Compositions and methods for the production of compounds
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.
1. A method of producing a compound, the method comprising:
(a) providing 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, wherein the LAL binding site is operably linked to an open reading frame encoding a compound-producing protein, and wherein binding of the recombinant LAL to the LAL binding site promotes expression of the compound-producing protein; and
(b) culturing the host cell under conditions suitable to allow expression of a compound by the compound-producing protein;
thereby producing a compound.
2. The method of claim 1 , wherein the host cell naturally lacks an LAL or the host cell naturally lacks an LAL binding site.
3. The method 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.
4. The method of claim 1 , wherein the recombinant LAL comprises a portion having the amino acid sequence of SEQ ID NO: 1.
5. The method of claim 1 , wherein the recombinant LAL has the amino acid sequence of SEQ ID NO: 1.
6. The method of claim 1 , 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 method 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 method 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 method of claim 8 , wherein at least one of the LAL binding sites is in a promoter and the promoter is a bidirectional promoter.
10. The method of claim 1 , 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.
11. The method of claim 10 , wherein the LAL comprises a portion having the amino acid sequence of SEQ ID NO: 1.
12. The method of claim 10 , wherein the LAL has the amino acid sequence of SEQ ID NO: 1.
13. The method of claim 10 , wherein the LAL binding site comprises the nucleic acid sequence of SEQ ID NO:2.
14. The method of claim 10 , wherein the LAL binding site comprises the nucleic acid sequence of SEQ ID NO:3.
15. The method 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.
16. The method of claim 15 , wherein at least one of the LAL binding sites is in a promoter.
17. The method of claim 16 , wherein the promoter is a bidirectional promoter.
18. The method of claim 1 , wherein the compound-producing protein is a polyketide synthase, a β-lactam compound-producing protein, or a non-ribosomal peptide synthase.
19. The method of claim 1 , wherein the compound is a polyketide, a β-lactam compound, or a non-ribosomal peptide.