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CN-122012422-A - Cytochrome P450 enzyme mutant and application thereof in hydroxylation

CN122012422ACN 122012422 ACN122012422 ACN 122012422ACN-122012422-A

Abstract

The invention discloses a cytochrome P450 enzyme mutant and application of hydroxylation thereof, relates to the technical fields of enzyme engineering and genetic engineering, and is generated by single-point or multi-point mutation on an amino acid sequence shown as SEQ ID NO. 2, wherein the mutant is selected from any one ：W72F、L75A、L75F、L78A、L82A、L82C、L82G、L82T、G85A、G87A、G87V、L181A、L437、G85S/S88L、G87A/S88T、G87A/S88T/Q188L. of the mutants disclosed by the invention, so that the conversion rate and the region selectivity (up to 95.1%) of 6 beta-hydroxylation reaction of various steroid substrates are obviously improved, a powerful biocatalytic tool is provided for efficiently and environmentally synthesizing 6 beta-hydroxylation steroid drug intermediates, and the application prospect is important.

Inventors

  • SONG YONGBO
  • ZHAO FAN
  • YE QI
  • XU WEIZHUO
  • SONG MING
  • YANG HONGTAO

Assignees

  • 沈阳药科大学

Dates

Publication Date
20260512
Application Date
20260202

Claims (8)

  1. 1. A cytochrome P450 enzyme mutant, which is produced by single or multiple point mutation in the amino acid sequence shown in SEQ ID No. 2, wherein the mutant is selected from any one of the following: W72F、L75A、L75F、L78A、L82A、L82C、L82G、L82T、G85A、G87A、G87V、L181A、L437、G85S/S88L、G87A/S88T、G87A/S88T/Q188L.
  2. 2. a DNA molecule encoding the nucleotide sequence of the cytochrome P450 enzyme mutant of claim 1 or the complement thereof.
  3. 3. A recombinant expression plasmid is characterized in that, the recombinant expression plasmid contains the DNA molecule according to claim 2.
  4. 4. A genetically engineered bacterium is characterized in that, the genetically engineered bacterium contains the recombinant expression plasmid of claim 3.
  5. 5. Use of a cytochrome P450 enzyme mutant according to claim 1 or a DNA molecule according to claim 2 or a recombinant expression plasmid according to claim 3 or a genetically engineered bacterium according to claim 4 for catalyzing the production of 6 beta-hydroxylation products of steroid compounds.
  6. 6. A method for the catalytic formation of a 6 β -hydroxylation product using a steroid, comprising: preparing a recombinant expression plasmid containing a mutant encoding P450BM 3; transferring the recombinant expression plasmid into a host strain to obtain genetically engineered bacteria; Taking steroid compound as a substrate, and carrying out whole-cell catalytic reaction to generate a corresponding 6 beta-hydroxylation product.
  7. 7. The method of claim 6, wherein the steroid is one of the following: 。
  8. 8. the method according to claim 6, wherein the whole cell catalytic reaction is carried out in a genetically engineered bacterium containing the mutant at a pH of 7.0 to 8.0 and a temperature of 16 ℃ to 25 ℃ for a reaction time of 10 to 24 hours.

Description

Cytochrome P450 enzyme mutant and application thereof in hydroxylation Technical Field The invention relates to the technical fields of enzyme engineering and genetic engineering, in particular to a mutant based on cytochrome P450 enzyme and application thereof in catalyzing a specific steroid compound to carry out 6 beta-position hydroxylation reaction. Background Steroid drugs are widely used clinically for treating various diseases because of their important pharmacological activities such as anti-inflammatory, anticancer, antiallergic, contraceptive, etc. The biological activity of the medicine is closely related to the molecular structure, wherein, realizing regional and stereoselective hydroxylation on a steroid skeleton is one of key steps for regulating and controlling the pharmacological functions of the medicine. Chemical hydroxylation generally has the problems of harsh reaction conditions, poor selectivity, serious environmental pollution and the like. In contrast, the use of cytochrome P450 (CYP) enzyme for biocatalysis has the remarkable advantages of mild reaction conditions, high regioselectivity and stereoselectivity, and the like, and has become an important strategy for synthesizing steroid drugs with high added value. Cytochrome P450 BM3 is a natural fusion monooxygenase derived from Bacillus megaterium (Bacillus megaterium), and is of great interest because of its high electron transfer efficiency and rapid catalytic rate. Recently, P450 BM3 mutant LG23 obtained by directed evolution has been reported to hydroxylate at the 7β position on a variety of steroid substrates (e.g. testosterone, nandrolone, etc.). However, for 6 beta-position hydroxylation reaction which has important value in steroid drug synthesis, the existing P450 enzyme has the problems of low catalytic activity, narrow substrate spectrum, insufficient selectivity and the like, and is difficult to meet the requirement of industrial production. There is no disclosure of any published technology disclosing P450 mutants with efficient 6 beta hydroxylation of specific steroid substrates such as 1, 4-Androstenedione (ADD), 4-androstenedione (4 AD) by single or multiple point mutations. Therefore, there is a need to develop a novel P450 enzyme mutant capable of catalyzing 6 beta-hydroxylation reaction of steroid compounds (such as 1, 4-androstenedione) with high efficiency and high selectivity so as to solve the bottleneck in the prior art. Disclosure of Invention The invention aims to provide a cytochrome P450 enzyme mutant and application thereof in catalyzing specific steroid compounds (such as ADD, 4AD and the like) to carry out 6 beta hydroxylation, and aims to solve the technical problems of low activity and poor selectivity of the existing P450 enzyme in catalyzing the specific steroid substrates to carry out 6 beta hydroxylation. The mutants are based on the LG23 backbone, by introducing specific substitutions at amino acid residues 72, 75, 78, 82, 85, 87, 88, 181 and 437 thereof, unique single or multiple point mutants are formed, the combination of mutation sites is not found in any disclosed technology for 6 beta hydroxylation of the specific steroid substrates, and experiments prove that the substrates exhibit unique and excellent 6 beta hydroxylation properties. In order to achieve the above purpose, the invention adopts the following technical scheme: The invention provides a cytochrome P450 BM3 mutant, which is based on a P450 BM3 mutant LG23 (with an amino acid sequence shown as SEQ ID NO: 2), and has single-point or multi-point mutation at 72, 75, 78, 82, 85, 87, 88, 181 and 437 amino acid residues. Specifically, the mutant is selected from any one of the following: W72F、L75A、L75F、L78A、L82A、L82C、L82G、L82T、G85A、G87A、G87V、L181A、L437、G85S/S88L、G87A/S88T、G87A/S88T/Q188L. The present invention provides a DNA molecule encoding a nucleotide sequence of a cytochrome P450 enzyme mutant as described above or a complement thereof. The present invention provides a recombinant expression plasmid containing a DNA molecule as described above. The invention provides a genetically engineered bacterium, which contains the recombinant expression plasmid. The invention provides application of the cytochrome P450 enzyme mutant, the DNA molecule, the recombinant expression plasmid or the genetically engineered bacterium in catalyzing steroid compounds to generate 6 beta-hydroxylation products. The invention also provides a method for catalyzing and generating 6 beta-hydroxylation products by using the steroid, which comprises the following steps: (1) Preparing a recombinant expression plasmid containing a mutant encoding P450 BM 3; (2) Transferring the recombinant expression plasmid into a host strain to obtain genetically engineered bacteria; (3) Using steroid compound as substrate, using gene engineering bacteria to make whole cell catalytic reaction to produce correspondent 6 beta-hydroxylation product. The steroid is selected fr