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 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 .