Process for fermentative production of L-methionine

一种甲硫氨酸、菌株的技术，应用在制造L-甲硫氨酸领域

Inactive Publication Date: 2009-07-08
WACKER CHEM GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The prior art also suggests that known methods ensuring increased synthesis of L-serine and L-cysteine ​​have a positive effect on the production of L-methionine

Method used

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  • Process for fermentative production of L-methionine
  • Process for fermentative production of L-methionine
  • Process for fermentative production of L-methionine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Cloning of Basic Vector pKP228

[0059] In order to place the yjeH gene under the control of a constitutive promoter, first construct a basic vector containing the constitutive GAPDH promoter of the gapA gene used by Escherichia coli glyceraldehyde 3-dehydrogenase. For this purpose, use primers

[0060] GAPDHfw: (SEQ ID NO: 3)

[0061] 5'GTC GAC GCG T GA GGC GAG TCA GTC GCG TAA TGC 3’

[0062] Mlu I

[0063] GAPDHrevl: (SEQ ID NO: 4)

[0064] 5'GAC CTT AAT TAA GAT CT C ATA TAT TCC ACC AGC TAT TTG TTA G 3’

[0065] PacI Bgl II

[0066] and chromosomal DNA polymerase chain reaction of Escherichia coli strain W3110 (ATCC27325). The resulting DNA fragments were purified by agarose gel electrophoresis and then separated (Chia Quick Gel Extraction Kit, Chiagen, Hilton, Germany). Afterwards, the fragment was treated with restriction enzymes PacI and MluI and cloned into the vector pACYC184-LH, similarly with PacI / NluI (August 18, 1995 under t...

Embodiment 2

[0067] Embodiment 2: Cloning of yjeH gene

[0068] The yjeH gene from E. coli W3100 strain was amplified by polymerase chain reaction. Use oligonucleotides

[0069] yjeH-fw: (SEQ ID NO: 5)

[0070] 5'-ATT GCT GGT TTG CTG CTT-3'

[0071] yjeH-rev: (SEQ ID NO: 6)

[0072] 5’-AGC ACA AAA TCG GGT GAA-3’

[0073] As specific primers and as template chromosomal DNA of E. coli strain W3110 (ATCC27325) was used. The prepared DNA fragments were purified and separated by agarose gel electrophoresis (Chia Quick Gel Extraction Kit, produced by Chiagen, Hilton, Germany). Cloning was performed by blunt-end ligation with a BglII-cleaved pKP228 vector whose 5'-overhang was filled in with Klenow enzyme. The procedure is to place the yjeH gene downstream of the GAPDH promoter in an appropriate manner so that transcription is initiated from there. The prepared vector was named pKP450.

Embodiment 3

[0074] Example 3: Combination of the yjeH gene with a feedback resistant metA allele

[0075] By polymerase chain reaction using template pKP466 (described in patent application DE-A-10247437) and primers

[0076] metA-fw: (SEQ ID NO: 7)

[0077] 5”-CGC CCA TGG CTC CTT TTA GTC ATT CTT-3'

[0078] NCOI

[0079] metA-rev: (SEQ ID NO: 8)

[0080] 5'-CGC GAG CTC AGT ACT ATT AAT CCA GCG-3’

[0081] SacI

[0082] The metA allele encoding a feedback-resistant O-homoserine transsuccinylase described in the patent application DE-A-10247437 of the same applicant on October 11, 2002 was amplified.

[0083] In this procedure terminal cleavage sites for the restriction endonucleases NcoI and SacI are generated. The resulting DNA fragment was digested with the same endonuclease, purified and cloned into the NcoI / SacI cleaved pKP450 vector. The resulting plasmid was named pKP451.

[0084] To create a control plasmid containing the metA allele but no yjeH gen...

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Abstract

Microorganism (A) for fermentative preparation of L-methionine (I), is prepared from a starting strain (B), and has higher activity than (B) of a product (II) of the yjeH gene or an homologous gene, is new. Independent claims are also included for: (1) plasmid that contains a yjeH gene linked to a promoter; (2) method for preparing (A) by introducing the plasmid of (1) into (B); and (3) method for preparing (I) by fermenting (A).

Description

technical field [0001] The present invention relates to a method for producing L-methionine by fermentation. Background technique [0002] The amino acid methionine plays an extremely important role in animal feeding. Methionine is one of several essential amino acids that cannot be produced biosynthetically in vertebrate metabolism. Therefore, in terms of animal feeding, it is necessary to ensure sufficient intake of methionine in the feed. However, the content of methionine in traditional feed plants (such as soybean or cereal plants) is often too low (especially for pigs and poultry), and to ensure optimal animal feeding, it is best to mix methionine in animal feed Amino acid as an additive. The particular importance of methionine for animal feeding can also be attributed to the fact that, in addition to L-cysteine ​​(or L-cystine), methionine is an important source of sulfur in the metabolism. Although animal metabolism can convert methionine to cysteine, the metabol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/00C12R1/19C12N15/09C12N15/00C12P13/12
CPCC12P13/12
Inventor 托马斯·迈尔克里斯托夫·温特哈尔特克斯廷·普法伊费尔
Owner WACKER CHEM GMBH
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