CN106011158A - Method using microorganism method to convert androstenedione so as to produce testosterone - Google Patents

Method using microorganism method to convert androstenedione so as to produce testosterone Download PDF

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CN106011158A
CN106011158A CN201610575007.5A CN201610575007A CN106011158A CN 106011158 A CN106011158 A CN 106011158A CN 201610575007 A CN201610575007 A CN 201610575007A CN 106011158 A CN106011158 A CN 106011158A
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testosterone
androstenedione
enzyme
hsd3
conversion
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饶志明
邵明龙
张显
杨套伟
徐美娟
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Jiangnan University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01051—3 (or 17)-Beta-hydroxysteroid dehydrogenase (1.1.1.51)
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

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Abstract

The invention relates to a method recombining Yarrowia lipolytica to efficiently and biologically convert androstenedione so as to produce testosterone and belongs to the field of genetic engineering. The method is characterized in that 17beta-hydroxyl steroid dehydrogenase from human and glucose dehydrogenase from saccharomyces cerevisiae are respectively cloned and excessively co-expressed in Y.lipolytica Polh, and the androstenedione is efficiently converted in the Yarrowia lipolytica to produce the testosterone for the first time at home and abroad by building an NADPH coenzyme circulation system. The enzyme activity determination and intracellular coenzyme level researches of the built genetic-engineering strains show that the recombinant strains can continuously and effectively convert the androstenedione to produce the testosterone. When the conversion temperature is 37 DEG C, conversion pH is 7.5, substrate cosolvent is methylated-beta-cyclodextrin, biomass is 200g/L and substrate concentration is 5g/L, 15g/L androstenedione is converted into 14.3g/L testosterone after three material-supplementing whole-cell conversions, the currently-reported highest level of testosterone production using microorganism conversion is achieved, and a foundation is provided for industrial testosterone production using microorganisms.

