COMPOSITIONS AND METHODS FOR BIOLOGICAL PRODUCTION OF LACTATE FROM C1 COMPOUNDS USING LACTATE DEHYDROGENASE TRANSFORMANTS

• US 20160369246A1
• Filed: 06/18/2014
• Published: 12/22/2016
• Est. Priority Date: 06/18/2013
• Status: Active Grant

• Current Assignee: Calysta Incorporated
• Original Assignee: Calysta Incorporated
• Inventors: Lee, Sungwon, Saville, Renee M., Regitsky, Drew D., Resnick, Sol M., Silverman, Joshua A.
• Granted Patent: US 10,450,548 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07B 2200/05 Isotopically modified compounds, e.g. labelled
• CPC Class Codes: C07B 59/001 Acyclic or carbocyclic compounds
• CPC Class Codes: C07C 59/08 Lactic acid
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/56 Lactic acid
• CPC Class Codes: C12Y 101/01027 L-Lactate dehydrogenase (1.1.1.27)
• CPC Class Codes: C12Y 101/01028 D-Lactate dehydrogenase (1.1.1.28)
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

The present disclosure provides non-naturally occurring c1 microorganisms useful for the production of lactate and related compositions, as well as methods for the biologically production of lactate. In specific embodiments, the present disclosure provides non-naturally occurring methanotrophic bacteria which are useful for producing lactate from c1 substrates.

Claims

1. A non-naturally occurring C₁ metabolizing microorganism comprising an exogenous nucleic acid encoding a lactate dehydrogenase (LDH), wherein the microorganism is a bacteria.

8. The non-naturally occurring C₁ metabolizing microorganism of any of claims 1, 3, and 5-7, wherein the C₁ metabolizing microorganism is a methylotrophic bacteria.

9. The non-naturally occurring C₁ metabolizing microorganism of claim 8, wherein the methylotrophic bacteria is a methanotrophic bacteria.

12. The non-naturally occurring C₁ metabolizing microorganism according to any of claims 1-11, wherein the non-naturally occurring C₁ metabolizing microorganism is selected from the group consisting of a Methylococcus, a Methylomonas, a Methylobacter, a Methylosinus, a Methylocystis, a Methylomicrobium, a Methanomonas, a Methylocella, a Methylacidiphilum, Methyloacida, a Methylibium, and a Methylocapsa.

13. The non-naturally occurring C₁ metabolizing microorganism of any of claims 1, 2, 4, and 9-11, wherein the methanotrophic bacteria is selected from the group consisting of Methylococcus capsulatus Bath strain, Methylomonas methanica 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-11,196), Methylomicrobium buryatense 5G, Methylosinus sporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylocella palustris (ATCC 700799), Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, Methylacidiphilum fumariolicum, Methyloacida kamchatkensis, Methylibium petroleiphilum, and Methylomicrobium alcaliphilum.

14. The non-naturally occurring C₁ metabolizing microorganism of any of claims 1-13, wherein the methanotrophic bacteria selected from the group consisting of Methylococcus capsulatus Bath strain, Methylosinus trichosporium OB3b (NRRL B-11,196), and Methylomicrobium buryatense 5G.

15. The non-naturally occurring C₁ metabolizing microorganism according to any of claims 1-14, wherein the exogenous nucleic acid encodes a lactate dehydrogenase comprising an amino acid sequence that is at least 85% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs:

34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, 52, 53, 54, 55, 56, 57, and 58.

16. The non-naturally occurring C₁ metabolizing microorganism of claim 15, wherein the reference amino acid sequence is selected from the group consisting of SEQ ID NOs:

34, 16, and 24.

17. The non-naturally occurring C₁ metabolizing microorganism of any of claims 1-14, wherein the exogenous nucleic acid encodes a lactate dehydrogenase having an amino acid sequence that corresponds to an amino acid sequence selected from the group consisting of SEQ ID NOs:

34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, 52, 53, 54, 55, 56, 57, and 58.

18. The non-naturally occurring C₁ metabolizing microorganism of any of claims 1-14, wherein the exogenous nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS:

33, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, and 47.

19. The non-naturally occurring C₁ metabolizing microorganism according to any of claims 1-14, wherein the exogenous nucleic acid molecule encodes an LDH from a microorganism selected from the group consisting of Listeria marthii, Actinomyces viscosus, Acinonyx jubatus, Archilochus colubris, Bacillus anthracis, Bacillus caldolyticus, Bacillus coagulans, Bacillus megaterium, Bacillus stearothermophilus (Q9p4b6) (also known as Geobacillus stearothermophilus), Bacillus subtilis, Bacillus thuringiensis, Bacteroides pectinophilus, Bifidobacterium longum, Bos taurus, Canis familiaris, Canis lupus, Deinococcus radiodurans, Enterococcus faecalis, Enterococcus faecium, Equus ferus, Felis catus, Kluyveromyces lactis, Kluyveromyces maxxianus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus coryniformis sp. torquens, Lactobacillus delbrueckii (including subsp. bulgaricus), Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Listeria monocytogenes, Plasmodium falciparum, Plasmodium ovale, Thermus thermophilus, Mus musculus, Oryctolagus cuniculus, Pediococcus acidilactici, Taeniopygia guttata, Rattus norvegicus, Rhizopus oryzae, Staphylococcus aureus, Streptococcus bovis, Streptococcus pasteurianus, Ruminococcus torques, Staphylococcus simiae, Staphylococcus vitulinus, Staphylococcus lentus, Macrococcus caseolyticus, Bacillus thuringiensis seovar konkukian str. 97-27, Bacillus thuringiensis serovar chinensis CT-43, and Bacillus mycoides,

