Microorganism producing o-phosphoserine and method of producing L-cysteine or derivatives thereof from O-phosphoserine using the same

Patent metadata

Field	Value
Publication number	US-9689009-B2
Application number	US-201113278106-A
Country	US
Kind code	B2
Filing date	Oct 20, 2011
Priority date	Oct 20, 2010
Publication date	Jun 27, 2017
Grant date	Jun 27, 2017

Abstract

The present invention provides methods for the production of cysteine or derivates thereof by culturing a microorganism having reduced activity of endogenous phosphoserine phosphatase and the activity of PhnC, PhnD, and PhnE is reduced, and enhanced activity of phosphoglycerate dehydrogenase and/or phosphoserine aminotransferase. The O-phosphoserine produced by such an organism can then be reacted with a sulfide in the presence of a sulfydrylase or a microorganism expressing a sulfhydrylase to produce cysteine or a derivative thereof. Microorganisms having these reduced and enhanced properties noted above are also provided herein.

First claim

The invention claimed is:

1. A method for producing cysteine or a derivative thereof, comprising:
(a) culturing a recombinant microorganism in which the activity of an endogenous phosphoserine phosphatase (SerB) and a phosphonate transporter (PhnCDE; phnC (ATP-binding component of phosphonate transport, EG 10713)-phnD (periplasmic binding protein component of Pn transporter, EG 10714)-phnE (integral membrane component of the alkylphosphonate ABC transporter, EG 11283)) is reduced, and the activity of a phosphoglycerate dehydrogenase (SerA) is enhanced, to produce O-phosphoserine (OPS);
wherein the level of endogenous SerB or PhnCDE activity is reduced by deletion of the endogenous SerB or PhnCDE gene
wherein the level of SerA activity is increased by increasing copy number of the SerA gene or changing an endogenous promoter into a strong promoter, wherein the phosphoserine phosphatase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and wherein the SerA having increased activity comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 6, and 7; and
(b) reacting the OPS of step (a) with a sulfide in presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing OPSS, to produce cysteine or derivatives thereof, wherein the OPSS comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10 and 12;
wherein the recombinant microorganism has been further modified to
(i) enhance the activity of a nucleotide transhydrogenase (PntAB), wherein the level of nucleotide transhydrogenase activity is enhanced by increasing the copy number of a gene encoding the nucleotide transhydrogenase or changing an endogenous promoter into a strong promoter; and/or
(ii) enhance the activity of at least one enzyme selected from the group consisting of o-acetylserine/cysteine efflux permease (YfiK), homoserine/homoserine lactone efflux protein (RhtB), and threonine/homoserine efflux protein (RhtC), wherein the level of enzyme activity is enhanced by increasing the copy number of a gene encoding the enzyme or changing an endogenous promoter into a strong promoter.

2. The method of claim 1 , wherein the recombinant microorganism is cultured in a medium containing glycine or serine.

3. The method of claim 2 , wherein the medium contains glycine in an amount of from 0.1 to 10 g/L.

4. The method of claim 2 , wherein the medium contains serine in an amount of from 0.1 to 5 g/L.

5. The method of claim 1 , wherein the SerA is resistant to serine feedback inhibition.

6. The method of claim 1 , wherein the recombinant microorganism has been further modified to enhance the activity a phosphoserine aminotransferase (SerC), wherein the level of phosphoserine aminotransferase activity is enhanced increasing the copy number of a gene encoding the phosphoserine aminotransferase or changing an endogenous promoter into a strong promoter, and wherein the SerC comprises the amino acid sequence of SEQ ID NO: 8.

7. The method of claim 1 , wherein the recombinant microorganism has been further modified to reduce the activity of an alkaline phosphatase (PhoA) or an acid phosphatase (AphA), wherein the level of enzyme activity is reduced by deletion of the endogenous gene.

