Glycogen-null methanotrophs and uses thereof

Patent metadata

Field	Value
Publication number	US-12234445-B2
Application number	US-201917293832-A
Country	US
Kind code	B2
Filing date	Dec 26, 2019
Priority date	Dec 27, 2018
Publication date	Feb 25, 2025
Grant date	Feb 25, 2025

Abstract

The present disclosure provides methanotrophic bacteria that are modified to produce less glycogen, and methods of using the modified methanotrophic bacteria to produce a desired product, such as protein(s) or metabolite(s).

First claim

What is claimed is:

1. A modified Methylococcus capsulatus , comprising:
(i) a chromosomal knock-out of a glgA2 isoform of a glycogen synthase gene, or
(ii) a chromosomal knock-out of an ADP-glucose pyrophosphorylase gene and a chromosomal knock-out of a glgA2 isoform of a glycogen synthase gene, wherein the modified Methylococcus capsulatus cultured under conditions comprising a non-limiting amount of a C 1 substrate produces:
at least about 30% less glycogen as compared to the parent Methylococcus capsulatus cultured under the same conditions; and/or
at least about 5% more crude protein as compared to the parent Methylococcus capsulatus cultured under the same conditions.

2. The modified Methylococcus capsulatus of claim 1 , wherein the Methylococcus capsulatus is Methylococcus capsulatus Bath.

3. The modified Methylococcus capsulatus of claim 1 or claim 2 , wherein the culture conditions further comprise the presence of a limiting amount of a nutrient or metabolite required for growth.

4. The modified Methylococcus capsulatus of claim 3 , wherein the limiting amount of the nutrient required for growth comprises a limiting amount of nitrogen.

5. The modified Methylococcus capsulatus of claim 1 , wherein the production of at least about 5% more crude protein as compared to the parent Methylococcus capsulatus further comprises a culture condition comprising from about 20% to about 80% nitrogen fixation.

6. The modified Methylococcus capsulatus of claim 1 , wherein the modified Methylococcus capsulatus has a lower ratio of utilized oxygen to utilized methane as compared to the parent Methylococcus capsulatus.

7. The modified Methylococcus capsulatus of claim 1 , wherein the chromosomal knock-out of the ADP-glucose pyrophosphorylase gene comprises an inactivating deletion comprising the polynucleotide sequence of SEQ ID NO:1.

8. The modified Methylococcus capsulatus of claim 1 , wherein the chromosomal knock-out of the glgA2 isoform of the glycogen synthase gene comprises an inactivating deletion comprising the polynucleotide sequence of SEQ ID NO:3.

9. The modified Methylococcus capsulatus of claim 1 , wherein the chromosomal knock-out comprises a knock-out of the ADP-glucose pyrophosphorylase gene and a knock-out of the glgA2 isoform of the glycogen synthase gene.

10. The modified Methylococcus capsulatus of claim 1 , wherein the modified Methylococcus capsulatus further comprises a chromosomal knock-out of a phosphoglucomutase gene and/or a glycogen branching enzyme gene.

11. The modified Methylococcus capsulatus of claim 10 , wherein the knock-out of the phosphoglucomutase gene is a knock-out of SEQ ID NO:4 or 5.

12. The modified Methylococcus capsulatus of claim 10 , wherein the knock-out of the glycogen branching enzyme gene is a knock-out of SEQ ID NO:6.

13. The modified Methylococcus capsulatus of claim 1 , wherein the modified Methylococcus capsulatus further comprises increased expression of an endogenous glycogen catabolism enzyme.

14. The modified Methylococcus capsulatus of claim 13 , wherein the glycogen catabolism enzyme is a glycogen phosphorylase, a glycogen debranching enzyme, an adenosine diphosphate sugar pyrophosphatase, or any combination thereof.

15. The modified Methylococcus capsulatus of claim 13 , wherein the increased expression of the endogenous glycogen catabolism enzyme is increased expression of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or any combination thereof.

16. The modified Methylococcus capsulatus of claim 1 , wherein the modified Methylococcus capsulatus further comprises a heterologous nucleic acid encoding a glycogen catabolism enzyme.