Description

A kind of microbial method converts the method that ANDROSTENEDIONE produces testosterone
Technical field
A kind of bioconversion ANDROSTENEDIONE produces the method for testosterone, belongs to genetic engineering and enzyme engineering field.
Background technology
Steroid drugs can be obtained by complete synthesis or to the degraded of natural steroid compound and its functional group conversion, and steroid drugs has There are the pharmacological actions such as the strongest infection, Gang Guomin, antiviral and shock.Along with the development in epoch, steroid drugs is Through becoming the second largest class medicine being only second to antibiotic, within 2000, steroid drugs has broken through 200 in the sales volume of whole world pharmaceutical market Hundred million dollars, account for the 6% of world's medicine total sales volume.
Steroid hormone class classification of drug: adrenocortical hormone, including hydrocortisone, prednisone etc..A Disenshi can be treated Disease, antiinflammatory, antiallergic, antishock etc.；Protein anabolic hormone, its main Physiological Function is suppression albumen alienation and promotes egg White synthesis, is mainly used in treating the disease that protein increases and synthesis deficiency causes；Gonadal hormone, including estrogen, male swash Element and progestogen.
The raw material of synthesizing steroid medicine extracting directly from animal tissue's liquid mostly in early days, owing to the content of these raw materials is low, source Less, complex synthetic route, the response rate are low, of a high price, it is impossible to meet the needs of production.The discovery of diosgenin and application, Industrialized production for steroid drugs provides abundant and cheap raw material, but production process needs to consume substantial amounts of Rhizoma Dioscoreae soap Element, makes Saponin price constantly rise, and adds production cost, and production process serious environment pollution.
Testosterone is the androgen produced by interstitial cell, and testosterone mainly has following aspect effect: 1. maintain Spermatogenic action, testosterone After Interstitial cell is secreted, can be through supporting that cell enters convoluted seminiferous tubule, testosterone can directly or first be changed into the dihydrotestosterone that activity is higher, Be combined with the androgen receptor of spermatogenic cell, promote the generation of sperm.2. stimulate the growth promoter of genitals, promote that male is secondary Levy appearance and maintain its normal condition；3. normal libido is maintained；4. the egg of protein synthesis, particularly muscle and genitals is promoted White matter synthesizes, and can also promote bone growth and calcium phosphorus precipitation and erythropoiesis etc. simultaneously.
17beta-Hydroxysteroid dehydrogenase (17 β-hydroxysteroid dehydrogenase, 17 β-HSD) is in gonadal hormone building-up process The catalyzing enzyme of final step.Its catalytic swashs the reduction between the ketone group on C17 position and alcohol radical and oxidation reaction, makes low biological alive Mutually convert between the estradiol of estrone, androstenedione and high bioactivity of property, testosterone.Derive from human body testis 17 β-HSD3, it is possible to narrow spectrum catalysis ANDROSTENEDIONE (AD) synthesis testosterone (TS), it, as one detection enzyme, is applied to Medical treatment detection field.Meanwhile, have, at aspects such as food development, health care, clinical monitoring, biological pesticides, the valency that is widely used Value.17 β-HSD are furtherd investigate the development to whole steroid drugs industry of far-reaching significance.Produce the testosterone of more high added value The most effective method is exactly with ANDROSTENEDIONE as substrate, is converted by microorganism one step.
The present invention passes through plasmid pINA1292 and pYLSC, it is achieved that derive from people's and saccharomyces cerevisiae 17 β-hsd3 and gdh Gene solubility overexpression in Yarrowia lipolytica Polh, enzyme is alive respectively reaches 7.35U/mg and 4.23U/mg, 15g/L AD is converted and produces 14.3g/L TS by 5L fermentation tank current adding substrate resting cell AD, 120h.Derive from people's 17 β-HSD3 express in Yarrowia lipolytica system, and NADPH coenzyme circular regeneration system and resting cell AD are Reported first, and the enzyme conversion ratio alive and AD that recombinant bacterium is expressed is the highest.Yarrowia lipolytica is as the industry of safety and stability Producing bacterial strain, condition of culture is simple, and fermentation period is short, becomes microorganism sterol fermentation industry potential production bacterial strain.
Summary of the invention
It is an object of the invention to provide: obtained micro-life with resting cell production TS ability by genetic engineering means The method of thing.The recombinant bacterial strain built is carried out enzyme activity and fermenting property carries out research and finds 17 β-HSD3 and GDH enzyme activity All improve a lot, and obtained higher conversion ratio.For the guidance that the industrialization offer of fermentable production TS is useful.
Technical scheme: be to build a strain resting cell cholesterol with genetic engineering for means to generate the restructuring solution of TS Fat Ye Shi yeast strain, with HPLC for the generation of product in method detection fermentation liquid, screens positive recombinant, passes through enzyme activity Detection with fermenting property determines vigor and the substrate conversion efficiency of its target enzyme.The present invention successfully constructs a strain with AD as the end Thing resting cell is the Yarrowia lipolytica engineered strain of the high yield TS of method, its 17 β-HSD3 and GDH enzyme activity and the end Thing conversion ratio relatively starting strain improves a lot.
