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Diaminobutyric acid acetyltransferase mutant for synthesizing Ectoine

A diaminobutyric acid acetyl transferase technology, applied in the field of metabolic engineering, can solve the problems of the high price of ecto, the difficulty of waste liquid treatment, and the impact on the life of equipment, so as to improve the economy, improve the enzyme activity, reduce the cost effect

Active Publication Date: 2022-03-04
上海邦林生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2008, Nagata et al. (Biotechnology and Bioengineering, 2008,99(4):941-948) found that Brevibacterium sp.JCM 6894 can not only synthesize ectoine, but also has the characteristics of not being easily broken under repeated impacts of high salt and low salt , but the output of ectoine is only 2.4g / L
However, high-salt fermentation has high requirements for bioreactor equipment, which not only affects the life of the equipment, but also makes subsequent waste liquid treatment very difficult, resulting in high prices of ectoin, which limits its application

Method used

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  • Diaminobutyric acid acetyltransferase mutant for synthesizing Ectoine
  • Diaminobutyric acid acetyltransferase mutant for synthesizing Ectoine
  • Diaminobutyric acid acetyltransferase mutant for synthesizing Ectoine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of wild-type ectABC gene cluster expression strain

[0041] Using the genome of Halomonas elongatus CGMCC 1.6329 (GenBank: FN869568.2) as a template and ABC-5 and ABC-3 as primers, PCR amplified about 2.5kb ectABC fragment and cloned it into the EcoRI / HindIII site of pTrc99A , to obtain the plasmid pTrc99A-ectABC. The primer sequences are as follows:

[0042] ABC-5: 5'-GGAATTCATGAACGCAACCACAGAGCC-3',

[0043] ABC-3: 5'-CCCAAGCTTTTACAGCGGCTTCTGGTCGT-3'.

[0044] Plasmid pTrc99A-ectABC was digested, identified by PCR and sequenced to verify that the insertion position, size and reading frame of the target gene ectABC were correct.

[0045] The recombinant plasmid pTrc99A-ectABC was transformed into Escherichia coli W3110 competent cells by electrotransformation to obtain strain ECT-1. The DNA sequence of the amplified ectABC gene cluster is shown in SEQ ID NO:1. The amino acid sequence of diaminobutyric acid acetyltransferase (ectA) in the exp...

Embodiment 2

[0046] Embodiment 2: Construction of ectA random mutant library by error-prone PCR method

[0047] Using the plasmid pTrc99A-ectABC as a template, the ectA random mutant library was constructed by error-prone PCR.

[0048] Design the following primer pair ECTA-5 / ECTA-3:

[0049] Forward primer ECTA-5: 5'-ATGAACGCAACCACAGAGCC-3',

[0050] Reverse primer ECTA-3: 5'-TCAGATCTGGTCGGTCTGGA-3'.

[0051] Using the plasmid pTrc99A-ectABC as a template, PCR amplification was carried out to obtain a DNA sequence of about 0.6kb transaminase mutant.

[0052] 50μL error-prone PCR reaction system includes: 10ng plasmid template, 50pmol pair of primers, 1×Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (Takara).

[0053] The PCR reaction conditions were: 95°C for 5 minutes; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 72°C for 10min.

[0054] Electrophoresis and gel recovery of PCR p...

Embodiment 3

[0056] Example 3: High throughput screening of EctA mutants

[0057] The EctA random mutation library obtained in Example 2 was transformed into Escherichia coli W3110 competent cells. Pick a single colony to a 96-well plate (each well contains 110 μL of liquid LB-Amp medium), and after incubating at 37°C and 400 rpm for 5 h, take out 60 μL of bacterial liquid from each well to a 96-well deep-well plate (per well The wells contained 240 μL of liquid ME-Amp-0.2mM IPTG), and incubated at 30°C and 400rpm for 12-16h. Centrifuge at 4°C and 4000 rpm for 10 min, take the supernatant, and detect the content of ectoine by HPLC.

[0058] The level of ectoine content in the fermentation broth was used as the standard for evaluating the activity of diaminobutyric acid acetyltransferase EctA. Select strains with significantly improved vitality, and entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to perform genome sequencing comparisons to determine the changes in amino acid sequences. ...

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Abstract

The invention discloses a diaminobutyric acid acetyltransferase mutant as shown in SEQ ID NO: 3, the diaminobutyric acid acetyltransferase mutant can efficiently catalyze the reaction of L-2, 4-diaminobutyric acid to generate N-acetyl-2, 4-diaminobutyric acid, and the N-acetyl-2, 4-diaminobutyric acid is further catalyzed by ectC enzyme to generate Ectoine.

Description

technical field [0001] The invention belongs to the field of metabolic engineering, and in particular relates to a diaminobutyric acid acetyltransferase mutant and its application for producing ectoine. Background technique [0002] Ectoin, the chemical name is (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, also known as tetrahydromethylpyrimidinecarboxylic acid, referred to as tetrahydro Pyrimidine, the molecular formula is C 6 h 10 o 2 N 2 , is an amino acid derivative with the following structural formula. [0003] [0004] Ectoin was originally discovered from halophilic and salt-tolerant microorganisms, which can protect cells from damage in environments such as high salt, high osmotic pressure, dryness, high temperature, and strong ultraviolet radiation. Further studies have found that ectoine can enhance the stability of cells or biomacromolecules, thereby performing self-repair. Studies have shown that ectoine also has a good repair and protect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12N15/70C12P17/12C12R1/19
CPCC12N9/1029C12Y203/01178C12N15/70C12N15/52C12P17/12Y02A50/30
Inventor 王金刚韦炎龙梁岩任亮
Owner 上海邦林生物科技有限公司
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