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Mutant enzyme CYP153A M228L and application thereof in synthesis of 10-hydroxy-2-decenoic acid

A technology of mutating enzymes and hydroxyl groups, applied in the field of biological fermentation, can solve the problems of not reaching the industrialized level, low synthesis efficiency, etc., and achieve the effect of improving the conversion rate and high-efficiency expression

Active Publication Date: 2021-07-13
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims at the synthesis pathway constructed by a single existing engineering bacterium. The synthesis efficiency of 10-HDA is extremely low and cannot reach the industrial level. On the basis of further optimizing the expression element and 10-HDA synthesis pathway, a two-step method is provided for the production of 10-hydroxy-2-decenoic acid using capric acid as a raw material using Escherichia coli engineering bacteria

Method used

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  • Mutant enzyme CYP153A M228L and application thereof in synthesis of 10-hydroxy-2-decenoic acid
  • Mutant enzyme CYP153A M228L and application thereof in synthesis of 10-hydroxy-2-decenoic acid
  • Mutant enzyme CYP153A M228L and application thereof in synthesis of 10-hydroxy-2-decenoic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] The fusion enzyme gene of alkane hydroxylase CYP153A of Marinobacter aquaeolei and P450NADH reductase of Bacillus megaterium, namely CYP153A-CPR BM3 Fusion enzyme gene, CYP153A M228L-CPR BM3 PCR amplification of fusion enzyme gene, acyl-CoA dehydrogenase gene PpFadE, acyl-CoA synthetase gene MaMACS, and acyl-CoA thioesterase gene ctydiI.

[0105] According to the fusion enzyme gene of alkane hydroxylase CYP153A of Marinobacter aquaeolei and P450NADH reductase of Bacillus megaterium, that is, CYP153A gene, CPR BM3 Gene, CYP153A M228L-CPR BM3 Fusion enzyme gene, acyl-CoA dehydrogenase gene PpFadE, acyl-CoA synthetase gene MaMACS, acyl-CoA thioesterase gene ctydiI design PCR amplification primers, the nucleotide sequences of the upstream primers are respectively as SEQID NO.1, 3, 5, 7, 9, and 11, the nucleotide sequences of the downstream primers are shown in SEQ ID NO.2, 4, 6, 8, 10, and 12;

[0106] Among them, the fusion enzyme gene of alkane hydroxylase CYP153A of M...

Embodiment 2

[0155] Digestion of recombinant plasmid vectors pET21b, pCDFDuet-1, pCDFDuet-1-MaMACS, pET28a-SUMO and gene ctYdiI

[0156] Extract the pET21b plasmid and perform a double enzyme digestion reaction. The reaction system is as follows:

[0157]

[0158] Reaction conditions: react at 37°C for 6h.

[0159] The pET21b plasmid vector was purified by 1% agarose gel electrophoresis after double digestion Figure 5 , and use the DNA gel recovery kit to recover the target fragment.

[0160] Extract the pCDFDuet-1 plasmid and perform a double enzyme digestion reaction, the reaction system is as follows:

[0161]

[0162] Reaction conditions: react at 37°C for 6h.

[0163] The pCDFDuet-1 plasmid vector was double digested and purified by 1% agarose gel electrophoresis, and the target fragment was recovered using a DNA gel recovery kit.

[0164] Extract pCDFDuet-1-MaMACS plasmid and perform double enzyme digestion reaction on it, the reaction system is as follows:

[0165]

...

Embodiment 3

[0173] Recombinant plasmid pET21b-CYP153A-CPR BM3 Multi-fragment seamless cloning, recombinant plasmid pET28a-SUMO-ctYdiI connection

[0174] Combine the double digested pET21b plasmid with CYP153A, CPR BM3 The PCR product is ligated, and the ligation reaction system is as follows:

[0175]

[0176] Mix the above ligation reaction system thoroughly and centrifuge for a few seconds, collect the droplet on the tube wall to the bottom of the tube, and connect at 37°C for 30 minutes to obtain the recombinant plasmid pET21b-CYP153A-CPR BM3 .

[0177] Ligate the digested pCDFDuet-1 plasmid with the MaMACS PCR product, and the ligation reaction system is as follows:

[0178]

[0179]The above ligation reaction system was thoroughly mixed and then centrifuged for a few seconds to collect the drop on the tube wall to the bottom of the tube, and reacted at 37°C for 37 minutes to obtain the recombinant plasmid pCDFDuet-1-MaMACS.

[0180] Ligate the digested pCDFDuet-1-MaMACS p...

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Abstract

The invention relates to a mutant enzyme CYP153A M228L and an application of the mutant enzyme CYP153A M228L in synthesis of 10-hydroxy-2-decenoic acid. The mutant enzyme CYP153A M228L is characterized in that amino acid at the 228th site of CYP153A enzyme is mutated into L from M; a method for biologically synthesizing 10-hydroxy-2-decenoic acid through a two-step method by taking decanoic acid as a raw material mainly comprises the following steps: constructing an optimized recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, an optimized recombinant plasmid pET21b-CYP153A M228L-CPRBM3, and an optimized recombinant plasmid pET28a-SUMO-ctYdiI; constructing escherichia coli recombinant bacteria to prepare resting cells, further culturing the resting cells to prepare the 10-hydroxy-2-decenoic acid. The conversion rate of the 10-hydroxy-2-decenoic acid is remarkably improved according to the technical scheme.

Description

technical field [0001] The invention relates to a mutant enzyme CYP153A M228L and its application in synthesizing 10-hydroxy-2-decenoic acid, belonging to the technical field of biological fermentation. Background technique [0002] 10-hydroxy-2-decenoic acid (10-hydroxy-2-decenoic acid, 10-HDA) is a monounsaturated fatty acid containing a hydroxyl group, the molecular formula is C 10 h 18 o 3 . So far, it has only been found in royal jelly and propolis in nature, so it is also called royal jelly acid. Studies have shown that 10-HDA has many important physiological functions such as antibacterial, immune regulation, anti-oxidation, anti-tumor, and lowering blood sugar. It has extremely high medical and health care values, and has a wide application prospect. The structure of the compound is as follows: [0003] [0004] In view of the extensive and important application value of 10-HDA, the research on finding efficient, convenient and low-cost production methods of ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12P7/64C12N1/21C12N15/70C12N15/60C12N15/54C12N15/55C12N15/31C12R1/19
CPCC12N9/0073C12P7/6409C12N15/70C12N9/0006C12N9/88C12N9/1029C12N9/16C12N9/001C07K14/245C12Y101/01035C12Y402/01017C12Y301/02C12Y103/08C12Y106/02004Y02A50/30
Inventor 苏静李岩王瑞明王丽王蕾蕾徐子淇汪俊卿刘孟连宋子昂
Owner QILU UNIV OF TECH
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