Main agents: AD and TS is purchased from SIGMA company of the U.S.
Recombinant bacterial strain construction method:
(1) 17 β-HSD3 and the clone of GDH gene complete sequence
The SD sequence (GI:21706851) of the 17 β-HSD3 of the people according to the announcement of GENBANK website and the GDH of saccharomyces cerevisiae Gene complete sequence, first pass around codon optimized after, respectively with on pINA1292 and pYLSC plasmid restriction enzyme site design 17 β-hsd3 and gdh gene primer.With the DNA of total gene synthesis as template, amplify 17 β-hsd3 and ZWF1 by PCR Complete sequence.
PCR reaction system: 10 × ExTaq Buffer 5 μ L, dNTP 4 μ L, template DNA 1 μ L, each 0.5 μ L of upstream and downstream primer, ExTaq enzyme 1 μ L, ddH2O polishing is to cumulative volume 50 μ L.PCR reaction condition: 94 DEG C of 5min, 94 DEG C of 30s, 65 DEG C 45s, 72 DEG C of 2min, circulate 35 times, 72 DEG C of 10min, 12 DEG C of 10min.
(2) structure of recombinant expression carrier pINA1292-17 β-hsd3 and pYLSC-gdh
Reclaim test kit description with reference to Shanghai JaRa company glue and reclaim PCR primer, glue reclaim product by a certain percentage with PMD18-T vector overnight connects, Transformed E .coli JM109 competent cell, uses amicillin resistance plate screening weight Group bacterium, recombiant plasmid discharges size respectively through BamH I/Nde I enzyme action and is about 2.7kb and 1.0kb and 2.7kb and 1.5kb Gene band, show construction of recombinant plasmid success, recombiant plasmid named pMD18-T-17 β-hsd3 and pMD18-T-gdh.
Extract plasmid pINA1292, pYLSC, pMD18-T-17 β-hsd3 of being stored in E.coli JM109 and PMD18-T-gdh, plasmid pINA1292 and pMD18-T-17 β-hsd3 and pYLSC and pMD18-T-gdh are respectively through BamH I/Not I and BstBI/XhoI double digestion, glue reclaims purification, T4DNA ligase overnight connects two fragments, will connect the most afterwards Thing thermal shock Transformed E .coli JM109 competent cell, uses kalamycin resistance plate screening positive transformant.Extract transformant Plasmid, recombiant plasmid discharges size respectively after BamH I/Not I and BstBI/XhoI double digestion respectively and is about 9.0kb and 1.0 The genetic fragment of kb and 3.6kb and 1.5kb, it was demonstrated that construction of recombinant plasmid success, recombiant plasmid named pINA1292-17 β-hsd3 And pYLSC-gdh.
(3) recombiant plasmid pINA1292-17 β-hsd3 and pYLSC-gdh electrotransformation convert bacterial strain Yarrowia lipolytica Polh
The recombiant plasmid pINA1292-17 β-hsd3 and pYLSC-gdh empirical tests successfully constructed is taken up in order of priority and turns with electrotransformation Change and express to type strain Y.lipolytica Polh.
(4) screening of recombinant bacterial strain Y.lipolytica Polh positive transformant；
Picking has the bacterium colony grown on YPD and hygromycin pressure flat, shake-flask culture, extracts chromosome and carries out PCR checking.
(5) restructuring Y.lipolytica Polh condition of culture, enzyme activity determination and HPLC analyze
Condition of culture: LB culture medium: cholesterol 1g/L, glucose 10g/L, peptone 10g/l, yeast extract 5g/L, NaCl 10g/L (solid medium adds 2% agar powder)
Flat board MD culture medium (g/L): yeast basic nitrogen source (YNB) 13.4, biotin 4 × 10-4, glucose 20, agar 15；
YPD culture medium (g/L): peptone 20, glucose 20, yeast powder 10, at YPD when being used for screening G418 resistance Solid medium adds the G418 of desired concn and i.e. obtains YPD/G418 flat board, at YPD solid when being used for screening hygromycin resistance Culture medium adds the hygromycin of desired concn and i.e. obtains YPD/HygB flat board；
Buffered Minimal Glycerol-complex Medium (BMGY) culture medium (g/L): YNB 13.4, glycerol 10, Biotin 4 × 10-4, constant volume uses 0.1mol/L kaliumphosphate buffer (pH 6.0)；
Buffered Minimal Methanol Medium (BMMY) culture medium (g/L): methanol 20, biotin 4 × 10-4, YNB 13.4, constant volume uses 0.1mol/L kaliumphosphate buffer (pH 6.0)；
Enzyme activity determination method: use HPLC method to measure.Concrete grammar is as follows: take cultivate 24h bacterium solution 50mL, 4 DEG C, 8,000r/min are centrifuged 5min collects thalline, with the Tris-HCl buffer solution twice of 50mL pH 7.0, is resuspended in 5ml This buffer in.In ice bath, 40% power ultrasonic crushes, work 2s interval 5s, working time 10min.10,000r/min Centrifugal 30min obtains supernatant, is destination protein in supernatant.Enzyme activity determination method is as follows: enzyme activity determination system 1mL Including 0.5mM NADPH, 200 μMs of AD (being dissolved in methanol), add appropriate enzyme liquid.Enzyme activity unit defines: 37 DEG C of temperature Under degree, 1min converts the enzyme amount of 1 μm ol AD generation TS and is defined as 1 enzyme unit (U) alive.G-6-P dehydrogenation Enzyme activity determination method: 1mL reaction system includes 50mM sodium phosphate buffer (pH 6.5), 10mM G-6-P With 1 μM of NADP+.According to the change detection vigor of NADPH light absorption value under 340nm, enzyme unit definition alive: per minute Produce the enzyme amount required for 1 μm ol NADPH.Glucose dehydrogenase vigour-testing method: 1mL reaction system includes 50mM Sodium phosphate buffer (pH 6.5), 10mM glucose and 1 μM of NADP+.According to NADPH light absorption value under 340nm Change detection vigor, enzyme unit definition alive: the enzyme amount required for generation 1 μm ol NADPH per minute.
