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Acyl CoA synthetase and application thereof

A technology of synthase and acyl group, which is applied in the field of acyl CoA synthase to achieve the effect of excellent catalytic performance

Pending Publication Date: 2022-07-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no acyl-CoA synthetase with high catalytic efficiency for different carboxylic acid substrates has been identified, and no acyl-CoA synthetase independent of Lys in Motif 10 has been reported.

Method used

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  • Acyl CoA synthetase and application thereof
  • Acyl CoA synthetase and application thereof
  • Acyl CoA synthetase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1. Sequence analysis of UkaQ

[0087] (1) Sequence analysis of UkaQ

[0088] The amino acid sequences of UkaQ and the analyzed acyl-CoA synthase were compared using the multiple sequence alignment software ClustalW, and the conserved motifs MotifA1-MotifA10 of UkaQ were found by combining the features of the conserved motifs MotifA1-MotifA10 of this type of enzymes. The acyl-CoA synthase sequences used for comparison are 4-coumaric acid-CoA ligase 3A9U from Populus tomentosa, 4-coumaric acid-CoA ligase 5U95 from tobacco, benzene from Rhodopseudomonas swampis Formate-CoA ligase 4EAT, malonyl CoAA3NYQ from Streptomyces coelicolor, phenylacetic acid-CoA ligase 2Y4O from Burkholderia neocepacia. Multiple sequence alignment results are as figure 1 As shown, the marked "Lysing position" is a key site in the conserved motif of acyl-CoA synthase (MotifA10) responsible for catalyzing the adenylation reaction.

[0089] The sequence alignment results show that the chara...

Embodiment 2

[0090] The expression vector construction and expression of embodiment 2.UkaQ

[0091](1) Use the following steps to obtain the gene encoding UkaQ

[0092] Using the amino acid sequence SEQ ID NO: 1 (GenBank: AQN08598.1) of UKaQ as a template, the nucleic acid sequence of the gene was converted into the gene through reverse translation, and the DNA of the UkaQ gene was fully synthesized and synthesized on the universal vector PUC57.

[0093] (2) Construction of expression vector p-UkaQ and recombinant strain

[0094] The UkaQ gene DNA and pET28a plasmid were digested with NdeI / XhoI and recovered respectively. After enzymatic ligation, they were transformed into EcoliDH5α competent cells, and the correct recombinant bacteria were screened. After successful verification, the plasmid was extracted and named p-UkaQ.

[0095] (3) Use the following steps to express p-UkaQ in vitro

[0096] The recombinant plasmid p-UkaQ in Example 1 was transformed into E.coli BL21 (DE3) competent...

Embodiment 3

[0097] Example 3: Biochemical function identification of UkaQ

[0098] The composition of the in vitro activity assay system of UkaQ is as follows: 1.5mM carboxylic acid substrate, 2mM ATP, 1.5mM COA, 5mM Mg 2 + , 50 mM Hepes Buffer (pH 7.5) and 1 μM UkaQ, the total volume was 200 μl, the reaction was carried out in a 1.5 mL EP tube, and the reaction without UkaQ or its mutant protein was used as a control. The reaction was incubated at 25 °C for 1 h, quenched by adding an equal volume of methanol, centrifuged at 12,000 rmp for 20 min to remove proteins, and 1 μl of the sample supernatant was taken for UPLC analysis. UPLC analysis of all samples was performed on an Eclipse XDB-C18 analytical column with a flow rate of 0.2 mL min-1 and the analysis procedure was as follows: T=0 min, 5%B; T=3min, 5%B; T=10min, 65% B; T=12 min, 5% B; T=15 min, 5% B (solvent A: H2O with 10 mM CH3COONH4, solvent B: CH3CN). For LC-HRMS analysis, the conditions were the same as for UPLC analysis. ...

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Abstract

The invention discloses an acyl CoA synthetase and application thereof, and relates to the field of gene engineering, enzyme engineering and combinatorial biosynthesis, the acyl CoA synthetase UkaQ and a mutant thereof are included, and the amino acid sequence of the acyl CoA synthetase UkaQ is as shown in SEQ ID NO: 1; the invention relates to an application of acyl CoA synthetase in efficient production of a natural product polyketone compound. The invention relates to a novel acyl-CoA synthetase which has efficient catalytic activity and wide substrate spectrum and is not regulated by acylation modification, and the identification of the acyl-CoA synthetase and the acquisition of mutants not only expand the diversity of adenosine synthetase superfamilies, but also increase the yield of the adenosine synthetase. Moreover, a powerful and universal biocatalyst is provided for synthesis and carbon skeleton modification of polyketone compounds and other natural products taking acyl CoA as precursors, and the method has important application and economic values for engineering modification and industrial large-scale production of metabolic processes of various natural products.

Description

technical field [0001] The invention relates to the fields of genetic engineering, enzyme engineering and combined biosynthesis, in particular to an acyl-CoA synthase and its application. Background technique [0002] During the biosynthesis of primary and secondary metabolites in organisms, acyl-CoAs act as precursors for different kinds of natural products, including fatty acids, polyketides, polyethers, polyenes, flavonoids ( flavonoids), stilbenes alkaloids and isoprenes, etc., the above-mentioned compounds are important sources of natural products with medicinal value. The synthesis of acyl-CoA in microbial organisms is generated by the activation of different carboxylic acid substrates by acyl-CoA synthetase, so acyl-CoA synthase is a key enzyme in the above-mentioned natural product synthesis pathway and is considered to be an ideal target for regulating the synthesis of related products Gene. [0003] Acyl-CoA synthases belong to the adenosine synthase family (ANL ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/76C12P17/08C12R1/465
CPCC12N9/93C12N15/76C12P17/08
Inventor 瞿旭东郑梦梦林芝
Owner SHANGHAI JIAO TONG UNIV
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