Recombinant synthesis of medium chain-length alkanes

• US 9,034,629 B2
• Filed: 01/27/2014
• Issued: 05/19/2015
• Est. Priority Date: 01/25/2013
• Status: Expired due to Fees

• Current Assignee: Joule Unlimited Technologies, Inc.
• Original Assignee: Joule Unlimited Technologies, Inc.
• Inventors: Skraly, Frank Anthony, Li, Ning
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US14/165,214
• Publication Number:
  US 20140213826A1
• Time in Patent Office: 477 Days
• US Class Current: 435/252.3
• CPC Class Codes: C07C 9/15 Straight-chain hydrocarbons
• CPC Class Codes: C10L 1/08 for compression ignition
• CPC Class Codes: C10L 2200/04 Organic compounds
• CPC Class Codes: C10L 2270/026 for diesel engines, e.g. automobiles, stationary, marine
• CPC Class Codes: C10L 2270/04 for turbines, planes, power generation
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• 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 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1288 Transferases for other substituted phosphate groups (2.7.8)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 5/02 acyclic C12P5/007 takes precedence
• CPC Class Codes: C12Y 102/99006 Carboxylate reductase (1.2.99.6)
• CPC Class Codes: C12Y 207/08007 Holo-[acyl-carrier-protein] synthase (2.7.8.7)
• CPC Class Codes: C12Y 301/02014 Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
• CPC Class Codes: C12Y 401/99005 Octadecanal decarbonylase (4.1.99.5)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present disclosure identifies methods and compositions for modifying photoautotrophic organisms as hosts, such that the organisms efficiently produce alkanes, and in particular the use of such organisms for the commercial production of alkanes and related molecules. Other materials, methods, and compositions are also described.

Claims

1. An engineered cyanobacterium, wherein the engineered cyanobacterium comprises recombinant genes encoding an alkane deformylative monooxygenase having EC number 4.1.99.5, a thioesterase having EC number 3.1.2.14, and a carboxylic acid reductase having EC number 1.2.99.6, wherein the alkane deformylative monooxygenase is encoded by adm, the thioesterase is encoded by fatB or fatB2, and the carboxylic acid reductase is encoded by carB, wherein the thioesterase converts C(_8-12) acyl-ACP to C(_8-12) fatty acid, wherein the carboxylic acid reductase converts C(_8-12) fatty acid to C(_8-12) aldehyde, wherein the alkane deformylative monooxygenase converts C(_8-12) aldehyde to C(_7-11) alkane, and wherein the engineered cyanobacterium produces at least one of heptane, nonane, and undecane in an amount greater than an otherwise identical cyanobacterium, cultured under identical conditions, but lacking recombinant genes encoding alkane deformylative monooxygenase, thioesterase, and carboxylic acid reductase.

2. The engineered cyanobacterium of claim 1, wherein the cyanobacterium further comprises one or more recombinant genes encoding a phosphopanthetheinyl transferase.

3. The engineered cyanobacterium of claim 1, wherein the cyanobacterium further comprises one or more recombinant genes encoding a phosphopanthetheinyl transferase having EC number 2.7.8.7.

4. The engineered cyanobacterium of claim 3, wherein the phosphopanthetheinyl transferase is encoded by entD.

5. The engineered cyanobacterium of claim 1, wherein the cyanobacterium is a thermotolerant cyanobacterium, or a Synechococcus species.

6. The engineered cyanobacterium of claim 1, wherein expression of an operon comprising one or more of the recombinant genes is controlled by a recombinant promoter, and wherein the promoter is constitutive or inducible.

7. The engineered cyanobacterium of claim 1, wherein the cyanobacterium produces alkanes 7 to 11 carbon atoms in length;

or alkanes 7, 8, 9, 10, or 11 carbon atoms in length.

8. A cell culture comprising a culture medium and the cyanobacterium of claim 1.

9. A method for producing hydrocarbons, comprising:

culturing an engineered cyanobacterium of claim 1 in a culture medium, wherein the engineered cyanobacterium produces increased amounts of alkanes relative to an otherwise identical cyanobacterium, cultured under identical conditions, but lacking the recombinant genes.

10. A method for producing hydrocarbons, comprising:

(i) culturing an engineered cyanobacterium of claim 1 in a culture medium; and

(ii) exposing the engineered cyanobacterium to light and inorganic carbon, wherein said exposure results in the conversion of said inorganic carbon by the cyanobacterium into alkanes, wherein the alkanes are produced in an amount greater than that produced by an otherwise identical cyanobacterium, cultured under identical conditions, but lacking the recombinant genes.

11. The engineered cyanobacterium of claim 1, wherein the cyanobacterium produces alkanes 9 carbon atoms in length.

12. The engineered cyanobacterium of claim 1, wherein the cyanobacterium produces alkanes 11 carbon atoms in length.

13. The engineered cyanobacterium of claim 1, wherein the cyanobacterium produces alkanes 9 and 11 carbon atoms in length.

14. The method of claim 9, wherein the alkanes produced are 9 or 11 carbons in length.

15. The method of claim 10, wherein the alkanes produced are 9 or 11 carbons in length.

16. The engineered cyanobacterium of claim 1, wherein the predominant alkane produced by the cyanobacterium is nonane.