WO2018028006A1 - 一种新金色分枝杆菌来源的甾酮c27-单加氧酶及其应用 - Google Patents

一种新金色分枝杆菌来源的甾酮c27-单加氧酶及其应用 Download PDF

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WO2018028006A1
WO2018028006A1 PCT/CN2016/097110 CN2016097110W WO2018028006A1 WO 2018028006 A1 WO2018028006 A1 WO 2018028006A1 CN 2016097110 W CN2016097110 W CN 2016097110W WO 2018028006 A1 WO2018028006 A1 WO 2018028006A1
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recombinant
monooxygenase gene
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monooxygenase
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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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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
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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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P33/02—Dehydrogenating; Dehydroxylating
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P33/12—Acting on D ring
    • C12P33/16—Acting at 17 position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13072—Methylsterol monooxygenase (1.14.13.72)

Definitions

  • the invention relates to a new mycobacterium breve-derived fluorenone C27-monooxygenase and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering.
  • Steroid hormone drugs play a very important role in regulating the body due to their various physiological functions, so they have a wide range of clinical applications. Steroid hormone drugs are widely used for anti-tumor, anti-inflammatory, anti-bacterial, anti-viral, anti-hormone and anti-allergy. In addition, various sex hormone drugs are the main drugs for the treatment of sexual organ degeneration and gynecological diseases, and are the main components of oral contraceptives. At the same time, steroid hormone drugs can also be used as an active ingredient in anti-obesity drugs for the prevention of coronary heart disease and as an inhibitor of HIV integrase, prevention of HIV infection and treatment for AIDS.
  • Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are important intermediates for steroid hormones and can be synthesized almost exclusively.
  • Steroid hormone drugs play an important role in the industrial production of carcass.
  • AD and ADD there are two main ways to prepare AD and ADD.
  • One is to extract diosgenin from wild Chinese herbal medicines such as turmeric and diatom, and then chemically synthesize it.
  • this method has problems of high production cost, complicated process and serious environmental pollution.
  • the animal and plant sterol which are abundant in nature, are used as raw materials, and the sterol side chain is selectively degraded by microbial fermentation technology to obtain the target product.
  • the microbial method has the advantages of low cost and mild environment, and has been paid more and more attention.
  • the preparation of steroids is currently carried out, and most of the animal and plant sterols are used as starting materials for microbial transformation, and steroidal intermediates are obtained, and further preparation is carried out.
  • the present invention screens for the first time in the process of degradation of the sterol side chain during the degradation of sterol side chain by the method of gene knockout and replenishment, three of the same enzymes, the ketone ketone C27-monooxygenase (SMO). Enzyme (SMO1, SMO2, SMO3). These three isozymes were enhanced in the high-yielding maleo-1,4-diene-3,17-dione (ADD) of New Mycobacterium, respectively. The yield of ADD was significantly improved, and the enhanced expression of SMO2 was more effective in increasing the yield of ADD.
  • SMO ketone ketone C27-monooxygenase
  • a first object of the present invention is to provide an indolone C27-monooxygenase having an amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 is shown.
  • a second object of the present invention is a recombinant M. aeruginosa strain having an increased ADD yield, which is a novel M. aeruginosa which overexpresses an indolinone C27-monooxygenase gene;