HPLC analyzes: AD and TS all has characteristic absorption peak under 240nm ultraviolet wavelength, so using HPLC method to measure Production concentration.Chromatographic condition: chromatographic column: DimosoilC18(5 μ l, 250mm × 4.6mm), flow phase: acetonitrile-water (V/V=85:15), Detector: UV Detector, detects wavelength: 210nm, column temperature: 30 DEG C, sample size: 10 μ L, flow velocity: 1.0ml/min.
Beneficial effects of the present invention: expand 17 complete synthesis β-HSD3 and GDH gene by genetic engineering means, by this reality Test the Yarrowia lipolytica expression system of the existing maturation in room, it is achieved that 17 β-HSD3 and G6PDH gene are at type strain Y. Overexpression in lipolytica Polh.This bacterial strain is inoculated in 100mL BMGY culture medium with 5% inoculum concentration, cultivates 24h, is then inoculated in the 5L fermentation tank equipped with 2L BMMY culture medium with 5% inoculum concentration, persistently adds methanol and lures Lead 4 days.Bacteria recovered by centrifugation, washes twice, and redissolves with Tris-HCl pH 7.5 buffer of a certain amount of 50mM, adds Substrate A D converts, and conversion condition is: conversion temperature is 37 DEG C, convert pH be 7.5, substrate cosolvent for the-β that methylates- Cyclodextrin (molar ratio is 1:1), Biomass is 200g/L, and concentration of substrate is 5g/L.After 3 feed supplement resting cells, 15g/L ANDROSTENEDIONE is converted into 14.3g/L testosterone, builds NADPH coenzyme indirect regeneration for domestic and international first passage ANDROSTENEDIONE production of testosterone by conversion is utilized, the final purpose realizing improving conversion rate, efficiently production testosterone in Pichia sp..
Substrate A D mono-step changes into the TS with more high additive value, and it has important physiology and medicine effect, is simultaneously Important medicine intermediate；Producing TS with microbial method, it has reaction condition gentleness, raw material availability than chemical production method The advantage, the most beneficially environmental conservation such as high, product purity high and technique is simple, easily controllable, it is easy to popularization and application.
Accompanying drawing explanation
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Detailed description of the invention
Embodiment 1: the structure of restructuring Y.lipolytica Polh bacterial strain
With the complete synthesis gene of laboratory as template, utilize PCR means to obtain the gene of this enzyme, connect cloning vehicle, it is achieved base A large amount of amplifications of cause.By the 17 β-hsd3 expanded in a large number and gdh genetic fragment, be connected respectively to after purification pINA1292 and On pYLSC carrier, after being proved to be successful, transformation mode bacterial strain Y.lipolytica Polh.Resistant panel is screened positive transformant, Inoculation shake flask fermentation, with product TS in HPLC detection fermentation liquid.Product TS detected, illustrate to have successfully constructed have turn Changing AD is the recombinant bacterial strain of TS.The present invention finally gives has the Y.lipolytica Polh bacterial strain that Efficient Conversion AD is TS.
Embodiment 2: the enzyme activity determination of recombinant bacterial strain
Enzyme activity determination method: use HPLC method to measure.Concrete grammar is as follows: take cultivate 24h bacterium solution 50mL, 4 DEG C, 8,000r/min are centrifuged 5min collects thalline, with the Tris-HCl buffer solution twice of 50mL pH 7.0, is resuspended in 5ml This buffer in.In ice bath, 40% power ultrasonic crushes, work 2s interval 5s, working time 10min.10,000r/min Centrifugal 30min obtains supernatant, is destination protein in supernatant.Enzyme activity determination method is as follows: enzyme activity determination system 1mL Including 0.5mM NADPH, 200 μMs of AD (being dissolved in methanol), add appropriate enzyme liquid.Enzyme activity unit defines: 37 DEG C of temperature Under degree, 1min converts the enzyme amount of 1 μm ol AD generation TS and is defined as 1 enzyme unit (U) alive.Glucose dehydrogenase vigor Assay method: 1mL reaction system includes 50mM sodium phosphate buffer (pH 6.5), 10mM glucose and 1 μM of NADP+。 According to the change detection vigor of NADPH light absorption value under 340nm, enzyme unit definition alive: generation 1 μm ol NADPH per minute Required enzyme amount.Enzyme is lived and is respectively reached 7.35U/mg and 4.23U/mg.
Embodiment 3: recombinant bacterial strain resting cell performance detects
This bacterial strain is inoculated in 100mL BMGY culture medium with 5% inoculum concentration, cultivates 24h, then connect with 5% inoculum concentration Plant in the 5L fermentation tank equipped with 2L BMMY culture medium, persistently add methanol and carry out inducing 4 days.Bacteria recovered by centrifugation, Wash twice, redissolve with Tris-HCl pH 7.5 buffer of a certain amount of 50mM, add substrate A D and convert, convert Condition is: conversion temperature is 37 DEG C, and converting pH is 7.5, and substrate cosolvent is methyl-β-cyclodextrin (molar ratio is 1:1), Biomass is 200g/L, and concentration of substrate is 5g/L.After 3 feed supplement resting cells, 15g/L ANDROSTENEDIONE converts For 14.3g/L testosterone, build NADPH coenzyme indirect regeneration for domestic and international first passage in Pichia sp., utilize androstane Alkene diketone production of testosterone by conversion, the final purpose realizing improving conversion rate, efficiently production testosterone.