8. The method of claim 1 , wherein the recombinant microorganism has been further modified to reduce the activity of at least one endogenous phosphoglycerate mutase isozyme selected from the group consisting of GpmA, GpmI and GpmB, wherein said activity is reduced by deletion of at least one endogenous gene encoding the at least one phosphoglycerate mutase isozyme.

9. The method of claim 1 , wherein the recombinant microorganism has been further modified to reduce the activity of an L-serine dehydratase I (SdaA), wherein said activity is reduced by deletion of the endogenous gene encoding the L-serine dehydratase I.

10. The method of claim 1 , wherein the recombinant microorganism has been further modified to reduce the activity of a 2-amino-3-ketobutyrate coenzyme A ligase (Kbl) or a transcription factor (IClR), wherein said activity is reduced by deletion of the endogenous gene encoding the 2-amino-3-ketobutyrate coenzyme A ligase or by deletion of the endogenous gene encoding the transcription factor.

11. The method of claim 1 , wherein the recombinant microorganism has been further modified to enhance the activity of at least one enzyme selected from the group consisting of acetyl-CoA synthetase (Acs), acetic acid kinase (AckA), phosphotransacetylase (Pta), malate synthase G (GlcB), malate dehydrogenase (MaeB), glutamate dehydrogenase (GdhA), glyoxylate carboligase (Glc), tartronate semialdehyde reductase 2 (GlxR) and glycerate kinase II (GlxK), wherein the level of enzyme activity is enhanced by increasing the copy number of a gene encoding the enzyme or changing an endogenous promoter into a strong promoter.

12. The method of claim 11 , wherein the recombinant microorganism is improved in sugar consumption and growth by enhancement of the activity of at least one enzyme selected from the group consisting of glyoxylate carboligase (Glc), tartronate semialdehyde reductase 2 (GlxR), and a glycerate kinase II (GlxK), wherein the level of enzyme activity is enhanced by increasing the copy number of a gene encoding the enzyme or changing an endogenous promoter into a strong promoter.

13. The method of claim 1 , wherein the recombinant microorganism is Escherichia sp. or Coryneform bacteria.

14. The method of claim 1 , wherein the sulfide of step (b) is selected from the group consisting of Na 2 S, NaSH, (NH 4 ) 2 S, H 2 S, Na 2 S 2 O 3 and a combination thereof.

15. The method of claim 1 , wherein the sulfide of step (b) is used at a molar concentration 0.1 to 3 times higher than that of OPS used in the enzymatic reaction.

16. The method of claim 1 , wherein the reaction of step (b) is carried out in presence of a cofactor selected from the group consisting of PLP (pyridoxal-5-phosphate), DTT (dithiothreitol), and a combination thereof, wherein the concentration of PLP in the reaction is 0.001 to 2 mM and the concentration of DTT in the reaction is 0.001 to 100 mM.

17. The method of claim 1 , further comprising isolating and purifying the cysteine or its derivative.

Assignees

• Chang Jin Sook
• Jo Jae Hyun
• Bae Hyun Ae
• Song Byeong Cheol
• Kim Sol
• Kim Hye Won
• Um Hye Won
• Jhon Sung Hoo
• Shin Yong Uk
• Yang Eun Bin
• Lee Kyoung Min
• Shin Soo An
• Cj Cheiljedang Corp

Inventors

• Chang Jin Sook
• Jo Jae Hyun
• Bae Hyun Ae
• Song Byeong Cheol
• Kim Sol
• Kim Hye Won
• Um Hye Won
• Jhon Sung Hoo
• Shin Yong Uk
• Yang Eun Bin
• Lee Kyoung Min
• Shin Soo An

Classifications

• C12N9/1085

  transferring alkyl or aryl groups other than methyl groups (2.5) · CPC title

• C12P13/12 Primary

  Methionine; Cysteine; Cystine · CPC title

• C12N15/70

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

• C12N15/77

  for Corynebacterium; for Brevibacterium · CPC title

• C12Y205/01065

  O-Phosphoserine sulfhydrylase (2.5.1.65) · CPC title