CN112921011A

CN112921011A - 3-sterone-delta 1-dehydrogenase mutant, engineering bacterium and application

Info

Publication number: CN112921011A
Application number: CN202110376599.9A
Authority: CN
Inventors: 许正宏; 李会; 薛苗苗; 史劲松; 刘伟; 张晓梅; 徐国强
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-07
Filing date: 2021-04-07
Publication date: 2021-06-08
Anticipated expiration: 2041-04-07
Also published as: CN112921011B

Abstract

The invention discloses a 3-sterone-delta 1-dehydrogenase mutant, engineering bacteria and application, and belongs to the technical field of enzyme engineering. The 3-sterone-delta 1-dehydrogenase mutant of the Mycobacterium (Mycobacterium) LY-1 of the invention is obtained by mutating the aspartic acid at the 320-position of the 3-sterone-delta 1-dehydrogenase into tyrosine to obtain a 3-sterone-delta 1-dehydrogenase mutant KstD 1; pET32a is used as an expression plasmid, Escherichia coli BL21 is used as an expression host, heterologous expression of a 3-ketosteroid-delta 1-dehydrogenase mutant in Mycobacterium LY-1 is realized, recombinant bacteria Escherichia coli BL21-pET32a-KstD1 can convert a high-concentration C1, 2-dehydrosteroid compound in a short time, the conversion rate reaches 98%, the production cost is reduced, and the method has an important industrial application value.

Description

3-sterone-delta 1-dehydrogenase mutant, engineering bacterium and application

Technical Field

The invention belongs to the technical field of enzyme gene engineering and enzyme engineering, and particularly relates to a 3-sterone-delta 1-dehydrogenase mutant, engineering bacteria and application thereof, wherein the mutant can dehydrogenate C1 and 2 sites of steroid compounds into C1 and 2-site dehydrogenated steroid compounds.

Background

Steroid hormone drugs have a wide range of therapeutic effects, are often used for treating diseases such as allergy, osteoporosis, hormone level disorder and the like, are now the second major class of drugs in the world after antibiotics, and the common steroid drugs include progesterone, hydrocortisone, dexamethasone, aldosterone and the like. Steroids are a family of terpenoid lipids widely distributed in nature, all of which generally have the carbon skeleton structure of cyclopentanoperhydrophenanthrene, i.e. the steroid parent nucleus. Dehydrogenation of steroid parent nucleus C1,2 site can improve affinity of steroid, thereby strengthening drug effect and action time of steroid, and the process is mainly used for corticosteroid drugs, such as prednisolone. The dehydrogenation reaction of C1 and 2 can be carried out by a chemical method, but the method has the defects of low yield, strong toxicity, environmental pollution and the like, and the microbial transformation method is a better choice and becomes a crucial one-step reaction in the synthesis of steroid hormone drugs.

The 3-sterone-delta 1-dehydrogenase (KstD) belongs to flavoproteinases, is mostly positioned on a cell membrane, can introduce a double bond between C1 and C2 of a 3-sterone compound, improves the anti-inflammatory activity of an original substrate, and plays an important role in the production of steroid drugs. The reactions in which the KstD enzymes participate are irreversible reactions, occurring in a variety of microorganisms, and often there are a variety of KstD isoenzymes, differing in their substrate preferences.

As the development of gene technology continues, more and more researchers have conducted more intensive studies on the KstD enzyme, and many examples of heterologous expression of 3-ketosteroid-delta 1-dehydrogenase and modification of the enzyme exist, but the ubiquitous transmembrane domain remains an important bottleneck limiting the expression of the activity thereof, and after heterologous expression, the activity of the enzyme is low, only a low concentration of substrate can be converted, and the conversion time is long.

