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Recombinant escherichia coli for producing L-valine and application thereof

A valine and amino acid technology, applied in the direction of recombinant DNA technology, microorganism-based methods, bacteria, etc., can solve the problems of unbalanced reducing power, etc., to reduce production costs, increase yield and conversion rate, and cell tolerance, The production process is stable and easy to operate

Active Publication Date: 2021-08-20
ANHUI HUAHENG BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The present invention solves the problems in the L-valine fermentation process by enhancing the amino acid dehydrogenase activity of the L-valine fermentation strain, and / or activating the Entner-Doudoroff (Entner-Doudoroff, ED) pathway. The problem of unbalanced reducing power, thereby improving the yield and conversion rate of L-valine produced by Escherichia coli, and realizing one-step anaerobic fermentation of L-valine

Method used

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  • Recombinant escherichia coli for producing L-valine and application thereof
  • Recombinant escherichia coli for producing L-valine and application thereof
  • Recombinant escherichia coli for producing L-valine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Knockout of methyladehane coding gene Mgsa in ATCC8739 strains

[0112] From E. coli ATCC8739, a two-step homologous reorganization method knockout methylene ethyl aldehyde enzyme encoding gene MGSA, the specific steps are as follows:

[0113] In the first step, 2719 bp DNA fragment I was amplified by PXZ-Cs Plasmid DNA using PXZ-CS plasmid DNA, and 2719 bp DNA fragment I was used to amplify the first step homologous recombination.

[0114] The amplification system is: 10 μl of NewenglandBiolabs, DNTP (10 mm per DNTP) 1 μL, DNA template 20 ng, 2 μl of the primer (10 μm), PHUSONHHIGH-FideEndNa polymerase (2.5 u / μl) 0.5 μL The distilled water was 33.5 μL, and the total volume was 50 μL.

[0115] The amplification conditions were 98 ° C for 2 minutes (1 cycle); 98 ° C denaturation for 10 seconds, 56 ° C for 10 seconds, 72 ° C for 2 minutes (30 cycles); 72 ° C extends for 10 minutes (1 cycle).

[0116] The DNA fragment 1 is used for the first homologous recombinati...

Embodiment 2

[0120] Example 2: Urna of the lactate dehydrogenase encoding gene LDHA

[0121] From SVAL002, the lactate dehydrogenase encoding gene LDHA is knocking through two-step homologous recombination method, and the specific steps are as follows:

[0122] In the first step, the PXZ-CS plasmid DNA was used as a template, and the 2719 BP DNA fragment I was amplified using primer LDHA-CS-UP / LDHA-CS-DOWN for the first step homologous recombination. The amplification system and amplification conditions are consistent with the first embodiment. The DNA fragment I is electrically rotated to SVAL002.

[0123] The DNA fragment 1 was used for the first homologous recombination: first converted to E. coli SVAL002 by electro-conversion method, and then electrically converted to Escherichia coli SVAL002 with PKD46.

[0124] The electric turn conditions and steps are consistent with the first step of the MGSA gene knockout as described in Example 1. The 200 μl of bacteria was coated with an LB plate...

Embodiment 3

[0127] Example 3: Phosphate Code Enzyme Coded Gene PTA and Kinase Coded Gene ACKA Knockout

[0128] From SVAL004, the specific steps are as follows: The specific steps are as follows: The specific steps are processed from SVAL004.

[0129] In the first step, 2719 bp DNA fragment I was amplified by PXZ-Cs Plasmid DNA using PXZ-CS plasmid DNA using primer ACKA-CS-UP / PTA-CS-DOWN for the first step homologous recombination. The amplification system and amplification conditions are consistent with the first embodiment. The DNA fragment I is electrically rotated to SVAL004.

[0130] The DNA fragment 1 was used for the first homologous recombination: first converted to E. coli SVAL004 by electro-conversion, and then electrically rotated DNA fragment I to Escherichia coli SVAL004 with PKD46.

[0131] The electric turn conditions and steps are consistent with the first step of the MGSA gene knockout as described in Example 1. The 200 μl of bacteria was coated with an LB plate containing ...

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Abstract

According to the invention, the amino acid dehydrogenase activity of an L-valine fermentation strain is enhanced, and / or an Entner-Doudoroff (ED) metabolism pathway is activated, such that the problem of unbalanced reducing power during the L-valine fermentation process is solved, therefore the yield and the conversion rate of the L-valine produced by the escherichia coli are improved, and the one-step method anaerobic fermentation of the L-valine is achieved.

Description

Technical field [0001] The present invention relates to the construction method of producing recombinant microorganisms of L-proline, and recombinant microorganisms obtained by the constructing method are specifically recombinant E. coli, and a method of producing L-proline by fermentation. [0002] Inventory background [0003] L-proline is one of the three branched-chainaminoacid, BCAA, belongs to the essential amino acid, and cannot be synthesized in people and animals, and can only be obtained from the outside world. At present, L-proline is widely used in the field of food and medicine, mainly including food additives, nutritional supplements and flavoring agents; widely used in cosmetic preparation, and used as antibiotics or herbicide precursors; Increased feed mass and ratio demand, the role of L-proline in the feed additive industry will become more important in the feed additive industry, the demand will become bigger and bigger, and the future market has great potential...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/08C12R1/19
CPCC12N9/0016C12N9/1205C12N9/0006C12P13/08C12Y104/01009C12Y207/0101C12Y101/01086C12Y104/01005C12N15/52C12R2001/19C12N1/205
Inventor 张学礼郭恒华刘萍萍张冬竹唐金磊韩成秀唐思青刘树蓬马延和
Owner ANHUI HUAHENG BIOTECH
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