Nucleic acids and vectors for use with methanotrophic bacteria

• US 10,443,060 B2
• Filed: 06/18/2015
• Issued: 10/15/2019
• Est. Priority Date: 06/18/2014
• Status: Active Grant

• Current Assignee: Calysta Incorporated
• Original Assignee: Calysta Incorporated
• Inventors: Saville, Renee M., Silverman, Joshua, Minshull, Jeremy, Ness, Jon Edward, Leonard, Effendi, Stumpe, Jana, Welch, Mark
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US15/319,312
• Publication Number:
  US 20170121718A1
• Time in Patent Office: 1,580 Days
• Field of Search: None
• 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 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Y 101/01244 Methanol dehydrogenase (1.1.1.244)

Abstract

The present disclosure provides nucleic acids and vectors for use with methanotrophic bacteria. Related host cells and methods for using such nucleic acids and vectors for expressing polypeptides or other genetic manipulation of methanotrophic bacteria are also provided. In one aspect, the present disclosure is directed to a non-naturally occurring nucleic acid molecule, comprising (1) a promoter that is functional in a methanotrophic bacterium, and (2) a native or altered methanol dehydrogenase (MDH) ribosomal binding sequence, provided that when the promoter is an MDH gene promoter, the nucleic acid comprises an altered MDH ribosomal binding sequence.

Claims

1. A non-naturally occurring nucleic acid molecule, comprising:

(1) a promoter that is functional in a methanotrophic bacterium, and

(2) an altered methanol dehydrogenase (MDH) or a native MDH ribosomal binding sequence, wherein the altered MDH ribosomal binding sequence comprises the polynucleotide sequence as set forth in any one of SEQ ID NOS.;

20, 22, 24, 26, 27, 32, and 41;

provided that when the promoter is an MDH gene promoter, the nucleic acid comprises an altered MDH ribosomal binding sequence.

2. The nucleic acid of claim 1, wherein the promoter is a constitutive promoter or an inducible promoter.

3. The nucleic acid of claim 1, comprising an altered MDH ribosomal binding sequence.

4. The nucleic acid of claim 3, wherein the promoter is an MDH promoter, 30S ribosomal protein S16 promoter, 30S ribosomal protein S12 promoter, 50S ribosomal protein L13 promoter, hexulose 6-phosphate synthase promoter, T5 promoter, or Trc promoter.

5. The nucleic acid of claim 4, wherein the MDH promoter comprises a promoter portion of SEQ ID NO.:

14, the 30S ribosomal protein S16 promoter comprises a promoter portion of SEQ ID NO.;

15, the 30S ribosomal protein S12 promoter comprises a promoter portion of SEQ ID NO.;

43, the 50S ribosomal protein L13 promoter comprises a promoter portion of SEQ ID NO.;

44, the hexulose 6-phosphate synthase promoter comprises a promoter portion of SEQ ID NO.;

16, the T5 promoter comprises a promoter portion of SEQ ID NO.;

17, and the Trc promoter comprises a promoter portion of SEQ ID NO.;

18.

6. The nucleic acid of claim 4, further comprising a nucleic acid molecule encoding a target polypeptide operably linked to the promoter sequence and the ribosomal binding sequence.

7. The nucleic acid of claim 6, wherein the target polypeptide is a marker, a reporter protein, or a protein capable of improving production of a desired chemical or metabolite.

8. The nucleic acid of claim 7, wherein the target polypeptide is a crotonase, crotonyl CoA thioesterase, 4-oxalocrotonate decarboxylase, lactate dehydrogenase, or antibiotic resistance protein.

9. The nucleic acid of claim 7, wherein the target polypeptide is an amino acid biosynthesis enzyme, a fatty acid converting enzyme, a fatty acid elongation pathway enzyme, or a carbohydrate biosynthesis enzyme.

10. The nucleic acid of claim 9, wherein the amino acid biosynthesis enzyme is a lysine biosynthesis enzyme, a threonine biosynthesis enzyme, a methionine biosynthesis enzyme, or a cysteine biosynthesis enzyme.

11. The nucleic acid of claim 9, wherein the fatty acid converting enzyme is a fatty acyl-CoA reductase, a fatty alcohol forming acyl-ACP reductase, and/or a carboxylic acid reductase.

12. The nucleic acid of claim 9, wherein the fatty acid elongation pathway enzyme is a β

-ketoacyl-CoA synthase, a β

-ketoacyl-CoA reductase, a β

-hydroxy acyl-CoA dehydratase, and/or an enoyl-CoA reductase.

13. The nucleic acid of claim 9, wherein the carbohydrate biosynthesis enzyme is a glucan synthase.

14. The nucleic acid of claim 6, wherein the target polypeptide is LacI repressor protein or a BenR activator protein.

15. The nucleic acid of claim 14, wherein the promoter is a 30S ribosomal protein S16 promoter.

16. The nucleic acid of claim 15, wherein the nucleic acid further comprises a second promoter, a lac operator to which LacI repressor protein is capable of binding, and a sequence encoding a second target polypeptide, wherein the second promoter and the lac operator are operably linked to the nucleic acid molecule encoding the second target polypeptide.

17. The nucleic acid of claim 16, wherein the second target polypeptide is a marker, a reporter protein, or a protein capable of improving production of a desired chemical or metabolite.

18. The nucleic acid of claim 17, wherein the second target polypeptide is a lactate dehydrogenase, crotonase, crotonyl CoA thioesterase, 4-oxalocrotonate decarboxylase, antibiotic resistance protein, amino acid biosynthesis enzyme, fatty acid converting enzyme, fatty acid elongation pathway enzyme, or carbohydrate biosynthesis enzyme.

19. The nucleic acid of claim 18, wherein the second promoter is an MDH promoter.

20. The nucleic acid of claim 19, further comprising a second ribosomal binding sequence, wherein the second ribosomal binding sequence comprises an altered or native methanol dehydrogenase (MDH) ribosomal binding sequence.

21. A vector, comprising the nucleic acid of claim 1.

22. A host cell, comprising the nucleic acid of claim 1.

23. The host cell of claim 22, wherein the host cell is Methylococcus capsulatus Bath, Methylomonas 16a, Methylosinus trichosporium OB3b, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylobacterium organophilum, Methylomonas sp AJ-3670, Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum, Methylacidiphilum fumariolicum, Methyloacida kamchatkensis, Methylibium petroleiphilum, or Methylomicrobium alcahphilum.

24. The host cell of claim 22, wherein the host cell is Methylococcus capsulatus Bath or Methylosinus trichosporium OB3b.