Therefore, the method combining genomics, bioinformatics analysis and metabolic engineering operation is adopted to excavate and identify more novel easily soluble expressed enzyme genes, modify the genes and screen out the KstD enzyme which can transform high substrate concentration or reduce transformation time, and the method has very important industrial significance.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a 3-ketosteroid-delta 1-dehydrogenase mutant of Mycobacterium LY-1, an engineering bacterium and application thereof, and particularly relates to an amino acid sequence of a 3-ketosteroid-delta 1-dehydrogenase KstD mutant from Mycobacterium (Mycobacterium) LY-1 and a nucleotide sequence for encoding the protein. The key active D320 site of the 3-sterone-delta 1-dehydrogenase is mutated into tyrosine to obtain a high-enzyme-activity mutant, and the mutant gene is heterologously expressed in host cells such as escherichia coli, so that the high-activity expression of the 3-sterone-delta 1-dehydrogenase mutant and the high-efficiency transformation of high-concentration steroid compounds are successfully realized, and a foundation is laid for further industrial application of the mutant.

The first purpose of the invention is to provide a 3-sterone-delta 1-dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO. 4.

In an alternative embodiment, the 3-sterone- Δ 1-dehydrogenase mutant kstD1 is obtained by replacing aspartic acid at position 320 of 3-sterone- Δ 1-dehydrogenase kstD of a parent Mycobacterium species having the amino acid sequence shown in SEQ ID NO.2 with tyrosine.

In an alternative embodiment, the gene for the 3-sterone-. DELTA.1-dehydrogenase is derived from Mycobacterium sp LY-1CGMCC No. 13031.

It is another object of the present invention to provide a gene encoding the mutant, which has a nucleotide sequence of any one of:

a) a base sequence shown as SEQ ID N0: 3;

b) encodes a protein consisting of the amino acid sequence shown as SEQ ID N0:4 or a nonsense mutant sequence thereof.

Another objective of the invention is to provide a recombinant expression plasmid for encoding a 3-ketosteroid-delta 1-dehydrogenase mutant of mycobacterium (Mcbacterium sp). The KstD1 gene of the 3-ketosteroid-delta 1-dehydrogenase mutant is used for constructing a recombinant expression plasmid pET32a-kstD1 by taking pET32a as a plasmid.

Another objective of the invention is to realize the high-efficiency expression of the 3-ketosteroid-delta 1-dehydrogenase mutant of mycobacterium LY-1 by taking competent cells including escherichia coli as an expression host, and transforming a plasmid containing the mutant KstD1 gene in a host microorganism or integrating the mutant KstD1 gene on the genome of the host microorganism to form a mutant KstD1 heterologous expression recombinant engineering strain.

In an alternative embodiment, the host microorganism comprises escherichia coli.

In an alternative embodiment, the host microorganism is escherichia coli BL 21.

The invention also aims to realize the efficient conversion of the substrate steroid compound to prepare the C1, 2-dehydrosteroid compound by using the mutant KstD1 heterologous expression recombinant engineering strain or/and crude enzyme solution secreted and expressed by the recombinant engineering strain. The steroid compounds include AD, T, progesterone and the like.

In an alternative embodiment, the concentration of the substrate steroid is 3 to 10 g/L.

In an optional embodiment, the recombinant engineering strain is transferred into a substrate steroid compound with the concentration of 3-10 g/L, the temperature is 32-37 ℃, and the temperature is 180-220 r.min^-1And (4) converting for 2-4 h to obtain the C1, 2-dehydrogenation steroid compound.

Another object of the present invention is to provide C1, 2-dehydrosteroid compounds prepared by transforming the KstD mutant heterologous expression recombinant engineering strain.

Has the advantages that: compared with the prior art, the 3-ketosteroid-delta 1-dehydrogenase gene found from Mycobacterium LY-1(Mycobacterium) is found to have homology of only less than 50% with the related reported 3-ketosteroid-delta 1-dehydrogenase through sequence comparison, and is a newly discovered gene, the enzyme is composed of two subunits of 34kD and 23kD and mainly exists in cytoplasm, so that heterologous expression in escherichia coli is easy to be carried out to form high-activity soluble enzyme protein, and compared with the enzyme with the same function, the enzyme obviously shortens the conversion time but has lower enzyme conversion rate. The amino acid of the active center of the 3-sterone-delta 1-dehydrogenase is determined, the site-directed mutation is carried out on the key amino acid residue, and the conversion rate of the mutated 3-sterone-delta 1-dehydrogenase is found to be improved to 98 percent, so that the method has important industrial value.

