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Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine

An alanine dehydrogenase, alanine technology, applied in application, fermentation, microorganism-based methods, etc., can solve problems such as limiting L-alanine production capacity

Active Publication Date: 2020-10-09
ANHUI HUAHENG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since alanine dehydrogenase is a key step in the production of L-alanine, the specificity and catalytic activity of this enzyme directly limit the production capacity of L-alanine in engineered strains

Method used

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  • Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine
  • Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine
  • Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine

Examples

Experimental program
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preparation example Construction

[0073] The preparation method of competent cells in the following examples is described in the following literature: Dower et al., 1988, Nucleic Acids Res 16: 6127-6145.

[0074] The pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad SciUSA 97:6640-6645) in the following examples is a product of the CGSC E. coli Collection Center of Yale University, USA, and the product catalog number is CGSC#7739. The MicroPulser electroporator is a product of Bio-Rad. pEASY Blunt simple vector is a product of Beijing Quanshijin Biotechnology Co., Ltd. Phusion 5× buffer is a product of NEB Company.

[0075] Escherichia coli (E.coli) ATCC 8739 in the following examples is preserved in American Type Culture Collection (ATCC for short, address: American Type Culture Collection (ATCC) 10801 University Boulevard Manassas, VA 20110 USA), and the public can obtain it from the U.S. The strain was obtained from the type culture collection bank. Escherichia coli (E. coli) ATCC 8739 is hereinaft...

Embodiment 1

[0085] The construction of embodiment 1, XZ-A51 bacterial strain

[0086] Starting from XZ-A12, the alanine dehydrogenase alaD gene from Geobacillus stearothermophilus XL-65-6 integrated at the ldhA site was knocked out by two-step homologous recombination. Specific steps are as follows:

[0087] 1. Construction of XZ-A50 strain

[0088] 1. Using the plasmid pXZ-CS (Tane et al., Appl Environ Microbiol. 2013, 9:4838-4844) containing the chloramphenicol resistance gene cat and the fructosucrose transferase gene sacB as a template, use the primer ldhA-cat -up / ldhA-sacB-down was amplified by PCR to obtain DNA fragment I for homologous recombination, with a total of 2719bp (sequence 1 in the sequence listing). DNA fragment I includes 50 bases upstream of the lactate dehydrogenase gene ldhA, a DNA fragment of cat-sacB, and 50 bases downstream of the ldhA gene.

[0089] Amplification system: NewEngland Biolabs Phusion 5X buffer 10μL, dNTP (each dNTP 10mM each) 1μL, DNA template 20...

Embodiment 2

[0097] Embodiment 2, the construction of alanine dehydrogenase coding gene alaD mutant library

[0098] The artificial promoter M1-46 (the promoter M1-46 is described in the following literature: construction of a stringent promoter on Escherichia coli chromosome) is connected to the commercialized pACYC184 plasmid to obtain the plasmid Palamut-1 for gene expression ( figure 1 ). Expression is performed by linking the DNA fragment of the alaD gene mutation library to the artificial promoter M1-46. Among them, the M1-46 artificial promoter is a weaker promoter, which is convenient for screening for mutants of alanine dehydrogenase with higher activity, and will not increase the activity of intracellular enzymes due to excessive transcription levels. Specific steps are as follows:

[0099] 1. Construction of Palamut-2 plasmid for alaD gene mutation

[0100] 1. Using the genomic DNA of XZ-A12 as a template, PCR amplification was performed using primers alaD-184up / alaD-184down...

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Abstract

The invention discloses an alanine dehydrogenase mutant and an application of the alanine dehydrogenase mutant in fermentation production of L-alanine. The alanine dehydrogenase mutant disclosed by the invention is a protein obtained by mutating lysine at the 327th site of an amino acid sequence of alanine dehydrogenase into asparagine. According to the invention, the alanine dehydrogenase mutantwith significantly improved activity is obtained based on a metabolic domestication screening technology, and the alanine dehydrogenase mutant is used for construction of L-alanine engineering strainsand efficient fermentation of L-alanine. The experiments prove that the L-alanine yield and the enzymatic activity of the L-alanine dehydrogenase of the strain provided by the invention are greatly improved and are improved by 88.2% compared with those of a strain for expressing wild alanine dehydrogenase, the fermentation capability and the L-alanine yield of an L-alanine engineering strain canbe effectively improved, and the mutant has important application value.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to an alanine dehydrogenase mutant and its application in fermentative production of L-alanine, in particular to an alanine dehydrogenase mutant with significantly improved activity and Its application in the construction of L-alanine engineering strains and the high-efficiency fermentation production of L-alanine. Background technique [0002] As a non-essential amino acid for human body, L-alanine has a wide range of uses in the food and pharmaceutical industries. L-alanine can improve the nutritional value of food in the field of food industry. Adding L-alanine can significantly improve the protein utilization rate in food and beverages. L-alanine can improve the taste of artificial sweeteners, making them like natural sweeteners. In addition, L-alanine can also improve the sour taste of organic acids, making it closer to the natural taste. L-alanine is often used as amino...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/0016C12N15/70C12Y104/01001C12P13/06
Inventor 张学礼郭恒华马延和张冬竹刘萍萍
Owner ANHUI HUAHENG BIOTECH
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