Nonribosomal peptide synthetases
The present disclosure is directed to the biosynthetic pathway for a nonribosomal peptide synthetase (NRPS) derived drug and analogs thereof. The invention provides polynucleotide sequences useful for heterologous expression in a convenient microbial host for the synthesis of the NRPS-derived drug, the polypeptides encoded by such polynucleotides, expression vectors comprising the polynucleotides, host cells comprising the polynucleotides or expression vectors, and kits comprising a host cell. Also provided is a method for the production of ET-743, the NRPS-derived drug.
1. A method for producing ET-743 or a metabolic intermediate thereof comprising:
growing a host cell transformed with one or more expression vectors comprising a polynucleotide encoding one or more polypeptides selected from the group consisting of SEQ ID NOs: 421, 288, 289, 290, 291, 420, and 350 under conditions to express the one or more polypeptides and producing ET-743 or the metabolic intermediate for producing ET-743.
2. The method of claim 1 wherein ET-743 or the metabolic intermediate thereof is isolated.
3. The method of claim 1 or claim 2 further comprising converting the intermediate to ET-743.
4. The method of claim 1 or claim 2 wherein the producing is completed in the same host cell.
5. The method of claim 1 wherein the host cell is transformed with at least one expression vector encoding at least one heterologous polypeptide of any one of SEQ ID NOs: 421, 288, 289, 290, 291, 420, or 350.
6. The method of any one of claims 1 , 2 or 5 wherein the host cell is a prokaryotic host cell.
7. The method of claim 6 wherein the prokaryotic host cell is Pseudomonas fluorescens.
8. The method of claim 1 wherein the host cell comprises a polynucleotide encoding a first polypeptide of SEQ ID NO: 421, 288, 289, 290, 291, 420, or 350.
9. The method of claim 8 wherein the host cell further comprises a polynucleotide encoding a second polypeptide of SEQ ID NO: 421, 288, 289, 290, 291, 420, or 350, wherein the first and second polypeptides are different.
10. The method of claim 9 wherein the host cell further comprises a polynucleotide encoding a third polypeptide of SEQ ID NO: 421, 288, 289, 290, 291, 420, or 350, wherein the first, second, and third polypeptides are different.
11. The method of claim 10 wherein the host cell further comprises a polynucleotide encoding a fourth polypeptide of SEQ ID NO: 421, 288, 289, 290, 291, 420, or 350, wherein the first, second, third and fourth polypeptides are different.
12. The method of claim 11 wherein the host cell further comprises a polynucleotide encoding a fifth polypeptide of SEQ ID NO: 421, 288, 289, 290, 291, 420, or 350, wherein the first, second, third, fourth and fifth polypeptides are different.
13. The method of claim 12 wherein the host cell further comprises a polynucleotide encoding a sixth polypeptide of SEQ ID NO: 421, 288, 289, 290, 291, 420, or 350, wherein the first, second, third, fourth, fifth and sixth polypeptides are different.