20. The non-naturally occurring C₁ metabolizing microorganism according to any one of claims 1-17 and 19, wherein the exogenous nucleic acid sequence encoding LDH is codon optimized for expression in the C₁ metabolizing microorganism.

21. The non-naturally occurring C₁ metabolizing microorganism according to any one of claims 1-20, wherein the exogenous nucleic acid molecule encoding the lactate dehydrogenase is operatively linked to an inducible promoter.

22. The non-naturally occurring C₁ metabolizing microorganism according to any one of claims 1-16 and 19-21, wherein the lactate dehydrogenase is L-lactate dehydrogenase.

23. The non-naturally occurring C₁ metabolizing microorganism according to any one of claims 1-16 and 19-21, wherein the lactate dehydrogenase is a D-lactate dehydrogenase.

24. A method of producing lactate, comprising culturing the non-naturally occurring C₁ metabolizing microorganism of any one of claims 1-23 in the presence of a carbon feedstock comprising a C₁ substrate under conditions sufficient to produce lactate.

25. The method of claim 24, wherein the C₁ substrate is selected from the group consisting of methane, carbon monoxide, and carbon dioxide.

26. The method of claim 24, wherein the C₁ substrate is methane.

27. The method of any of claims 24-26, wherein the carbon feedstock is natural gas.

28. The method of any one of claims 20-28, wherein conditions sufficient to produce lactate comprise culturing the non-naturally occurring C₁ metabolizing microorganism in a fermentation medium that comprises a buffer.

29. The method of any one of claims 24-28, further comprising the step of separating the lactate from the non-naturally occurring C₁ metabolizing microorganism.

30. The method of any one of claims 24-29, further comprising the step of purifying the lactate.

31. The method of any one of claims 24-30, wherein the lactate is an L-lactate.

35. A lactate composition produced by the method of any one of claims 24-30.

2. A non-naturally occurring C₁ metabolizing microorganism comprising an exogenous nucleic acid encoding a lactate dehydrogenase, wherein the C₁ metabolizing microorganism is a methanotroph.

10. The non-naturally occurring C₁ metabolizing microorganism of any one of claims 2 and 9, wherein the methanotrophic bacteria selected from the group consisting of a Type I methanotroph, a Type II methanotroph, and a Type X methanotroph.

11. The non-naturally occurring C₁ metabolizing microorganism of any of claims 2, 3, 5-7, 9 and 10, wherein the non-naturally occurring C₁ metabolizing microorganism is selected from the group consisting of a facultative methanotroph and an obligate methanotroph.

3. A non-naturally occurring C₁ metabolizing microorganism comprising an exogenous nucleic acid encoding a lactate dehydrogenase, wherein the C₁ metabolizing microorganism is capable of converting a carbon feedstock into lactate, and wherein the carbon feedstock is selected from the group consisting of methane, carbon dioxide, carbon monoxide, syngas, and natural gas.

6. The non-naturally occurring C₁ metabolizing microorganism of claim 3, wherein the carbon feedstock is methane.

7. The non-naturally occurring C₁ metabolizing microorganism of claim 3, wherein the carbon feedstock is natural gas.

4. A non-naturally occurring C₁ metabolizing microorganism comprising an exogenous nucleic acid encoding a lactate dehydrogenase, wherein the C₁ metabolizing microorganism is not a yeast.

5. A non-naturally occurring C₁ metabolizing microorganism comprising an exogenous nucleic acid encoding a lactate dehydrogenase, wherein the non-naturally occurring C₁ metabolizing microorganism is capable of producing more lactate as compared to that of a corresponding reference C₁ metabolizing microorganism when cultured in the presence of a C₁ substrate under at least one set of culture conditions.

32. A lactate composition, wherein the lactate is a compound selected from the group consisting of lactic acid, a lactic acid salt, a lactic acid anhydride, lactic acid anion, a lactic acid oligomer or a lactic acid ester having one or more CH₃C(OH)C(O)—

group(s), and wherein the δ

¹³C of the CH₃C(OH)C(O)—

group(s) is less than about −

30‰

.

33. The lactate composition according to claim 32, wherein the δ

¹³C of the CH₃C(OH)C(O)—

group(s) ranges from about −

70‰

to about −

30‰

.

34. The lactate composition according to claim 33, wherein the δ

¹³C of the CH₃C(OH)C(O)—

group(s) ranges from about −

60‰

to about −

40‰

.