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Aureobasidium pullulans malate-CoA ligase as well as recombinant expression vector and application thereof

A technology of malyl coenzyme and Aureobasidium pullulans, which is applied in the field of genetic engineering, can solve the problems that the sequence has not been reported, the polymalic acid polymerization pathway and related genes have not been elucidated, and achieve the effect of increasing yield

Inactive Publication Date: 2014-10-15
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, so far, the polymerization pathway and related genes for the production of polymalic acid by polymerizing malic acid as a monomer have not been elucidated.
Based on previous literature research, the polymerization pathway of polymalic acid may involve two enzymes, malate-CoA ligase (Mcl) and polymalic acid synthase (Pas), to participate in the reaction, but the related genes And the sequence has not been reported in A. pullulans, malyl-CoA ligase, with the participation of ATP and coenzyme A, realizes the acylation of monomeric malic acid, and polymalate synthase catalyzes the polymerization reaction

Method used

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  • Aureobasidium pullulans malate-CoA ligase as well as recombinant expression vector and application thereof
  • Aureobasidium pullulans malate-CoA ligase as well as recombinant expression vector and application thereof
  • Aureobasidium pullulans malate-CoA ligase as well as recombinant expression vector and application thereof

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Embodiment 1

[0024] Embodiment 1, the cloning of malyl-CoA ligase gene

[0025] Using bioinformatics to analyze the reported malyl-CoA ligase gene sequences in NCBI, the sequences were from Phaeospirillum molischianum (WP_002730336); Brucella (Brucell, WP_008503553a); Capsular Rhodobacter (Rhodobacter capsulatus , WP_013066464); Rhodospirillum rubrum (WP_011388966); Paracoccus denitrificans (Paracoccus denitrificans, WP_01174690); after the sequences were compared and analyzed, a demerger primer was designed in the conserved region, and the upstream primer was: 5'-argghggtatggacattgagg- 3' (SEQ ID NO.1), the downstream primer is: 5'-ccrccraaratgttgacraag-3' (SEQ ID NO.2), wherein r=a / g, y=c / t, m=a / c, k= g / t, s=c / g, w=a / t, h=a / c / t, b=c / g / t, v=a / c / g, d=a / g / t, n= a / c / g / t), and then use Aureobasidium pullans CCTCC NO: M2012223 genome as a template for PCR amplification. The amplification conditions are: 94°C pre-denaturation for 5 minutes; Anneal for 30s, extend at 72°C for 30s, 30 cycles; e...

Embodiment 2

[0031] Example 2 Construction of malyl-CoA ligase cDNA recombinant expression vector

[0032] In order to realize expression in Escherichia coli, the total RNA of Aureobasidium pullulans was extracted with a fungal RNA extraction kit (purchased from OMEGA Company, product number R6840-01), and then reverse transcriptase kit (purchased from Fermentas company, product number is K1621) to synthesize the first strand of cDNA, and use this as a template, the upstream primer is: 5'-atgttcaagctcgcccgcag-3' (SEQ ID NO.11), and the downstream primer is: 5'-ttagataccgagagagaactcgacac-3' (SEQ ID NO. 12) Perform PCR amplification. The PCR amplification conditions are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 57°C for 50 seconds, extension at 72°C for 2 minutes, and 30 cycles; extension at 72°C for 10 minutes; and finally, incubation at 25°C for 10 minutes. The amplified product was connected to pMD19-T Vector, then transformed into E.coli D...

Embodiment 3

[0033] Example 3 Expression and purification of malyl-CoA ligase

[0034] The constructed recombinant expression vector pET-Mcl was transferred into E.coli BL21(DE3), and after pre-cultivation and 0.5mmol / L IPTG induction for 6h, the bacteria were collected, resuspended in the cell lysate, and used The cells were lysed by sonication, and the cell supernatant was obtained by centrifugation; the protein was purified from the cell supernatant by Ni-NTA affinity chromatography. Then use SDS-PAGE to detect the protein, the results are as follows figure 1 shown. Depend on figure 1 It can be seen that a single band appears at about 68kDa on lane 1. Since the carrier contains four protein tags, Trx.Tag, S.Tag and 2 His.Tag tags, the molecular weight of the purified protein is greater than 48kDa. It indicated that malyl-CoA ligase was successfully expressed, and the protein purification effect was better.

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Abstract

The invention discloses aureobasidium pullulans malate-CoA ligase. An amino acid sequence of aureobasidium pullulans malate-CoA ligase is represented as SEQ ID NO.10, and aureobasidium pullulans malate-CoA ligase comprises 439 amino acids; a cDNA (complementary desoxyribonucleic acid) sequence is represented as SEQ ID NO.9; a genomic sequence is s represented as SEQ ID NO.8; and after the cDNA sequence is subjected to recombinant expression, recombinant protein with molecular weight of about 68 kDa can be obtained. The enzymatic property detection shows that the activity of malate-CoA ligase subjected to the recombinant expression is 0.176 U, the optimal reaction temperature is 25 DEG C, the optimal pH is 8.0, and the optical ATP (adenosine triphosphate) substrate concentration is 0.2 mM; and further, the malate-CoA ligase has wider substrate selectivity and can catalyzing different monomers such as malic acid, oxalic acid, oxaloacetic acid, succinic acid, citric acid, butyric acid or malonic acid for reaction, and the malic acid yield is increased.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the Aureobasidium pullulans malyl-CoA ligase gene, and also relates to a recombinant expression vector and application for expressing the malyl-CoA ligase. Background technique [0002] Polymalic acid (PMA) is a new type of fully biodegradable polyester polymer. Due to its good water solubility, biodegradability and biocompatibility, it can be used as a drug carrier and microcapsule material. , biomedical materials, water-absorbing materials, cosmetics, food packaging and other materials, have a wide range of application prospects. [0003] Polymalic acid is mainly produced by fermentation of Aureobasidium pullulans. The study on the biosynthetic pathway of polymalic acid shows that polymalic acid is synthesized using malic acid produced by the TCA cycle as a substrate. However, so far, the polymerization pathway and related genes of polymalic acid produced by poly...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/70C12N1/15C12P19/32
CPCC12N9/93C12Y602/01009
Inventor 邹祥吴小燕涂光伟
Owner SOUTHWEST UNIVERSITY
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