Cell-Based Production of Nonulosonates

Patent metadata

Field	Value
Publication number	US-2017145459-A1
Application number	US-201615354603-A
Country	US
Kind code	A1
Filing date	Nov 17, 2016
Priority date	Apr 20, 2010
Publication date	May 25, 2017
Grant date	—

Abstract

The present invention relates to the cell-based production of bacterial nonulosonates and their biosynthetic precursors. Specifically, the present invention provides recombinant cells for the production of pseudaminic acid, legionaminic acid, UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose, and UDP-2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose. Methods for producing the sugars are also provided.

First claim

1 . A recombinant cell for the production of legionaminic acid, comprising an inactivated GlcNAc-6-P deacetylase gene, a gene encoding a PglF enzyme function, a gene encoding a PglE enzyme function, a gene encoding a PglD enzyme function, a gene encoding a LegI enzyme function, a gene encoding a LegG enzyme function, a gene encoding a GlcNAc-6-P mutase, and a gene encoding a GlcNAc-1-P uridyltransferase.

2 . The recombinant cell of claim 1 , wherein the PglF, PglE, and PglD enzyme functions are genes from C. jejuni , the LegI enzyme function is from C. jejuni or L. pneumophila , the LegG enzyme function is a gene from L. pneumophila LegG, and the GlcNAc-6-P mutase and GlcNAc-1-P uridyltransferase genes are from S. cerevisiae.

3 . The recombinant cell of claim 1 , further comprising at least one of an inactivated sialic acid transporter gene, an inactivated sialic acid aldolase gene.

4 . The recombinant cell of claim 1 , further comprising at least one of an inactivated nanT sialic acid transporter gene, an inactivated nanA sialic acid aldolase gene, an inactivated wecA undecaprenyl-P/UDP-GlcNAc transferase gene, an inactivated nanE ManNAc-6-P epimerase gene or a gene encoding an acetyl-CoA synthase.

5 . The recombinant cell of claim 1 , wherein the cell is an E. coli cell.

6 . The recombinant cell of claim 1 , wherein the recombinant cell is an E. coli cell further comprising an inactivated nanT sialic acid transporter gene, an inactivated nanA sialic acid aldolase gene, an inactivated nagA GlcNAc-6-P deacetylase gene, a PglF gene encoding SEQ ID NO:6, a PglE gene encoding SEQ ID NO:7, a PglD gene encoding SEQ ID NO:8, a LegI gene encoding SEQ ID NO:9 or SEQ ID NO:10, a LegG gene encoding SEQ ID NO:11, the agm1 GlcNAc-6-P mutase gene, and the uap1 GlcNAc-1-P uridyltransferase gene.

7 . The recombinant cell of claim 1 , further comprising at least one of an inactivated ManNAc-6-P epimerase gene or an inactivated undecaprenyl-P/UDP-GlcNAc transferase gene.

8 . The recombinant cell of claim 7 , wherein the ManNAc-6-P epimerase gene is nanE, and the undecaprenyl-P/UDP-GlcNAc transferase gene is wecA.

9 . A method for the production of legionaminic acid, comprising growing the recombinant cell of claim 1 and recovering the produced legionaminic acid.

10 . The method of claim 9 , wherein growth medium for the recombinant cell is supplemented with palmitate.

11 . A recombinant cell for the production of UDP-2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose (UDP-BacdiNAc), comprising an inactivated GlcNAc-6-P deacetylase gene, a gene encoding a PglF enzyme function, a gene encoding a PglE enzyme function, a gene encoding a PglD enzyme function, a gene encoding a GlcNAc-6-P mutase, and a gene encoding a GlcNAc-1-P uridyltransferase.

12 . The recombinant cell of claim 11 , wherein the PglF, PglE, and PglD genes are from C. jejuni , and the GlcNAc-6-P mutase and GlcNAc-1-P uridyltransferase genes are from S. cerevisiae.

13 . The recombinant cell of claim 11 , further comprising at least one of an inactivated sialic acid transporter gene or an inactivated sialic acid aldolase gene.

14 . The recombinant cell of claim 11 , further comprising at least one of an inactivated nanT sialic acid transporter gene, an inactivated nanA sialic acid aldolase gene, an inactivated wecA undecaprenyl-P/UDP-GlcNAc transferase gene or a gene encoding acetyl-CoA synthase.

15 . The recombinant cell of claim 11 , wherein the recombinant cell is an E. coli cell further comprising an inactivated nanT sialic acid transporter gene, an inactivated nanA sialic acid aldolase gene, an inactivated nagA GlcNAc-6-P deacetylase gene, a PglF gene encoding SEQ ID NO:6, a PglE gene encoding SEQ ID NO:7, a PglD gene encoding SEQ ID NO:8, the agm1GlcNAc-6-P mutase gene, and the uap1 GlcNAc-1-P uridyltransferase gene.

16 . The recombinant cell of claim 11 , wherein the cell is an E. coli cell.

17 . The recombinant cell for the production of UDP-BacdiNAc of claim 11 , wherein the cell is that of IDAC deposit No. 060411-01.

18 . A method for the production of UDP-2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose (UDP-BacdiNAc), comprising growing the recombinant cell of claim 11 and recovering the produced UDP-BacdiNAc.

19 . The method of claim 18 , wherein growth medium for the recombinant cell is supplemented with palmitate.

Assignees

• Nat Res Council Canada
• Univ Ottawa

Inventors

• Whitfield Dennis M
• Logan Susan M
• Schoenhofen Ian C
• Boddy Christopher N
• Lundgren Benjamin R

Classifications

• C12Y306/01045

  UDP-sugar diphosphatase (3.6.1.45) · CPC title

• C12P19/26 Primary

  Preparation of nitrogen-containing carbohydrates · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y305/01025

  N-Acetylglucosamine-6-phosphate deacetylase (3.5.1.25) · CPC title

• C12N15/70

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