Compositions and methods for biological production of lactate from C1 compounds using lactate dehydrogenase transformants

• US 10,450,548 B2
• Filed: 06/18/2014
• Issued: 10/22/2019
• Est. Priority Date: 06/18/2013
• Status: Active Grant

• Current Assignee: Calysta Incorporated
• Original Assignee: Calysta Incorporated
• Inventors: Saville, Renee M., Lee, Sungwon, Regitsky, Drew D., Resnick, Sol M., Silverman, Joshua A.
• Primary Examiner(s): Ekstrom, Richard C
• Application Number:
  US14/898,948
• Publication Number:
  US 20160369246A1
• Time in Patent Office: 1,952 Days
• Field of Search: None
• 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 methanotrophic bacteria comprising a heterologous nucleic acid encoding a lactate dehydrogenase (LDH), wherein methanotrophic bacteria is capable of converting methane into lactate.

2. The methanotrophic bacteria of claim 1, wherein the methanotrophic bacteria is selected from the group consisting of a Type I methanotroph, a Type II methanotroph, and a Type X methanotroph.

3. The methanotrophic bacteria of claim 1, wherein the methanotrophic bacteria is an obligate methanotroph.

4. The methanotrophic bacteria according to claim 1, wherein the methanotrophic bacteria 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.

5. The methanotrophic bacteria of claim 1, wherein the methanotrophic bacteria is selected from the group consisting of Methylococcus capsulatus Bath, Methylomonas methanica 16a , Methylosinus trichosporium OB3b, Methylomicrobium buryatense 5G, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylobacterium organophilum, Methylomonas sp AJ-3670 , Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, Methylacidiphilum fumariolicum, Methyloacida kamchatkensis, Methylibium petroleiphilum, and Methylomicrobium alcaliphilum.

6. The methanotrophic bacteria of claim 1, wherein the methanotrophic bacteria is selected from the group consisting of Methylococcus capsulatus Bath strain, Methylosinus trichosporium OB3b, and Methylomicrobium buryatense 5G.

7. The methanotrophic bacteria according to claim 1, wherein the heterologous 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, 53, 54, 55, 56, 57, and 58.

8. The methanotrophic bacteria of claim 7, wherein the reference amino acid sequence is selected from the group consisting of SEQ ID NOs:

34, 16, and 24.

9. The methanotrophic bacteria of claim 1, wherein the heterologous nucleic acid encodes a lactate dehydrogenase having 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, 53, 54, 55, 56, 57, and 58.

10. The methanotrophic bacteria of claim 1, wherein the heterologous 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.

11. The methanotrophic bacteria according to claim 1, wherein the heterologous nucleic acid molecule encodes an LDH from a microorganism selected from the group consisting ofListeria 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.

12. The methanotrophic bacteria according to claim 1, wherein the heterologous nucleic acid sequence encoding LDH is codon optimized for expression in the methanotrophic bacteria.

13. The methanotrophic bacteria according to claim 1, wherein the heterologous nucleic acid molecule encoding the lactate dehydrogenase is operatively linked to an inducible promoter.

14. The methanotrophic bacteria according to claim 1, wherein the lactate dehydrogenase is L-lactate dehydrogenase.

15. The methanotrophic bacteria according to claim 1, wherein the lactate dehydrogenase is a D-lactate dehydrogenase.

16. A method of producing lactate, comprising culturing the methanotrophic bacteria of claim 1 in the presence of a carbon feedstock comprising methane under conditions sufficient to produce lactate, wherein the methanotrophic bacteria convert methane to lactate.

17. The method of claim 16, wherein the carbon feedstock is natural gas.

18. The method of claim 16, wherein conditions sufficient to produce lactate comprise culturing the methanotrophic bacteria in a fermentation medium that comprises a buffer.