Drawings

FIG. 1 shows a Western Blot of an intracellular protein of a mutant recombinant strain of Escherichia coli (the size of a target protein LY-KstD1 is about 57kDa),

m: standard protein molecular weight;

KstD 1: escherichia coli BL21-pET32a-kstD1 holoprotein sample.

FIG. 2 is a schematic diagram showing the principle of the KstD enzyme activity assay.

FIG. 3 is a line graph showing the time course of transformation efficiency of the mutant recombinant bacteria obtained in example 7 to transform steroid AD into corresponding C1, 2-dehydrosteroid ADD, wherein KstD is 3-sterone- Δ 1-dehydrogenase and KstD1 is a 3-sterone- Δ 1-dehydrogenase mutant.

FIG. 4 is a liquid phase diagram of the mutant recombinant bacteria obtained in example 8 transforming steroid compound T into corresponding C1, 2-dehydrogenation steroid compound.

FIG. 5 is a liquid phase diagram of the mutant recombinant bacteria obtained in example 8 transforming steroid progesterone into corresponding C1, 2-dehydrosteroid.

Detailed Description

Examples

The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

The amplification of wild type 3-ketosteroid-delta 1-dehydrogenase gene with the DNA sequence of mycobacterium LY-1 as template is shown in SEQ ID NO 1, and the amino acid sequence is shown in SEQ ID NO 2. The 320 th aspartic acid of the wild 3-ketosteroid-delta 1-dehydrogenase gene is mutated into tyrosine, a recombinant expression plasmid pET32a-KstD1 is constructed by taking pET32a as a plasmid, and escherichia coli BL21 as an expression host, so that the high-efficiency expression of the 3-ketosteroid-delta 1-dehydrogenase mutant KstD1 of mycobacterium LY-1 is realized.

The deduced amino acid sequence of the complete mutant gene is shown in SEQ ID NO. 4.

The cloning and expression method of the mycobacterium LY-1 strain 3-sterone-delta 1-dehydrogenase mutant KstD1 comprises the following steps:

example 1: DNA extraction of Mycobacterium LY-1

Strain of Mycobacterium LY-1 in enzyme production Medium (NaNO)₃5.4 g/L; 15g/L of yeast powder; 2g/L of glycerol; (NH)₄)₂HPO₄0.6g/L) for 6-7 days at a temperature of 30 ℃. And (4) centrifugally collecting the thalli, extracting the DNA of the mycobacterium LY-1 according to the bacterial DNA extraction kit process, and using the obtained DNA for subsequent tests.

Example 2: cloning of wild-type 3-sterone-delta 1-dehydrogenase Gene of Mycobacterium LY-1 Strain

All gene information related to a mycobacterium LY-1 strain is obtained by whole genome sequencing in the early stage, a wild type KstD gene is obtained according to the whole gene annotation result of the mycobacterium LY-1, a primer is designed according to the kstD gene sequence obtained by identification to amplify a target gene, and the primer sequence is as follows:

primer Yl CGAGCTCGTGACCGCCACCAGCCACAAGA

Primer Y2: CGAGCTCGTGTTCTACATGACTGGACAGG

And using the obtained DNA sequence as a template and the nucleotide sequence as a primer to perform PCR amplification on the coding gene of the wild type 3-sterone-delta 1-dehydrogenase kstD. The PCR reaction was carried out in a 25. mu.L system under the following conditions: beginning circulation after pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 64 ℃ for 30s, and extension at 72 ℃ for 2min for 34 cycles; final extension at 72 ℃ for 5 min.

The KstD gene complete sequence (1704bp) is obtained by sequencing, gene walk-through and gene splicing the obtained PCR product, and is shown as SEQ ID NO: 1. By alignment with the existing kstD amino acid sequence, the homology is only 43%, and thus, a new kstD gene is identified.

Example 3: construction of recombinant expression plasmids

The expression plasmid adopted in the research is escherichia coli expression plasmid pET32a, which is an integrative expression plasmid, PCR products of plasmid pET32a and 3-sterone-delta 1-dehydrogenase kstD are subjected to double enzyme digestion respectively and then are subjected to gel tapping recovery, T4 ligase is used for connecting at 16 ℃ overnight, the connecting product is used for transforming E.coli JM109 competent cells, the cells are cultured on an LB plate containing aminobenzyl resistance (100mg/L) overnight, positive transformants are screened, and the plasmids are extracted after enrichment culture and named as pET32 a-kstD.