17. The modified Methylococcus capsulatus of claim 16 , wherein the glycogen catabolism enzyme is a glycogen phosphorylase gene, a glycogen debranching enzyme gene, an adenosine diphosphate sugar pyrophosphatase gene, or any combination thereof.

18. The modified Methylococcus capsulatus of claim 1 , wherein the modified Methylococcus capsulatus further comprises a heterologous nucleic acid encoding a glycolysis pathway enzyme.

19. The modified Methylococcus capsulatus of claim 1 , wherein the modified Methylococcus capsulatus further comprises a heterologous nucleic acid encoding a biosynthesis enzyme to produce a desired product.

20. The modified Methylococcus capsulatus of claim 19 , wherein the encoded biosynthesis enzyme is an amino acid biosynthesis enzyme selected from the group consisting of a lysine biosynthesis enzyme, a tryptophan biosynthesis enzyme, a methionine biosynthesis enzyme, a cysteine biosynthesis enzyme, and a threonine biosynthesis enzyme.

21. The modified Methylococcus capsulatus of claim 20 , wherein the encoded biosynthesis enzyme is a lysine biosynthesis enzyme selected from the group consisting of a lysine-sensitive aspartokinase III, an aspartate kinase, an aspartate-semialdehyde dehydrogenase, a dihydrodipicolinate synthase, a dihydrodipicolinate reductase, a 2,3,4,5-tetrahydropyridine-2,6-carboxylate N-succinyltransferase, an acetylornithine/succinyldiaminopimelateaminotransferase, a succinyl-diaminopimelate desuccinylase, a succinyldiaminopimelate transaminase, a diaminopimelate epimerase, a diaminopimelate dicarboxylase, or any combination thereof.

22. The modified Methylococcus capsulatus of claim 21 , wherein the Methylococcus capsulatus expresses a deregulated endogenous aspartokinase activity.

23. The modified Methylococcus capsulatus of claim 22 , wherein the deregulated endogenous aspartokinase activity is an aspartokinase mutant that is resistant to feedback inhibition by one or more of lysine, threonine, and methionine.

24. The modified Methylococcus capsulatus of claim 22 , wherein the deregulated endogenous aspartokinase activity is encoded by a mutant thrA gene, metL gene, lysC gene or any combination thereof, each comprising a spontaneous mutation, random mutation, site specific mutation, or any combination thereof.

25. The modified Methylococcus capsulatus of claim 19 , wherein the heterologous nucleic acid encoding the biosynthesis enzyme further comprises a heterologous control element that activates or increases expression of one or more biosynthesis enzymes as compared to the biosynthesis enzyme comprising its native control element.

26. The modified Methylococcus capsulatus of claim 25 , wherein the heterologous control element comprises a modified, endogenous promoter that regulates dihydrodipicolinate synthase, wherein the modification to the endogenous promoter increases lysine production.

27. The modified Methylococcus capsulatus of claim 20 , wherein the encoded biosynthesis enzyme is a tryptophan biosynthesis enzyme selected from the group consisting of a chorismate-pyruvate lyase, an anthranilate synthase component I, an anthranilate synthase component II, an anthranilate phosphoribosyltransferase, a phosphoribosylanthranilate isomerase, a tryptophan biosynthesis protein, an N-(5′phosphoribosyl) anthranilate isomerase, an indole-3-glycerol phosphate synthase, a tryptophan synthase alpha chain, a tryptophan synthase beta chain, and any combination thereof.

28. The modified Methylococcus capsulatus of claim 20 , wherein the encoded biosynthesis enzyme is a methionine methionine biosynthesis enzyme selected from the group consisting of a homoseri

Assignees

• Calysta Inc

Inventors

• Saville Renee M
• Silverman Joshua A
• Tang Vincent
• Luning Eric G
• Rueda Paloma
• Hsi Megan
• Stegantseva Yelena

Classifications

• C12Y504/02002

  Phosphoglucomutase (5.4.2.2) · CPC title

• C12Y207/07027

  Glucose-1-phosphate adenylyltransferase (2.7.7.27), i.e. ADP-glucose pyrophosphorylase · CPC title

• C12Y204/01011

  Glycogen(starch) synthase (2.4.1.11) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title