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3-phosphoglycerate dehydrogenase variants whose inhibition by L-serine is reduced, and genes encoding them

一种磷酸甘油酸脱氢酶、磷酸甘油酸的技术，应用在基因工程、植物基因改良、酶等方向，能够解决酶活性损失等问题

Active Publication Date: 2009-08-12
WACKER CHEM GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this case, too, the deletion resulted in a significant loss of some enzyme activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cloning of the serA gene

[0050] The serA gene was amplified by polymerase chain reaction from E. coli strain W3110 (American Type Culture Collection, ATCC 27325). Nucleotides

[0051] serA-fw: (SEQ ID NO: 3)

[0052] 5'-GAA TTC CAT ATG GCAAAG GTA TCG CTG GAG-3’

[0053] Nd

[0054] with

[0055] serA-rev: (SEQ ID NO: 4)

[0056] 5'-AGA AAG CTT TTA TTA GTA CAG CAG ACG GGC-3’

[0057] Hind III

[0058] as a specific primer.

[0059] The resulting DNA fragment was digested with restriction enzymes NdeI and HindIII, and the 5' overhangs were filled in with Klenow enzyme. The DNA fragments were then purified by agarose gel electrophoresis and isolated using the GeneClean method (GeneClean kit BIO101, PO Box 2284, La Jolla, CA, 92038-2284). The serA fragment obtained in this way was cloned into the expression vector pQE-70 (Qiagen, Hilden, Germany). To achieve this, the vector was first digested with SphI and BamHI, the 3' ove...

Embodiment 2

[0060] Example 2: Site-directed mutagenesis of the serA gene

[0061] Site-directed mutagenesis at codons 349 and 372 of the serA gene was performed using reverse transcription polymerase chain reaction. The vector pFL209 described in Example 1 was used as a template. Primer

[0062] serA40-mut (SEQ ID NO: 5)

[0063] 5’-GAA AAC CGT CCG NNN GTG CTA ACT GCG-3’

[0064] N=G, A, T or C

[0065] and

[0066] serA40-rev (SEQ ID NO: 6)

[0067] 5’-GTG GAT GTG CAT CAG ACG-3’

[0068] Used to mutagenize codon 349.

[0069] The resulting PCR product was ligated into a circle and transformed into E. coli strain JM109. Finally, sequence to confirm the mutation at codon 349 and check the rest of the sequence for correctness.

[0070] In principle, the same procedure was used for mutagenesis of codon 372, but the primers used were

[0071] serA20-mut2 (SEQ ID NO: 7)

[0072] 5’-CAA TAT CTG CAA NNN TCC GCC CAG ATG GG-3’

[0073] N=G, A, T or C

[0074] and

[0075] serA20-rev ...

Embodiment 3

[0077] Embodiment 3: Determine PGD activity and inhibitor constant K i

[0078] In order to determine the influence of PGD enzyme activity and L-serine on its activity, the LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) of 100 ml volume, wherein In each case 2 ml of an overnight culture carrying a plasmid encoding the serA allele was inoculated, additionally containing 100 mg / l of ampicillin, and grown in a shaker at 30°C and 150 rpm. At an optical density of 1.0, 0.4 mM isopropyl-β-thiogalactoside was added in each case to induce serA expression, and the cultures were grown for an additional 3 hours. Cells were collected by centrifugation, washed and resuspended in 2 ml of buffer (100 mM potassium phosphate, pH 7.0; 10 mM MgCl 2 ; 1 mM dithiothreitol). Cells were lysed using a French press (Spectronic Instruments, Inc., Rochester, NY, USA) at a pressure of 18000 psi. The crude extract was clarified by centrifugation at 30,000 g and PGD activity was determin...

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PUM

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Abstract

A 3-phosphoglyceraldehyde dehydrogenase (I) that, compared with the wild-type enzyme of Escherichia coli, has lower sensitivity to serine, is new, where it differs from the wild-type sequence (410 amino acids, given in the specification) by having 349Gly and / or 372Thr replaced by other amino acids. Independent claims are also included for the following: (1) the serA allele (II) that encodes (I), differing from the wild-type serA (1233 base pair (bp) sequence, given in the specification) by having codons for 349Gly and / or 372Thr replaced by codons for other amino acids; (2) a vector that contains (II) under control of a promoter; (3) method for preparing a microbial strain by introducing a vector of (2); (4) microbial strain, suitable for fermentative production of amino acids of the phosphoglycerate family, their derivatives or compounds, or of compounds derived from C1 metabolism, that contains (I); and (5) producing amino acids of the phosphoglycerate family, their derivatives or compounds, or compounds derived from C1 metabolism by culturing strains of (4).

Description

technical field [0001] The present invention relates to a variant of 3-phosphoglycerate dehydrogenase whose inhibitory property of L-serine is reduced and its coding gene. Background technique [0002] At present, microbial fermentation is mainly used to prepare 20 natural, protein-forming amino acids. As such, the application is based on the fact that microorganisms have suitable biosynthetic pathways for the synthesis of natural amino acids. [0003] In wild strains, however, such biosynthetic pathways are tightly controlled to ensure that amino acids are produced only to meet the internal needs of the cell. An example of an important control mechanism in many biosyntheses is the phenomenon of feedback inhibition (or end product inhibition). In feedback inhibition, usually an enzyme in a biosynthetic pathway that catalyzes the initial enzymatic reaction of the biosynthetic pathway is inhibited by the end product of the biosynthetic pathway. This inhibition often results...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12P13/04C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12P13/06
CPCC12Y101/01095C12P13/04C12N9/0006C12P13/06
Inventor 托马斯·迈尔雷娜特·弗林斯帕施
Owner WACKER CHEM GMBH
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