Example 4: enzyme activity determination of 3-sterone-delta 1-dehydrogenase and mutants thereof

The reaction mixture comprises 50mM Tris-HCl, pH 7.0, 1.5mM PMS, 40 μm DCPIP, substrate dissolved in 2% methanol with different concentrations (2.5 μm-400 μm), and proper amount of crude enzyme solution. The measurement wavelength was 600nm, the reaction temperature was 30 ℃, and the reduction amount nmoL of dcppi per minute was measured by a microplate reader (e.600 nm ═ 18.7 × 10)³cm^-1·M^-1). The reaction principle is shown in figure 2, the dehydrogenation amount of C1(2) of the steroid substrate is as follows: the reduction amount of dcppi is 1: 1. the enzyme activity of different KstD mutants can be determined by this method.

Enzyme activity: the amount of enzyme required for the catalytic reduction of 1. mu. mol of DCPIP per minute was defined as 1 enzyme activity unit (U).

Example 5: effect of mutation of active site of 3-sterone-delta 1-dehydrogenase on expression of enzyme activity

Using a site-directed mutagenesis kit to design primers D320-F and D320-R as shown in Table 1, using constructed pET32a-kstD as a template to perform PCR, respectively replacing 320 th aspartic acid D of 3-sterone-delta 1-dehydrogenase with glutamine Q, tyrosine Y, valine V, glycine G, alanine A and leucine L, and correspondingly obtaining mutantsComprises the following steps: KstD1(D320Q), KstD1(D320Y), KstD1(D320V), KstD1(D320G), KstD1(D320A), KstD1 (D320L). The PCR reaction conditions are as follows: 95 ℃ for 3min, 34 cycles (95 ℃ for 30S, 58 ℃ for 30S, 72 ℃ for 1.5min), 72 ℃ for 10 min. PCR amplification System: 1 μ L of template, 1 μ L of upstream and downstream primers, Prime Star Max (Premix) DNA20 μ L, ddH₂O17. mu.L. And purifying and recovering the PCR product by using a gel recovery kit, and carrying out electrophoresis test on the concentration of the recovered product. This was transformed into competent E.coil JM109, spread on ampicillin LB plates, and positive colonies were picked. After shaking table overnight culture at 37 ℃, extracting plasmid, and then transferring into escherichia coli BL21 to obtain the recombinant strain with site-directed mutagenesis.

TABLE 1 primer sequences

Note: the underlined sequence represents the mutated amino acid codon sequence: A/T/C/G

And (3) carrying out ultrasonic cell disruption on different mutant recombinant bacteria which grow well through fermentation, carrying out ultrasonic cell disruption for 20min, centrifuging at 8000rpm for 20min, and taking the supernatant to obtain a crude enzyme solution. And (3) using the supernatant crude enzyme solution for enzyme activity determination.

The enzyme activity determination result is shown in table 2, the enzyme activity of the mutant 2, namely KstD1(D320Y), is highest, the 320 th aspartic acid of 3-sterone-delta 1-dehydrogenase is mutated into tyrosine, the mutant recombinant strain is named as BL21-pET32a-kstD1, and the specific enzyme activity of the mutant recombinant strain is improved by 168% compared with the wild type 3-sterone-delta 1-dehydrogenase. In addition, the enzyme activity of the mutant 4, namely the KstD1(D320G) is basically kept unchanged, and is slightly increased but not obvious compared with that of a wild-type non-mutant strain; after site-directed mutagenesis is carried out on the other mutant strains including the mutant 1, the mutant 3, the mutant 5 and the mutant 6, the enzyme activities of the mutant strains are all inhibited, and particularly the enzyme activity reduction rates of the mutant 5 and the mutant 6 are nearly half. Therefore, the mutant forms of site-directed mutagenesis have completely different influences on the enzymatic activity characteristics of the strains, and the mutant strains with the best enzymatic activity are screened out in the embodiment through different mutation comparison, and the best mutation mode, namely mutant 2, KstD1(D320Y), is preferably selected.

