Process

Patent metadata

Field	Value
Publication number	US-10920256-B2
Application number	US-201716077281-A
Country	US
Kind code	B2
Filing date	Feb 10, 2017
Priority date	Feb 12, 2016
Publication date	Feb 16, 2021
Grant date	Feb 16, 2021

Abstract

The present invention relates to processes to make neosaxitoxin, and analogues and variants thereof, and intermediates in the production of neosaxitoxin in recombinant host cells. Neosaxitoxin and the analogues and variants thereof may be used in the production of pharmaceutical compositions.

First claim

The invention claimed is:

1. A process for producing neosaxitoxin in a host cell, the process comprising the steps:
(A) culturing a host cell which comprises nucleic acid molecules encoding a phosphopantetheinyltransferase (PPTase) and encoding the Sxt polypeptides A, B, D, G, H, I, S, T, U, V, W and X in a culture medium in the presence of the substrates:
(i) S-adenosylmethionine,
(ii) arginine
(iii) acetyl-CoA, malony-CoA or propionyl-CoA, and
(iv) carbamoyl phosphate,

wherein the host cell is a recombinant prokaryotic cell or a recombinant yeast cell,
and wherein the host cell does not comprise nucleic acid molecules encoding the Sxt polypeptides C, F, J, K, L, M, P, Q, R and ORF24,
under conditions which are suitable for the production of neosaxitoxin; and optionally
(B) isolating and/or purifying neosaxitoxin from the host cells or from the culture medium.

2. A process for producing neosaxitoxin or an analogue or variant thereof, the process comprising the steps:
(A) contacting the substrates:
(i) S-adenosylmethionine,
(ii) arginine
(iii) acetyl-CoA, malony-CoA or propionyl-CoA, and
(iv) carbamoyl phosphate,

with Sxt A, B, D, G, H, I, S, T, U, V, W and X polypeptides, in a reaction medium, and optionally
(B) isolating and/or purifying neosaxitoxin or an analogue or variant thereof from the reaction medium.

3. A process as claimed in claim 2 , wherein the reaction medium additionally comprises a PPTase.

4. A process for producing neosaxitoxin or an analogue or variant thereof in a host cell, the process comprising the steps:
(A) culturing a host cell which comprises nucleic acid molecules encoding the Sxt polypeptides A, B, D, G, H, I, S, T, U, V, W and X in a culture medium in the presence of the substrates:
(i) S-adenosylmethionine,
(ii) arginine
(iii) acetyl-CoA, malony-CoA or propionyl-CoA, and
(iv) carbamoyl phosphate,

wherein the host cell is a recombinant prokaryotic cell or a recombinant yeast cell,
and wherein the host cells do not comprise nucleic acid molecules encoding one or more or all of Sxt polypeptides Q, R and ORF24,
under conditions which are suitable for the production of neosaxitoxin or an analogue or variant thereof; and optionally
(B) isolating and/or purifying neosaxitoxin or an analogue or variant thereof from the host cells or from the culture medium.

5. A process as claimed in claim 4 , wherein the host cell additionally comprises a nucleic acid molecule encoding a PPTase.

6. A process as claimed in claim 4 , wherein the host cells do not comprise nucleic acid molecules encoding one or more or all of the Sxt polypeptides C, J and K.

7. A process as claimed in claim 4 , wherein the host cells do not comprise nucleic acid molecules encoding any of the Sxt polypeptides in one or more of (a)-(c)
(a) C, Q, R and ORF24;
(b) L, Q, R and ORF 24
(c) J, K, L, Q, R and ORF 24.

8. A process as claimed in claim 4 , wherein the host cells do not comprise nucleic acid molecules encoding one or more or all of Sxt polypeptides F, M and P.

9. A process as claimed in claim 4 , wherein the host cell additionally comprises nucleic acid molecules encoding one or more of Sxt polypeptides C, E, J, K, and L (preferably C and/or E).

10. A process as claimed in claim 4 , wherein the host cells do not comprise nucleic acid molecules encoding one or more or all of Sxt polypeptides F, M, N, O, P, Y, Z, ORF3, ORF4, ORF29, ORF34, OMPR or HISA.

11. A process as claimed in claim 4 , wherein the host cell is a bacterial cell, or an E. coli cell.

12. A process as claimed in claim 4 , wherein the host cell is a heterotroph.

13. A process as claimed in claim 4 , wherein the neosaxitoxin or analogue or variant thereof, or a pharmaceutically acceptable salt thereof, is formulated into a pharmaceutical composition.

14. A process as claimed in claim 13 , wherein the formulating step comprises admixing isolated or purified neosaxitoxin or an analogue or variant thereof with one or more pharmaceutically-acceptable carriers, adjuvants and/or excipients.

15. A host cell which comprises nucleic acid molecules coding for the Sxt polypeptides A, B, D, G, H, I, S, T, U, V, W and X, wherein the host cell does not comprise nucleic acid molecules coding for:
(i) one or more or all of the Sxt polypeptides C, J or K;
(ii) one or more or all of the Sxt polypeptides Q, R and ORF24;
(iii) one or more or all of the Sxt polypeptides C, Q, R and ORF24;
(iv) one or more or all of the Sxt polypeptides L, Q, R and ORF 24;
(v) one or more or all of the Sxt polypeptides J, K, L, Q, R and ORF 24; or
(vi) one or more or all of the Sxt polypeptides F, M and P,

wherein the host cell is a recombinant prokaryotic cell or a recombinant yeast cell.

16. A host cell as claimed in claim 15 , wherein the host cell additionally comprises nucleic acid molecules coding for one or more Sxt polypeptides selected from the group consisting of Sxt C, E, J, K, L, and/or R (preferably C and/or E).

17. A host cell as claimed in claim 15 , wherein the host cell does not comprise nucleic acid molecules coding for one or more or all of the Sxt polypeptides selected from the group consisting of F, M, N, O, P, Y, Z, ORF3, ORF4, ORF29, ORF34, OMPR or HISA.

18. A host cell as claimed in claim 15 , wherein the host cell is a bacterial cell, or an E. coli cell.

Assignees

• Vestlandets Innovasjonsselskap As
• Newsouth Innovations Pty Ltd
• Vestlandets Innovasjonsseiskap AS

Inventors

• Kellmann Ralf
• Neilan Brett

Classifications

• C12R2001/19

  Escherichia coli · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12P17/182 Primary

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

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title