Natural promoters for gene expression in C1 metabolizing bacteria

• US 7,098,005 B2
• Filed: 10/14/2005
• Issued: 08/29/2006
• Est. Priority Date: 10/21/2002
• Status: Expired due to Fees

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Wang, Tao, Seip, John E., Picataggio, Stephen K., Ye, Rick W., Ni, Hao, Dicosimo, Deana J.
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US11/251,304
• Publication Number:
  US 20060036088A1
• Time in Patent Office: 319 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Abstract

Genes have been identified in the Methylomonas genome that are responsive to various metabolic and growth conditions. The identified responsiveness of these genes allows for the use of their promoters in regulated gene expression in C1 metabolizing bacteria. In particular, the hps promoter, which in its native state drives the expression of 3-hexulose-6-phosphate synthase (HPS), was found to be useful for directing expression of heterolgous coding regions (e.g., crtZ) in the obligate methanotroph Methylomonas sp. 16a.

Claims

1. A promoter region highly expressed in the presence of methane or methanol having the nucleic acid sequence as set forth in SEQ ID NO:

18.

2. A method for the expression of a coding region of interest in a C1 metabolizing bacteria comprising:

a) providing a transformed C1 metabolizing bacterial cell having a chimeric gene comprising;

1) a promoter region having the nucleic acid sequence as set forth in SEQ ID NO;

18; and

2) a coding region of interest expressible in a C1 metabolizing bacteria, wherein the promoter region is operably linked to a coding region of interest; and

b) growing the transformed C1 metabolizing bacteria cell of step (a) in the presence of a C1 carbon source selected from the group consisting of methane and methanol wherein the chimeric gene of step (a) is expressed.

3. A method according to claim 2 wherein the C1 metabolizing bacterial host cell is selected from the group consisting of methanotrophs and methylotrophs.

4. A method according to claim 3 wherein the C1 metabolizing bacterial host cell is a methylotroph selected from the group consisting of Methylomonas, Methylobacter, Mehtylococcus, Methylosinus, Methylocyctis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and Pseudomonas.

5. The method according to any claim 2 wherein the coding region of interest is selected from the group consisting of genes encoding:

transaldolase, fructose bisphosphate aldolase, keto deoxy phosphogluconate aldolase, phosphoglucomutase, glucose-6-phosphate isomerase, phosphofructokinase, 6-phosphogluconate dehydratase, 6-phosphogluconate-6-phosphate-1 dehydrogenase, dxs, dxr, ispA, ispD, ispE, ispF, crtE, crtX, crtY, crtI, crtB, crtZ, crtD, crtO, crtW, genes encoding limonene synthase, ugp, gumD, wza, espB, espM, waaE, espV, gumH, genes encoding glycosyltransferase genes, aroG, aroB, aroQ, aroE, aroK, 5-enolpyruvylshikimate-3-phosphate synthase, aroC, trpE, trpD, trpC, trpB, pheA, tyrAc, pds, phaC, phaE, efe, pdc, adh, pinene synthase, bornyl synthase, phellandrene synthase, cineole synthase, sabinene synthase, and taxadiene synthase.