The nucleotide sequence of the complete mutant gene is shown as SEQ ID NO. 3, and the deduced mutant amino acid sequence according to the complete mutant gene is shown as SEQ ID NO. 4.

TABLE 2 wild type and mutant specific enzyme activities

Example 6: induced expression and purification of mutant recombinant bacteria

The mutant recombinant bacteria BL21/pET32a-kstD1 obtained in example 5 are streaked in a glycerol tube, a single colony is picked up to 10mL of LB liquid culture medium and cultured at 37 ℃ overnight, then the single colony is inoculated in 50mL of LB liquid culture medium with the inoculation amount of 1 percent and cultured at 37 ℃ to OD₆₀₀0.5mM inducer IPTG was added to the medium at nm of 0.6-0.8, and the culture was continued for 12 hours. After a certain period of induction expression, 8000r min at 4 DEG C^-1Centrifuge for 10min, discard the supernatant, wash the mycelia twice with 5mL of PBS buffer. Carrying out ultrasonic disruption for 20min by using a cell ultrasonic disruptor, wherein the disruption period is 4s ultrasonic and 6s ultrasonic. The thallus crushing liquid is at 4 deg.C and 10000 r.min^-1Centrifugation was carried out for 10min, and the supernatant was collected for Western Blot analysis. As shown in FIG. 1, the lane is a whole protein disrupted supernatant sample of E.coli BL21-pET32a-kstD1, the theoretical size of the target protein LY-kstD1 is about 57kDa, and it can be seen from the figure that the correct band appears at the corresponding position, which proves that the enzyme protein can be expressed in E.coli in a soluble manner.

Example 7: analysis of mutant recombinant bacterium transformed steroid compound AD

The mutant recombinant bacteria BL21-pET32a-kstD1 are induced to express for 12 hours, and the bacteria are collected by centrifugation and washed twice by 10mL of PBS buffer solution. 3g/L AD was added to 50mL PBS buffer, and then a solution prepared by adding a solution prepared by mixing the substrate and the solution in a molar ratio of 1: the cyclodextrin of 1 facilitates the dissolution of the substrate. Resuspending the washed mutant recombinant bacteria with PBS (phosphate buffer solution) after substrate dissolution at 37 ℃ for 220 r.min^-1The conversion was carried out for 4h and samples were taken at 0h, 1h, 2h, 3h, 4h for HPLC analysis. Calculating to obtain KstD1 converted steroid compound ADThe corresponding C1, 2-dehydrosteroid ADD reached 98% conversion in 3 hours and remained stable thereafter. As shown in fig. 3, the time-course line graph of the conversion rate of the mutant recombinant bacterium obtained in this example to convert the steroid compound AD into the corresponding C1, 2-dehydrosteroid compound ADD shows that the conversion rate of the mutant recombinant bacterium is always higher than that of the wild type, and the conversion rate is stabilized at 98% after 3 hours, so that it can be found that the 320 th amino acid of the enzyme is located at the active center, which has a great influence on the converted steroid compound.

Example 8: analysis of mutant recombinant bacteria for transforming other steroid compounds

In addition to the high efficiency of transforming steroid compound AD in a short time, the present example attempted to transform other high concentration substrates such as T and progesterone by using mutant recombinant bacterium BL21-pET32a-kstD1, the specific method is the same as example 8.

The transformation result shows that the mutant recombinant bacterium BL21-pET32a-kstD1 can also perform C1 and 2-position dehydrogenation on T and progesterone with the concentration of 3g/L, as shown in figures 4 and 5, compared with T standard and progesterone standard, the substrate concentration is obviously reduced after the mutant recombinant bacterium BL21-pET32a-kstD1 is transformed, a C1 and 2-position dehydrogenation steroid compound product is correspondingly obtained, the concentration of the product is obviously improved, and the conversion rate of the substrate is over 98% within 3 h.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Sequence listing

