides · 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