Compositions and methods for the production of compounds

Patent metadata

Field	Value
Publication number	US-10907188-B2
Application number	US-201716093074-A
Country	US
Kind code	B2
Filing date	Apr 12, 2017
Priority date	Apr 12, 2016
Publication date	Feb 2, 2021
Grant date	Feb 2, 2021

Abstract

The present disclosure provides nucleic acids encoding a Large ATP-binding regulator of the LuxR family (LAL) of transcription factors, vectors and host cells including such nucleic acids, and methods for producing compounds (e.g., polyketides or β-lactam compounds) with such nucleic acids, vectors, and/or host cells.

First claim

What is claims is:

1. A genetically modified host cell comprising:
(i) a nucleic acid encoding a recombinant Large ATP-binding regulator of the LuxR family (LAL) that is heterologous to the host cell; and
(ii) a nucleic acid comprising an LAL binding site that is heterologous to the host cell.

2. The host cell of claim 1 , wherein the host cell naturally lacks an LAL or the host cell naturally lacks an LAL binding site.

3. The host cell of claim 1 , wherein the LAL binding site is operably linked to an open reading frame.

4. The host cell of claim 3 , wherein the open reading frame encodes a compound-producing protein.

5. The host cell of claim 1 , wherein:
the recombinant LAL comprises a portion having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1;
the recombinant LAL comprises a portion having the amino acid sequence of SEQ ID NO: 1; or
the recombinant LAL has the amino acid sequence of SEQ ID NO: 1.

6. The host cell of claim 4 , wherein the host cell has been modified to enhance expression of the compound-producing protein by (i) deletion of an endogenous gene cluster which expresses an endogenous compound-producing protein; (ii) insertion of a heterologous gene cluster which expresses a heterologous compound-producing protein; (iii) exposure of the host cell to an antibiotic challenge; and/or (iv) introduction of a heterologous promoter that results in an at least 2-fold increase in expression of a compound produced by the compound-producing protein compared to the expression of the compound when the homologous promoter has not been replaced.

7. The host cell of claim 1 , wherein:
the nucleic acid further comprises one or more additional LAL binding sites;
at least one of the LAL binding sites is in a promoter; or
the nucleic acid further comprises a gene encoding an LAL.

8. The host cell of claim 7 , wherein:
the gene encoding an LAL is under the control of a promoter comprising an LAL binding site; or
at least one of the LAL binding sites is in a promoter.

9. The host cell of claim 8 , wherein at least one of the LAL binding sites is in a promoter and the promoter is a bidirectional promoter.

10. A nucleic acid comprising an LAL binding site and a sequence encoding an LAL, wherein the LAL binding site comprises a sequence having no more than one insertion, deletion, or substitution with respect to the nucleic acid sequence of SEQ ID NO:2 and/or comprises the nucleic acid sequence of SEQ ID NO: 3, and wherein
the LAL comprises a portion having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1;
the LAL comprises a portion having the amino acid sequence of SEQ ID NO: 1; or
the LAL has the amino acid sequence of SEQ ID NO: 1.

11. The nucleic acid of claim 10 , wherein the nucleic acid lacks a TTA inhibitory codon in at least one open reading frame.

12. The nucleic acid of claim 10 , wherein the LAL binding site comprises the nucleic acid sequence of SEQ ID NO:2.

13. The nucleic acid of claim 10 , wherein the nucleic acid further comprises an open reading frame.

14. The nucleic acid of claim 13 , wherein the open reading frame encodes a compound-producing protein.

15. The nucleic acid of claim 14 , wherein the compound-producing protein is a polyketide synthase, a β-lactam compound-producing protein, or a non-ribosomal peptide synthase.

16. The nucleic acid of claim 10 , wherein:
the nucleic acid further comprises one or more additional LAL binding sites; or
the gene encoding the LAL is under the control of a promoter comprising an LAL binding site.

17. The nucleic acid of claim 16 , wherein at least one of the LAL binding sites is in a promoter.

18. The nucleic acid of claim 17 , wherein the promoter is a bidirectional promoter.

19. An expression vector comprising a nucleic acid of claim 10 .

20. A host cell comprising the nucleic acid of claim 10 .

Assignees

• Warp Drive Bio Inc
• Ginkgo Bioworks Inc

Inventors

• Bowman Brian R
• Blodgett Joshua A V
• Verdine Gregory L
• Gray Daniel C
• Morgenstern Jay P
• Foulston Lucy
• Robison Keith

Classifications

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12Y114/00

  Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• G01N2333/36

  from Actinomyces; from Streptomyces (G) · CPC title