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gagaacgccg gcacacccgt ggacagcatg tggatcgtct tcgaccagca gtaccgcaac 1200

agctatgtct tcgccgccga actgtttccg cgcatgccga taccgcagac ctggtacgac 1260

gccggaatcg cagtcaaggc agacaacttc gatgagctgg caaccaaaat gcaggttccg 1320

gtcgacaatt tcgaggcaac tgttacccgg ttcaacgaaa acgctttcgc gggagaggac 1380

cccgacttcg agcggggccg cagtgcctac gaccgctact acggtgatcc cacgatcacg 1440

ccgaacccga acctgcggcc actggtcaaa ggaccgttct acgcggtcaa gatggtactg 1500

agcgacctcg gcacctgcgg cggcctccga gccgacgacc atgcccgagt cctgcgtgaa 1560

gacggcaccg tcatcgacgg actgtatgcg atcggtaaca cggcagccaa cgcgttcggc 1620

aaaacctatc ccggtgccgg cgcgaccatc gcgcaggggc tggtcttcgg ctacatcgcc 1680

gctcgcgacg cggccgaagc gtag 1704

<210> 4

<211> 567

<212> PRT

<213> mutant of 3-sterone-. DELTA.1-dehydrogenase ()

<400> 4

Met Thr Ala Thr Ser His Lys Ser Ile Pro Ala Gly Leu Thr Val Ala

1 5 10 15

Ala Thr Glu Val Asp Leu Leu Val Val Gly Ser Gly Thr Gly Leu Ala

20 25 30

Ala Ala Leu Ala Ala His Glu Gln Gly Leu Ser Val Leu Val Val Glu

35 40 45

Lys Ser Ser Tyr Val Gly Gly Ser Thr Ala Arg Ser Gly Gly Ala Leu

50 55 60

Trp Leu Pro Ala Ser Pro Val Ile Glu Asp Cys Gly Gly Asn Asp Pro

65 70 75 80

Val Ser Arg Ala His Thr Tyr Leu Glu Ser Val Val Gly Asn Ser Ala

85 90 95

Pro Pro Glu Arg Ser Ala Ala Tyr Leu Glu Asn Leu Pro Ala Thr Val

100 105 110

Glu Met Leu Arg Arg Thr Thr Pro Met Lys Leu Phe Trp Ala Lys Glu

115 120 125

Tyr Ser Asp Tyr His Pro Glu Ala Pro Gly Gly Ser Ala Ala Gly Arg

130 135 140

Thr Cys Glu Cys Arg Pro Leu Asn Thr Ser Ile Leu Gly Glu Tyr Leu

145 150 155 160

Pro Asp Leu Arg Pro Gly Val Met Glu Val Ser Ile Pro Met Pro Thr

165 170 175

Thr Gly Ala Asp Tyr Arg Trp Leu Asn Leu Met Ser Arg Val Pro Arg

180 185 190

Lys Gly Leu Pro Thr Ile Ile Lys Arg Leu Ala Gln Gly Ile Gly Gly

195 200 205

Leu Ala Leu Gly Arg Arg Tyr Ala Ala Gly Gly Gln Ala Leu Ala Ala

210 215 220

Gly Leu Phe Ala Gly Val Ile Arg Ala Gly Ile Pro Ile Trp Leu Asp

225 230 235 240

Thr Ala Leu Thr Glu Leu Val Thr Glu Gly Ser Arg Val Thr Gly Ala

245 250 255

Val Val Glu His Gly Gly Glu Arg Val Thr Val Thr Ala Arg Arg Gly

260 265 270

Val Val Leu Ala Ala Gly Gly Phe Asp His His Met Glu Met Arg Arg

275 280 285

Lys Phe Gln Ser Asp Ser Leu Gly Ser Asn Leu Ser Leu Gly Ala Glu

290 295 300

Ser Asn Thr Gly Asp Ala Ile Arg Leu Gly Gln Asp Val Gly Gly Tyr

305 310 315 320

Ile Ala Leu Met Asp Gln Ser Trp Trp Phe Pro Ala Val Ala Pro Leu

325 330 335

Pro Gly Ala Ala Pro Ala Val Met Leu Ala Glu Arg Ser Leu Pro Gly

340 345 350

Ser Phe Ile Val Asp His Asn Gly His Arg Phe Ala Asn Glu Ser Ala

355 360 365

Asp Tyr Met Ser Phe Gly Gln Arg Ile Leu Asp Leu Glu Asn Ala Gly

370 375 380

Thr Pro Val Asp Ser Met Trp Ile Val Phe Asp Gln Gln Tyr Arg Asn

385 390 395 400

Ser Tyr Val Phe Ala Ala Glu Leu Phe Pro Arg Met Pro Ile Pro Gln

405 410 415

