Hybrid polyketide synthases

Patent metadata

Field	Value
Publication number	US-9334514-B2
Application number	US-201113882099-A
Country	US
Kind code	B2
Filing date	Oct 31, 2011
Priority date	Oct 29, 2010
Publication date	May 10, 2016
Grant date	May 10, 2016

Abstract

The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

First claim

What is claimed is:

1. A non-naturally occurring hybrid polyketide synthase (PKS) which synthesizes adipic acid, wherein the PKS comprises:
(a) a first module that is a loading module that incorporates succinyl-CoA, wherein the first module comprises an aspartate-specific adenylation (A) domain from the non-ribosomal peptide synthetase (NRPS) from the calcium-dependent antibiotic (CPA) pathway from Saccharopolyspora erythraea linked to a peptidylcarrier protein (PCP) domain from the bleomycin pathway from Streptomyces verticillus ; wherein the A domain comprises a glutamine substitution for the conserved Asp235 residue of the substrate-binding pocket as defined in the gramicidin S synthase phenylalanine-activating domain; and
a second module comprising at least one extension module and a TE domain; or
(b) a first module that is a loading module that incorporates succinyl-CoA, wherein the first module comprises a loading domain and ACP domain of a chondrochloren PKS in which a succinyl-CoA synthetase replaces a CoA ligase domain and
a second module comprising at least one extension module and a TE domain; or
(c) an etnangien loading module which incorporates succinate using a trans-AT domain; and
(i) at least one heterologous extension module, and a TE domain: or
(ii) extension module 1 from Sorangium cellulosum PKS in which DH-ER-KR domains are inserted between the ACP2 and ACP3; and a TE domain; or
(iii) wherein the etnangien loading module has inactivated the KS domain joined to the KS domain of extension module 2 from Sorangium cellulosum PKS linked to a malonyl-specific AT domain joined to DH-ER-KR-ACP-TE domains; or

(d) a loading module and at least three extension modules as set forth in FIG. 14 , wherein the PKS produces the intermediates [a], [b], [c], and [d] set forth in FIG. 14 , wherein the 9-carbon backbone of [d] is released from the ACP by the TE domain and converted to adipic and propionic acid by an esterase, an alcohol dehydrogenase, a flavin-binding family monooxygenase, and an aldehyde dehydrogenase as set forth in FIG. 13 .

2. The non-naturally occurring hybrid PKS of claim 1 , wherein the PKS comprises the modules set forth in (a), wherein the at least one extension module comprises a KS domain joined to an AT-DH-ER-KR-ACP from nystatin module 5 or nystatin module 15 that incorporates malonyl-CoA and fully reduces a corresponding β-carbonyl group; and a TE domain fused to the ACP from the nystatin module 5 or 15.

3. The non-naturally occurring hybrid PKS of claim 2 , wherein the KS domain is from the bleomycin pathway from Streptomyces verticillus and the TE domain is from the erythromycin pathway.

4. The PKS of claim 2 , wherein the A domain is directly linked to the PCP domain.

5. The non-naturally occurring hybrid PKS of claim 1 , wherein the PKS comprises the modules set forth in (b) and wherein the at least one extension module comprises a nystatin module 5; and a TE domain.

6. The non-naturally occurring hybrid PKS of claim 1 , wherein the PKS comprises the etnangien loading module, the at least one heterologous extension module, and the TE domain as set forth in (c) (i).

7. The non-naturally occurring hybrid PKS of claim 1 , wherein the PKS comprises a loading module and three extension modules as set forth in FIG. 14 , wherein the PKS produces the intermediates [a], [b], [c], and [d] set forth in FIG. 14 , wherein the 9-carbon backbone of [d] is released from the ACP by the TE domain and converted to adipic and propionic acid by an esterase, an alcohol dehydrogenase, a flavin-binding family monooxygenase, and an aldehyde dehydrogenase as set forth in FIG. 13 .

8. The non-naturally occurring hybrid PKS of claim 1 , wherein the PKS comprises the modules set forth in FIG. 11 , and wherein the A domain is a variant of protein DhbE from Bacillus subtilis.

9. A recombinant nucleic acid encoding the non-naturally occurring hybrid polyketide synthase (PKS) of claim 1 .

10. A replicon comprising the recombinant nucleic acid of claim 9 , wherein the replicon is capable of stably maintained in a host cell.

11. The replicon of claim 10 , wherein the replicon is a plasmid or vector.

12. The replicon of claim 11 , wherein the vector is an expression vector.

13. A host cell comprising the recombinant nucleic acid of claim 9 or the replicon of claim 10 .

14. The host cell of claim 13 , wherein the host cell when cultured produces adipic acid.

15. A method of producing adipic acid comprising: culturing the host cell of claim 14 in a suitable culture medium such that adipic acid is produced.

16. The method of claim 15 , further comprising isolating the adipic acid.

17. The method of claim 16 , further comprising reacting the adipic acid with a diamine to produce a nylon.

Assignees

• Fortman Jeffrey L
• Hagen Andrew
• Katz Leonard
• Keasling Jay D
• Poust Sean
• Zhang Jingwei
• Zotchev Sergey
• Univ California

Inventors

• Fortman Jeffrey L
• Hagen Andrew
• Katz Leonard
• Keasling Jay D
• Poust Sean
• Zhang Jingwei
• Zotchev Sergey

Classifications

• C12P13/005

  Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12N9/93 Primary

  Ligases (6) · CPC title

• C12Y604/00

  Ligases forming carbon-carbon bonds (6.4) · CPC title

• C12P7/46

  Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid · CPC title