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L-aspartic acid-alpha-decarboxylase mutant and application thereof

A technology of aspartic acid and decarboxylase, applied in the field of genetic engineering, can solve the problem of less PanD, etc., and achieve the effect of increasing the level of self-shearing and increasing the yield

Active Publication Date: 2021-09-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Further studies have found that the cleavage patterns of PanD from different sources are quite different. First, PanD protein is mainly expressed as an inactive π protein in E.coli, and then PanZ-AcCoA is formed under the action of the activator PanZ complex, promotes PanD activation through selection of the reactive conformation, but less activated PanD in E. coli due to insufficient intracellular PanZ

Method used

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  • L-aspartic acid-alpha-decarboxylase mutant and application thereof
  • L-aspartic acid-alpha-decarboxylase mutant and application thereof
  • L-aspartic acid-alpha-decarboxylase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: analysis of panD gene molecular evolution relationship and selection pressure analysis

[0054] (1) Analysis of molecular evolution relationship of panD gene

[0055] In this study, we explored the sequence differentiation of the panD gene in different organisms through the method of molecular evolution. Using the Escherichia coli gene EcopanD (SAMN02604091) as the original template, a comprehensive search was performed on the sequence of the panD gene family through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The confirmation standard is that the sequences with E value 40% are members of the panD gene family. Finally, the PanD amino acid sequences of 24 microorganisms were obtained, namely Mycobacterium tuberculosis (MtupanD), Mycobacterium africanum (MafpanD), Mycobacterium canettii (McapanD), Mycobacterium bovis (MbopanD), Bacillus cereus (BcepanD), Mycobacterium liflandii (MlipanD), Streptomycesfradiae ( SfrpanD), Rhodococcus opacus(RoppanD), Cory...

Embodiment 2

[0063] Example 2: Construction of L-aspartic acid-α-decarboxylase mutants by site-directed mutagenesis of the whole plasmid

[0064] (1) Primer

[0065] Further, on the basis of the EcoPanD protein and the enantiosite in Class III as a control, directed evolution was carried out on the above three sites to form a library of L-aspartic acid-α-decarboxylase EcoPanD mutants, which are as follows: (1) Lysine at the 14th amino acid position shown in SEQ ID NO.1 is mutated into threonine (EcoPanD K14T , the nucleotide sequence of the mutant is shown in SEQID NO.2); (2) the amino acid 44th isoleucine shown in SEQ ID NO.1 is mutated into valine (EcoPanD I44V , the nucleotide sequence of the mutant is shown in SEQ ID NO.3); (3) the amino acid 85th valine shown in SEQ ID NO.1 is mutated into leucine (EcoPanD V85L , the nucleotide sequence of the mutant is shown in SEQ ID NO.4). The PCR primer sequences used in this embodiment are listed in Table 2. In vitro amplification of the Ecopa...

Embodiment 3

[0073] Example 3: Screening and Fermentation of Mutant Library

[0074] The transformant that obtains of embodiment 2 is carried out as follows:

[0075] 1. Sequencing verification: use a sterilized pipette tip to select 3 transformants on the plate and inoculate them in LB liquid medium containing 100 μg / mL kanamycin. 100μg / mL Kana antibiotics in LB liquid medium. 37°C, shake culture at 200r / min for 12h, sequence and preserve bacteria. The constructed strains are shown in Table 3.

[0076] The strains constructed in Table 3 Example 3

[0077]

[0078] 2. Induced expression of mutants: Under sterile conditions, take 1 mL of seed liquid from the LB liquid medium in step 1, transfer to 50 mL of LB medium (final concentration is 100 μg / mL Kana), 37 ° C, 200 r / mL Shake culture for about 2 hours until OD 600 0.6 to 0.8, the final concentration of 0.3mM IPTG was added and shake cultured for 12h for expression. Crude protein verification steps are as follows: Take 1mL of ind...

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Abstract

The invention provides an L-aspartic acid-alpha-decarboxylase mutant and an application of the L-aspartic acid-alpha-decarboxylase mutant in synthesis of beta-alanine. According to the L-aspartic acid-alpha-decarboxylase mutant and the application thereof, L-aspartic acid-alpha-decarboxylase is modified by whole plasmid site-directed mutagenesis, and amino acid sites at the 14th site, the 44th site and the 85th site of the L-aspartic acid-alpha-decarboxylase which has an amino acid sequence of SEQ ID NO.1 and is derived from Escherichia coli are replaced, so that a mutant strain influencing the self-shearing of the L-aspartic acid-alpha-decarboxylase is obtained. According to the L-aspartic acid-alpha-decarboxylase mutant and the application thereof, by comparing mutants EcoPanDK14T, EcoPanDI44V and EcoPanDV85L with a wild type, it is found that compared with the wild type, the self-shearing level of the mutant is remarkably increased, and the yield of beta-alanine is remarkably increased after fermentation.

Description

(1) Technical field [0001] The invention relates to an L-aspartic acid-alpha-decarboxylase mutant and its application in the synthesis of beta-alanine in microorganisms, and belongs to the technical field of genetic engineering. (2) Background technology [0002] β-Alanine (β-Alanine, C 3 h 7 NO 2 ), soluble in water, is the only β-type amino acid that exists in nature. It is an isomer with α-alanine, one of the 20 amino acids that make up human protein. As a non-protein amino acid, β-alanine can be synthesized by itself in some bacteria, fungi, plants and animals, while mammals need to ingest it from the external environment. β-alanine is widely used in the fields of medicine, food, chemical industry and environment, and has broad market prospects. First, many industrially important compounds such as: 3-hydroxypropionic acid, poly-3-hydroxypropionate, pantothenic acid, camosine, pamidronate Sodium acid (balsalazide) and balsalazide (balasalazide) are synthesized using...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12R1/01
CPCC12N9/88C12Y401/01011C12N15/70
Inventor 赵嫚刘薇彭莉王美南
Owner ZHEJIANG UNIV OF TECH
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