Use of inducible promoters in the production of methionine

Patent metadata

Field	Value
Publication number	US-9988655-B2
Application number	US-201715646940-A
Country	US
Kind code	B2
Filing date	Jul 11, 2017
Priority date	Dec 14, 2009
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

Abstract

The present invention relates to use of inducible promoters in the production of methionine by fermentation. The present invention concerns a method for the production of methionine, its precursors or derivatives in a fermentative process comprising the following steps:
culturing a modified microorganism in an appropriate culture medium comprising a source of carbon, a source of sulphur and a source of nitrogen, and recovering methionine and/or its derivatives from the culture medium, wherein in said modified microorganism, the expression of at least one gene involved in methionine production is under the control, direct or indirect, of a heterologous inducible promoter.

The invention also concerned the modified microorganism used in the method.

First claim

The invention claimed is:

1. A microorganism modified for an improved production of methionine or its derivatives in a fermentative process, wherein in said modified microorganism, expression of five, six, or seven copies of homoserine dehydrogenase (thrA) and serine acetyltransferase (cysE) genes involved in methionine production is under direct control of a heterologous temperature-inducible promoter.

2. The microorganism of claim 1 , wherein said temperature-inducible promoter is selected from the group consisting of promoters regulated by a modified repressor of phage lambda, the promoter PR or a derivative of PR, the promoter PL or a derivative of PL and a modified lac promoter regulated by a temperature sensitive Lac repressor.

3. The microorganism of claim 2 , wherein said modified repressor of phage lambda is the lambda repressor allele cI857 or any other temperature labile allele of the lambda repressor cI.

4. The microorganism of claim 2 , wherein in the modified microorganism, the gene recA is deleted.

5. The microorganism of claim 1 , wherein said expression of thrA and cysE genes involved in methionine production is under indirect control of said heterologous temperature-inducible promoter, said genes being transcribed by a heterologous RNA polymerase, having an expression that is under control of an inducible promoter.

6. The microorganism of claim 5 , wherein said heterologous RNA polymerase is selected from the group consisting of T7 and T3 polymerase.

7. The microorganism of claim 1 , wherein said microorganism further comprises at least one gene whose expression is under control, direct or indirect, of a heterologous inducible promoter selected from the group consisting of cysteine synthase (cysK), ORF upstream of cysK (cysZ), ATP sulfurylase (cysN), sulfate adenylyltransferase (cysD), adenylylsulfate kinase (cysC), Periplasmic sulfate-binding protein (sbp), phosphoenolpyruvate carboxylase (ppc), phosphoenolpyruvate synthase (pps), pyruvate carboxylase (pyc), acetyl-CoA synthetase (acs), homoserine O-transsuccinylase (metA), cystathionine gamma-synthase (metB), cystathionine beta-lyase (metC), 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (metE), 5,10-methylenetetrahydrofolate reductase (metF), B12-dependent homocysteine-N5-methyltetrahydrofolate transmethylase (metH), methionine adenosyltransferase (metK), aspartokinase II/homoserine dehydrogenase II (metL), aspartate-semialdehyde dehydrogenase (asd), aspartate aminotransferase (aspC), aspartokinase III (lysC), pyruvate kinase I (pykA), pyruvate kinase II (pykF) formyltetrahydrofolate deformylase (purU), the operons cysPUWAM (periplasmic sulphate binding protein, a component of sulphate ABC transporter, a membrane bound sulphate transport protein, a sulphate permease and an 0-acetyl serine sulfhydralase), cysJIH (alpha and beta subunits of a sulfite reductase and an adenylylsulfate reductase) and gcvTHP (Tetrahydrofolate dependent aminomethyl transferase, a glycine cleavage, carrier of aminomethyl group and a glycine dehydrogenase), phosphoglycerate dehydrogenase (serA), phosphoserine phosphatase (serB), phosphoserine aminotransferase (serC), serine hydroxymethyl transferase (glyA), acetate kinase (ackA), phosphotransacetylase (pta), pyruvate dehydrogenase E1 (ace), pyruvate dehydrogenase E2 (aceF), lipoamide dehydrogenase (Ipd), succinyl-CoA synthetase beta subunit (sucC), succinyl-CoA synthetase alpha subunit (sucD), phosphoenolpyruvate carboxykinase (pck), malate dehydrogenase (maeB), pyruvate oxidase (poxB), acetohydroxy acid synthase I large subunit (ilvB), acetohydroxy acid synthase I small subunit (ilvN), acetohydroxy acid synthase II large subunit (ilvG), acetohydroxy acid synthase II small subunit (ilvM), acetohydroxy acid synthase III large subunit (ilvI), acetohydroxy acid synthase III small subunit (ilvH), DAHP synthetase (aroF), DAHP synthetase (aroG), DAHP synthetase (aroH), homoserine kinase (thrB), threonine synthase (thrC), serine deaminase (sdaA), serine deaminase (sdaB), S-Adenosylmethionine decarboxylase (speD), ornithine decarboxylase (speC), arginine succinyltransferase (astA), dihydrodipicolinate synthase (dapA), malate dehydrogenase (mdh), malate dehydrogenase FAD/NAD(P)-binding domain (mqo), citrate synthase (gltA).

8. The microorganism of claim 1 , wherein the gene thrA is a thrA allele having reduced feedback sensitivity to threonine.

9. The microorganism of claim 7 , wherein the gene metA is a metA allele encoding enzyme with reduced feedback sensitivity to methionine and S-adenosylmethionine.

Assignees

• Evonik Degussa Gmbh

Inventors

• Figge Rainer
• Vasseur Perrine

Classifications

• C12P13/12 Primary

  Methionine; Cysteine; Cystine · CPC title