17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, coding gene and engineering bacteria

A technology of hydroxysteroids and mutant enzymes, applied in the field of enzyme engineering and bioengineering, can solve the problems of low enzyme activity, unfavorable large-scale preparation, and low expression, and achieve the effect of improving catalytic activity

Active Publication Date: 2020-07-28
TIANJIN UNIV
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  • Description
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  • Application Information

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Problems solved by technology

However, the 17β-hydroxysteroid hydroxylase derived from microorganisms has the following problems: ① The 17β-hydroxysteroid hydroxylase from this source is a reversible oxidoreductase that catalyzes the reaction between the C17-keto group and the C17-alcohol group on the steroid nucleus. The reversible reduction/oxidation reaction is not suitable for the high-efficiency production of testosterone; ② During the process of catalyzing the conversion of 4-AD into testosterone, it will be accompanied by some by-products of C19 steroids, such

Method used

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  • 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, coding gene and engineering bacteria
  • 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, coding gene and engineering bacteria
  • 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, coding gene and engineering bacteria

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Experimental program
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Embodiment 1

[0030] Rational Design of Mutation Sites of 17β-Hydroxysteroid Hydroxylase 3

[0031] (1) Simulation of the spatial structure of 17β-hydroxysteroid hydroxylase 3: Using the same family enzyme 17β-hydroxysteroid hydroxylase 1 (PDB: 1FDT) as a model, it was found that its structure has a set of typical features: ① consists of 8 α-helices and 8 β sheets constitute a barrel-shaped structure; ②The nicotinamide ring of NADPH and the nearby 17 residues form an oval pocket at the C-terminus; ③Two channels are found from the crystal structure for substrate entry and product release;④ Key amino acids Phe306 and Trp227 play an important role in substrate recognition. Combined with the above information, the SWISS-MODEL online server was used to simulate the spatial structure of 17β-hydroxysteroid hydroxylase 3 (SEQ ID NO.3); the structural model of homology modeling was evaluated and optimized by Verify_3D to obtain a more accurate Analog structure. From this, the rational design idea ...

Embodiment 2

[0035] Preparation of recombinant plasmids pPIC3.5K-17β-HSD3 and pPICZαA-G6PDH

[0036] A recombinant plasmid pPIC3.5K-17β-HSD3 is obtained by linking the codon-optimized 17β-hydroxysteroid dehydrogenase gene (SEQ ID NO.4) to the pPIC3.5K plasmid.

[0037] A recombinant plasmid, pPICZαA-G6PDH, will be able to catalyze NADP + The enzyme for synthesizing NADPH, such as the glucose-6-phosphate dehydrogenase gene (SEQ ID NO. 18) derived from Saccharomyces cerevisiae, is obtained by linking to pPICZαA plasmid.

[0038] Wherein, the source of the 17β-hydroxysteroid dehydrogenase gene: Homo sapiens (GI: 21706851), the nucleotide sequence of the gene is shown in SEQ ID NO.17; the human source 17β-hydroxysteroid hydroxylase 3 After codon optimization of the gene, a codon-optimized 17β-hydroxysteroid dehydrogenase gene (SEQID NO.4) suitable for expression in Pichia pastoris host was obtained;

[0039]The pPIC3.5K plasmid and pPICZαA plasmid were respectively purchased from Thermo Fish...

Embodiment 3

[0055] Contains a mutant enzyme encoding 17β-hydroxysteroid hydroxylase 3 (17β-HSD3 G186R / Y195W ) gene recombinant plasmid (abbreviation: recombinant plasmid containing mutant gene) preparation

[0056] Based on the mutation site rationally designed in Example 1, the primers for site-directed mutagenesis were designed and synthesized as follows:

[0057] G186R-F: (introducing a mutation at position 186) (SEQ ID NO.13)

[0058] G186R-R: (introducing a mutation at position 186) (SEQ ID NO.14)

[0059] Y195W-F: (Introduction of mutations at positions 186 and 195) (SEQ ID NO.15)

[0060] Y195W-R: (Introduction of mutations at positions 186 and 195) (SEQ ID NO.16)

[0061] According to the requirements of the seamless recombination kit, the recombinant plasmid containing the mutant gene was constructed by seamless connection and reverse PCR technology, which is mainly divided into two steps: (1) using the recombinant plasmid pPIC3.5K-17β-HSD3 as a template, G186R-F ...

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Abstract

The invention discloses a 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, a coding gene and engineering bacteria. The 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme is abbreviated as 17 beta-HSD3<G186R/Y195W>, and an amino acid sequence of the 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme is as shown in SEQ ID NO.1. 17 beta-hydroxysteroid hydroxylase 3 is subjected to molecular modification by adopting a rational design technology, the 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme with improved affinity with a substrate is obtained through methods of homologous modeling, molecular docking, binding energy calculation and the like, the enzyme activity is improved by 1.66 times as compared with the original enzyme activity, and by utilizing the strategy, the catalytic activity of the 17 beta-hydroxysteroid hydroxylase 3 can be obviously improved. The 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme is coded, so that the yield of testosterone is up to 3.95 g/L, and technical support and data reference are provided for large-scale biological preparation of the testosterone which is a male hormone drug.

Description

technical field [0001] The invention belongs to the technical fields of enzyme engineering and bioengineering, and specifically relates to a 17β-hydroxysteroid hydroxylase 3 mutant enzyme, a coding gene, a plasmid containing the coding gene, and an engineering bacterium. [0002] technical background [0003] Steroids are widely used as anti-inflammatory, diuretic, anabolic, contraceptive, androgenic, progestogenic and anticancer drugs, so they are an invaluable resource in the pharmaceutical industry with a global market of over $100 billion per year, Testosterone (TS) is one of the very important male hormones. Testosterone is a key sex hormone that regulates male fertility, muscle mass and fat distribution. It is clinically used to treat male hypogonadism, maintain male secondary sexual characteristics and sexual function. At the same time, testosterone is also a very useful steroid precursor , commonly used in the synthesis of high-performance steroid drugs such as 5α-di...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0036C12Y106/00C12N15/70
Inventor 齐崴尤生萍张伟丁娟娟钱建武苏荣欣
Owner TIANJIN UNIV
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