Biosynthetic pathway for heterologous expression of a nonribosomal peptide synthetase drug and analogs

Patent metadata

Field	Value
Publication number	US-9611491-B2
Application number	US-201414467851-A
Country	US
Kind code	B2
Filing date	Aug 25, 2014
Priority date	Apr 15, 2010
Publication date	Apr 4, 2017
Grant date	Apr 4, 2017

Abstract

The present invention is directed to the biosynthetic pathway for a nonribosomal peptide synthetase (NRPS) derived drug and analogs thereof. The invention also discloses polynucleotide sequences useful for heterologous expression in a convenient microbial host for the synthesis of the NRPS derived drug.

First claim

What is claimed is:

1. A host cell transformed with a polynucleotide encoding (i) the EtuA1 polypeptide (SEQ ID NO: 1) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 1 having EtuA1 activity, and (ii) the EtuA3 polypeptide (SEQ ID NO: 3) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 3 having EtuA3 activity.

2. The host cell of claim 1 further transformed with a polynucleotide encoding the EtuF3 polypeptide (SEQ ID NO: 9) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 9 having EtuF3 activity.

3. The host cell of claim 2 further transformed with a polynucleotide encoding the EtuO polypeptide (SEQ ID NO: 16) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 16 having EtuO activity.

4. The host cell of claim 3 further transformed with a polynucleotide encoding (i) the EtuH polypeptide (SEQ ID NO: 10) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 10 having EtuH activity (ii) the EtuM2 polypeptide (SEQ ID NO: 12) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 12 having EtuM2 activity or (iii) the EtuM1 polypeptide (SEQ ID NO: 11) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 11 having EtuM1 activity.

5. The host cell of claim 1 further transformed with a polynucleotide encoding EtuO (SEQ ID NO: 16) or a protein 95% identical to SEQ ID NO: 16 having EtuO activity.

6. The host cell of claim 5 further transformed with a polynucleotide encoding (i) EtuF3 (SEQ ID NO: 9) or a protein 95% identical to SEQ ID NO: 9, having EtuF3 activity or (ii) the EtuA2 polypeptide (SEQ ID NO: 2) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 2, having EtuA2 activity.

7. The host cell of claim 6 further transformed with a polynucleotide encoding EtuH (SEQ ID NO: 10) or a protein 95% identical to SEQ ID NO: 10 having EtuH activity, (ii) EtuM2 (SEQ ID NO: 12) or a protein 95% identical to SEQ ID NO: 12 having EtuM2 activity, or (iii) EtuM1 (SEQ ID NO: 11) or a protein 95% identical to SEQ ID NO: 11 having EtuM1 activity.

8. The host cell of claim 5 further transformed with a polynucleotide encoding a protein selected from the group consisting of: (a) the EtuD1 polypeptide (SEQ ID NO: 4) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 4 having EtuD1 activity, (b) the EtuD2 polypeptide (SEQ ID NO: 5) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 5 having EtuD2 activity, (c) the EtuD3 polypeptide (SEQ ID NO: 6) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 6 having EtuD3 activity, (d) the EtuF1 polypeptide (SEQ ID NO: 7) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 7 having EtuF1 activity, (e) the EtuF2 polypeptide (SEQ ID NO: 8) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 8 having EtuF2 activity, (f) the EtuN1 polypeptide (SEQ ID NO: 13) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 13 having EtuN1 activity, (g) the EtuN2 polypeptide (SEQ ID NO: 14) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 14 having EtuN2 activity, (h) the EtuN3 polypeptide (SEQ ID NO: 15) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 15 having EtuN3 activity, (i) the EtuP1 polypeptide (SEQ ID NO: 17) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 17 having EtuP1 activity, (j) the EtuP2 polypeptide (SEQ ID NO: 18) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 18 having EtuP2 activity, (k) the EtuR1 polypeptide (SEQ ID NO: 19) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 19 having EtuR1 activity, (l) the EtuR2 polypeptide (SEQ ID NO: 20) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 20 having EtuR2 activity, (m) the EtuR3 polypeptide (SEQ ID NO: 21) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 21 having EtuR3 activity, (n) the EtuT polypeptide (SEQ ID NO: 22) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 22 having EtuT activity, (o) the EtuU1 polypeptide (SEQ ID NO: 23) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 23 having EtuU1 activity, (p) the EtuU2 polypeptide (SEQ ID NO: 24) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 24 having EtuU2 activity, or (q) the EtuU3 polypeptide (SEQ ID NO: 25) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 25 having EtuU3 activity.

9. The host cell of claim 5 further transformed with a polynucleotide encoding a protein selected from the group consisting of: (a) the EtuD1 polypeptide (SEQ ID NO: 4) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 4 having EtuD1 activity, (b) the EtuD2 polypeptide (SEQ ID NO: 5) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 5 having EtuD2 activity, (c) the EtuD3 polypeptide (SEQ ID NO: 6) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 6 having EtuD3 activity, (d) the EtuF1 polypeptide (SEQ ID NO: 7) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 7 having EtuF1 activity, (e) the EtuF2 polypeptide (SEQ ID NO: 8) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 8 having EtuF2 activity, (f) the EtuN1 polypeptide (SEQ ID NO: 13) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 13 having EtuN1 activity, (g) the EtuN2 polypeptide (SEQ ID NO: 14) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 14 having EtuN2 activity, (h) the EtuN3 polypeptide (SEQ ID NO: 15) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 15 having EtuN3 activity, (i) the EtuP1 polypeptide (SEQ ID NO: 17) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 17 having EtuP1 activity, (j) the EtuP2 polypeptide (SEQ ID NO: 18) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 18 having EtuP2 activity, (k) the EtuR1 polypeptide (SEQ ID NO: 19) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 19 having EtuR1 activity, (l) the EtuR2 polypeptide (SEQ ID NO: 20) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 20 having EtuR2 activity, (m) the EtuR3 polypeptide (SEQ ID NO: 21) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 21 having EtuR3 activity, (n) the EtuT polypeptide (SEQ ID NO: 22) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 22 having EtuT activity, (o) the EtuU1 polypeptide (SEQ ID NO: 23) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 23 having EtuU1 activity, (p) the EtuU2 polypeptide (SEQ ID NO: 24) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 24 having EtuU2 activity, and (q) the EtuU3 polypeptide (SEQ ID NO: 25) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 25 having EtuU3 activity.

10. The host cell of claim 9 transformed with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 polynucleotides individually encoding a polypeptide selected from the group consisting of: (a) the EtuD1 polypeptide (SEQ ID NO: 4) of a nonribosomal peptide synthetase or a protein 95% identical to SEQ ID NO: 4 having EtuD1 activity, (b) the EtuD2 polypeptide (SEQ ID NO: 5) of a nonribosomal peptide synthetas

Assignees

• Univ Michigan Regents
• Allegheny-Singer Res Inst
• Univ Colorado State Res Found

Inventors

• Sherman David H
• Ehrlich Garth D
• Janto Benjamin
• Williams Robert M
• Rath Christopher M

Classifications

• C12P17/188 Primary

  Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms (ergot-alcaloids C12P17/183) · CPC title

• C12P17/182

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title