A kind of l-aspartic acid-alpha-decarboxylase mutant and its application

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

Active Publication Date: 2022-06-21
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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  • A kind of l-aspartic acid-alpha-decarboxylase mutant and its application
  • A kind of l-aspartic acid-alpha-decarboxylase mutant and its application
  • A kind of l-aspartic acid-alpha-decarboxylase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0054] (1) Molecular evolution relationship analysis of panD gene

[0055] This study explored the sequence differentiation of panD gene in different organisms by means of molecular evolution. Using the E. coli gene EcopanD (SAMN02604091) as the original template, a comprehensive search for the sequence of the panD gene family was performed through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). Confirmation criteria are that sequences with E value 40% are members of the panD gene family. The PanD amino acid sequences of 24 microorganisms were finally 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), Corynebacterium amycolatum(CampanD), Coryne...

Embodiment 2

[0063] Example 2: Construction of L-aspartate-α-decarboxylase mutants using whole plasmid site-directed mutagenesis

[0064] (1) Primers

[0065] Further, based on the EcoPanD protein and the enantiomeric sites in Class III as the control, the above three sites were further subjected to directed evolution to form the L-aspartate-α-decarboxylase EcoPanD mutant library, which are as follows: (1) The lysine at position 14 of the amino acid shown in SEQ ID NO.1 was mutated to threonine (EcoPanD K14T , the nucleotide sequence of the mutant is shown in SEQ ID NO.2); (2) the 44th isoleucine of the amino acid 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 85th amino acid shown in SEQ ID NO.1 is mutated from valine to leucine (EcoPanD V85L , the nucleotide sequence of the mutant is shown in SEQ ID NO.4). The PCR primer sequences used in this example are listed in Table 2. The EcopanD gene (derived f...

Embodiment 3

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

[0074] The transformants obtained in Example 2 were carried out as follows:

[0075] 1. Sequencing verification: Use sterilized pipette tips to select 3 transformants on the plate and inoculate them in LB liquid medium containing 100 μg / mL kanamycin, and at the same time, E.coli W3110 / pTrc99A-EcopanD was inoculated as a control in a 100 μg / mL Kana antibiotic in LB liquid medium. 37°C, 200r / min shaking culture for 12h, sequencing and bacteria preservation. The constructed strains are shown in Table 3.

[0076] Table 3 Strain constructed in Example 3

[0077]

[0078] 2. Induced expression of mutants: Under sterile conditions, take 1 mL of seed solution from the LB liquid medium in step 1, transfer it to 50 mL of LB medium (final concentration is 100 μg / mL Kana), 37 ° C, 200 r / Min shaking culture for about 2h until OD 600 0.6-0.8, add IPTG with a final concentration of 0.3mM, shake and culture for 12h fo...

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Abstract

The invention provides an L-aspartic acid-α-decarboxylase mutant and its application in synthesizing β-alanine. The present invention transforms L-aspartic acid-α-decarboxylase through site-directed mutagenesis of the whole plasmid by substituting the 14th part of L-aspartic acid-α-decarboxylase derived from Escherichia coli with an amino acid sequence of SEQ ID NO.1 position, 44 and 85 amino acid positions, and a mutant strain affecting the self-cleavage of L-aspartic acid-α-decarboxylase was obtained. The present invention compares the mutant EcoPanD K14T 、EcoPanD I44V 、EcoPanD V85L Compared with the wild type, it was found that the self-shearing level of the mutant was significantly increased, and the production of β-alanine was significantly increased after fermentation.

Description

(1) Technical field [0001] The invention relates to an L-aspartate-α-decarboxylase mutant and its application in synthesizing β-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 ), easily soluble in water, is the only β-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 of all, many industrially important compounds such as: 3-hydroxypropionic acid (3-hydroxypropionic acid), poly-3-hydroxypropionate (Poly-3-hydroxypropionate), pantothenic acid (pantothenic acid), carnosine (camosine), pamidronate ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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