Compositions and methods for the production of compounds

Patent metadata

Field	Value
Publication number	US-11479797-B2
Application number	US-201716345445-A
Country	US
Kind code	B2
Filing date	Oct 27, 2017
Priority date	Oct 28, 2016
Publication date	Oct 25, 2022
Grant date	Oct 25, 2022

Abstract

The present disclosure provides proteins, nucleic acids, vectors, and host molecules useful for the production of compounds of interest, and methods for their use.

First claim

The invention claimed is:

1. A method of producing a compound, the method comprising:
(a) providing a parent nucleic acid encoding a parent polyketide synthase;
(b) modifying at least a first codon of a ketoreductase domain of the parent nucleic acid to produce a modified nucleic acid encoding a modified polyketide synthase capable of producing a compound, wherein modifying the first codon produces a modified ketoreductase domain comprising at least one of the following modifications:
(i) a substitution of an amino acid other than tyrosine at a position corresponding to a tyrosine in a conserved YAAAN (SEQ ID NO: 97) catalytic motif and a deletion of a region corresponding to a conserved αFG helix of SEQ ID NO: 1,
(ii) a substitution of a glutamic acid residue at a position corresponding to alanine 6632 of an S9-pksA ORF of SEQ ID NO: 2, or
(iii) a deletion of amino acids corresponding to amino acids 3386 to 3516 of a WT S12-pksB ORF of SEQ ID NO: 3; and
wherein the one or more modifications decrease the enzymatic activity of the modified ketoreductase domain in comparison to the enzymatic activity of the ketoreductase domain of the parent polyketide synthase
(c) introducing the modified nucleic acid to one or more host cells; and
(d) culturing the one or more host cells under conditions suitable to allow expression of the compound by the modified polyketide synthase;
thereby producing the compound.

2. The method of claim 1 , wherein (b) further comprises modifying at least a second codon of the parent nucleic acid,
wherein modifying the second codon produces a second modified domain, and
wherein the second modified domain is a β-ketone processing domain.

3. The method of claim 2 , wherein the β-ketone processing domain is a ketoreductase, a dehydratase, or an enoylreductase.

4. The method of claim 3 , wherein the β-ketone processing domain is a ketoreductase, wherein the ketoreductase (a) comprises a substitution of an amino acid other than tyrosine at a position corresponding to a tyrosine in a conserved YAAAN (SEQ ID NO: 97) catalytic motif and a deletion of a region corresponding to a conserved αFG helix of SEQ ID NO: 1; (b) comprises a substitution of a glutamic acid residue at a position corresponding to alanine 6632 of an S9-pksA ORF of SEQ ID NO: 2; or (c) comprises a deletion of amino acids corresponding to amino acids 3386 to 3516 of a WT S12-pksB ORF of SEQ ID NO: 3.

5. The method of claim 3 , wherein the β-ketone processing domain is a dehydratase, wherein the dehydratase comprises (a) a substitution of an aspartic acid at a position corresponding to a glycine at position 4288 in pksB of an S679-pksB ORF in a conserved HXXXGXXXXP (SEQ ID NO: 98) motif of SEQ ID NO: 4; (b) a substitution in a conserved LPFXW (SEQ ID NO: 99) motif at a position corresponding to position 3066 to 3070 in an S12-pksB ORF of SEQ ID NO: 5; (c) a deletion corresponding to a region between Pro 6844 and Trp 6874 of an S679-pksA ORF of SEQ ID NO: 6; or (d) a substitution or deletion at the positions corresponding to A, B, C, and D of SEQ ID NO: 7.

6. The method of claim 3 , wherein the β-ketone processing domain is an enoylreductase, wherein the enoylreductase comprises a substitution or deletion of a lysine at a position corresponding to position 1546 of an S12-pksB ORF of SEQ ID NO: 8 and/or a substitution or deletion of an aspartic acid at a position corresponding to position 1568 of an S12-pksB ORF of SEQ ID NO: 8 or SEQ ID NO: 9.

7. The method of claim 1 , wherein the modified nucleic acid further encodes a Large ATP-binding regulator of the LuxR family (LAL).

8. The method of claim 7 , wherein the LAL comprises the amino acid sequence of SEQ ID NO: 38.

9. The method of claim 1 , wherein the modified nucleic acid further comprises an LAL binding site, wherein the nucleic acid sequence encoding the LAL binding site is operatively linked to the nucleic acid sequence encoding the first modified polyketide synthase.

10. The method of claim 9 , wherein the LAL binding site comprises the nucleic acid sequence of SEQ ID NO: 39.

11. The method of claim 9 , wherein the LAL binding site comprises the nucleic acid sequence of SEQ ID NO: 40.

12. The method of claim 10 , wherein binding of an LAL to the LAL binding site promotes the expression of the first modified polyketide synthase.

13. The method of claim 1 , wherein the modified nucleic acid further encodes a nonribosomal peptide synthase.

14. The method of claim 1 , wherein the modified nucleic acid further encodes a P450 enzyme.

15. The method of claim 1 , wherein the modified ketoreductase domain is functionally inactive.

Assignees

• Ginkgo Bioworks Inc

Inventors

• Gray Daniel C
• Bowman Brian R
• Verdine Gregory L
• Sowa Mathew Edward

Classifications

• C12N9/1029 Primary

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12P19/62 Primary

  the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title