Claims (5)

1. the 17beta-Hydroxysteroid dehydrogenase gene that a codon optimizes, it is characterised in that its nucleotide sequence such as SEQ ID Shown in NO.1.
2. a recombiant plasmid pINA1292-17 β-hsd3, it is characterised in that 17 beta-hydroxysteroids described in claim 1 are taken off Hydrogenase gene is connected acquisition with carrier pINA1292.
3. the structure of a transformation system, it is characterised in that NADP can be catalyzed+The enzyme such as glucose dehydrogenase of synthesis NADPH GDH is connected the recombiant plasmid pINA1292-17 β-hsd3-GDH that acquisition is new with the recombiant plasmid described in claim 2, and will weight Group plasmid pINA1292-17 β-hsd3-GDH coexpression in Yarrowia lipolytica Polh obtains.
4. the application of transformation system described in claim 3, it is characterised in that be applied to convert ANDROSTENEDIONE synthesis testosterone.
5. can be catalyzed NADP described in claim 3+The enzyme of synthesis NADPH, it is characterised in that preferably, but be not limited to select Glucose dehydrogenase.
CN201610575007.5A 2016-07-20 2016-07-20 Method using microorganism method to convert androstenedione so as to produce testosterone Pending CN106011158A (en)

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CN109306342A (en) * 2017-07-26 2019-02-05 中国科学院天津工业生物技术研究所 A kind of new 17 beta-hydroxy steroid dehydrogenase types, its gene and application
CN111471737A (en) * 2020-04-26 2020-07-31 江南大学 Method for preparing sterol derivative by virtue of Burkholderia transformation and application

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108796021A (en) * 2017-04-28 2018-11-13 沈阳药科大学 A kind of engineering bacteria and its purposes for being used to prepare stosterone
CN109306342A (en) * 2017-07-26 2019-02-05 中国科学院天津工业生物技术研究所 A kind of new 17 beta-hydroxy steroid dehydrogenase types, its gene and application
CN109306342B (en) * 2017-07-26 2021-12-10 中国科学院天津工业生物技术研究所 17 beta-hydroxy steroid dehydrogenase, gene and application thereof
CN111471737A (en) * 2020-04-26 2020-07-31 江南大学 Method for preparing sterol derivative by virtue of Burkholderia transformation and application

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