Thr Trp Tyr Asp Ala Gly Ile Ala Val Lys Ala Asp Asn Phe Asp Glu

420 425 430

Leu Ala Thr Lys Met Gln Val Pro Val Asp Asn Phe Glu Ala Thr Val

435 440 445

Thr Arg Phe Asn Glu Asn Ala Phe Ala Gly Glu Asp Pro Asp Phe Glu

450 455 460

Arg Gly Arg Ser Ala Tyr Asp Arg Tyr Tyr Gly Asp Pro Thr Ile Thr

465 470 475 480

Pro Asn Pro Asn Leu Arg Pro Leu Val Lys Gly Pro Phe Tyr Ala Val

485 490 495

Lys Met Val Leu Ser Asp Leu Gly Thr Cys Gly Gly Leu Arg Ala Asp

500 505 510

Asp His Ala Arg Val Leu Arg Glu Asp Gly Thr Val Ile Asp Gly Leu

515 520 525

Tyr Ala Ile Gly Asn Thr Ala Ala Asn Ala Phe Gly Lys Thr Tyr Pro

530 535 540

Gly Ala Gly Ala Thr Ile Ala Gln Gly Leu Val Phe Gly Tyr Ile Ala

545 550 555 560

Ala Arg Asp Ala Ala Glu Ala

565

Claims

1. A mutant 3-sterone- Δ 1-dehydrogenase characterized by: the amino acid sequence is shown as SEQ ID NO. 4.

2. A mutant 3-sterone- Δ 1-dehydrogenase according to claim 1, wherein: the 3-ketosteroid-delta 1-dehydrogenase mutant is obtained by mutating the 320 th aspartic acid of 3-ketosteroid-delta 1-dehydrogenase of parent mycobacterium with the starting amino acid sequence shown as SEQ ID NO.2 into tyrosine.

3. A mutant 3-sterone- Δ 1-dehydrogenase according to claim 1 or 2, characterized in that: the gene of the 3-sterone-delta 1-dehydrogenase is derived from Mycobacterium sp LY-1CGMCC No. 13031.

4. A gene of a mutant 3-sterone- Δ 1-dehydrogenase characterized in that: the nucleotide sequence is any one of the following: a) 3, b) encodes a protein consisting of the amino acid sequence shown as SEQ ID No.4 or a nonsense-mutated sequence thereof.

5. A recombinant expression plasmid for encoding a 3-ketosteroid- Δ 1-dehydrogenase mutant of mycobacterium (Mcobacterium sp.) LY-1, wherein: contains the nucleotide sequence of claim 4 and pET32a as expression vector.

6. An engineered strain highly expressing 3-sterone- Δ 1-dehydrogenase, wherein the engineered strain incorporates the gene of the 3-sterone- Δ 1-dehydrogenase mutant according to claim 4, or comprises the recombinant expression plasmid according to claim 5.

7. The engineered strain of claim 6, for use in the preparation of C1, 2-dehydrosteroid compounds, said preparation of C1, 2-dehydrosteroid compounds is the dehydrogenation of C1, 2-position of steroid compounds, said steroid compounds including AD, T, progesterone.

8. The use of the engineered strain according to claim 7, wherein the steroid is used as a substrate at a concentration of 3-10 g/L.

9. The application of the engineering strain according to claim 7, wherein the engineering strain is transferred to 3-10 g/L steroid at 32-37 ℃ and 180-220 r-min^-1And (4) converting for 2-4 h to obtain the C1, 2-dehydrogenation steroid compound.

10. Use of an engineered strain according to any one of claims 7 to 9 to produce a C1, 2-dehydrosteroid.