  • the monooxygenase gene is any one or two or more of the genes Smo1, Smo2, and Smo3.
  • amino acid sequence of the indolone C27-monooxygenase gene Smo1 is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO.
  • amino acid sequence of the indolone C27-monooxygenase gene Smo2 is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO.
  • amino acid sequence of the indolone C27-monooxygenase gene Smo3 is shown in SEQ ID NO. 6, and the nucleotide sequence is shown in SEQ ID NO.
  • the New Mycobacterium tuberculosis recombinant strain overexpresses the indolinone C27-monooxygenase gene with a strain of Mycobacterium tuberculosis.
  • the starting bacteria may be any one of the following: Mycobacterium neoaurum ATCC 25795, Mycobacterium neoaurum ZJUVN (CGMCC 5477), Mycobacterium neoaurum NwIB-01 (CCTCC M 209094), Gordonia neofelifaecis NRRL B- 59395 (CCTCCAB-209144), Arthrobacter simplex (TCCC 11037), Mycobacterium fortuitum subsp. fortuitum (MTCC 929) or Mycobacterium neoaurum JC-12.
  • Mycobacterium neoaurum ATCC 25795 has been in the literature Yao K, Xu LQ, Wang FQ, Wei DZ (2014) Characterization and engineering of 3-ketosteroid- ⁇ 1-dehydrogenase and 3-ketosteroid-9 ⁇ -hydroxylase in Mycobacterium neoaurum ATCC 25795to produce9 ⁇ -hydroxy -4-androstene-3, 17-dione through the catabolism of sterols. Metab Eng 24(0): 181-191 doi: http://dx.doi.org/10.1016/j.ymben.2014.05.005 is disclosed.
  • Mycobacterium neoaurum ZJUVN (CGMCC 5477) is already in the literature Zhang Xy, Peng Y, Su Zr, Chen Qh, Ruan H, He Gq (2013) Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08.Journal of Zhejiang University SCIENCE B 14(2): 132-143 doi: 10.1631/jzus. B1200067.
  • Mycobacterium neoaurum NwIB-01 (CCTCC M 209094) is already in the literature Wei W, Fan SY, Wang FQ, Wei DZ (2014) Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid ⁇ 1-dehydrogenase in Mycobacterium neoaurum NwIB-01.World J Microbiol Biotechnol 30(7): 1947-1954 doi: 10.1007/s11274-014-1614-3 is disclosed.
  • Arthrobacter simplex (TCCC 11037) is already in the literature Wang M, Zhang LT, Shen YB, Ma YH, Zheng Y, Luo JM (2009) Effects of hydroxypropyl- ⁇ -cyclodextrin on steroids 1-en-dehydrogenation biotransformation by Arthrobacter simplex TCCC 11037. J Mol Catal, B Enzym 59(1–3): 58-63 doi: http://dx.doi.org/10.1016/j.molcatb. 2008.12.017 is disclosed.
  • Mycobacterium fortuitum subsp.fortuitum (MTCC 929) is already in the literature GullaV, Banerjee T, Patil S (2010) Bioconversion of soysterols to androstenedione by Mycobacterium fortuitum subsp. fortuitum NCIM 5239, a mutant derived from total sterol degrader strain. J Chem Technol Biotechnol 85 (8): 1135-1141 doi: 10.1002/jctb.2410 is disclosed.
  • Mycobacterium neoaurum JC-12 is already in the literature Shao ML, Zhang X, Rao ZM, Xu MJ, Yang TW, Li H, Xu ZH (2015) Enhanced production of androst-1,4-diene-3, 17-dione by Mycobacterium neoaurum JC-12 using three-stage fermentation strategy.
  • PLoS ONE 10(9): e0137658 is disclosed.
  • the overexpression specifically, the fluorenone C27-monooxygenase gene is ligated to the plasmid pMV261 to obtain a recombinant plasmid, and then the recombinant plasmid is transformed into the starting bacterium.
  • a third object of the present invention is to provide a method for increasing the yield of ADD by overexpressing an indolinone C27-monooxygenase gene by using the novel Mycobacterium tuberculosis recombinant strain of the present invention as a production strain
  • the ADD is produced by fermentation; the recombinant strain overexpresses the indolinone C27-monooxygenase gene; and the anthrone C27-monooxygenase gene is any one or more of the genes Smo1, Smo2, and Smo3.
  • amino acid sequence of the indolone C27-monooxygenase gene is set forth in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO.
  • the fermentation production is based on phytosterols or/and cholesterol.
  • the fermentation medium component for fermentation cholesterol 20 g/L, glucose 20 g/L, peptone 10 g/L, beef extract 6 g/L, K 2 HPO 4 3 g/L, MgSO 4 7H 2 O 0.5 g/L, MnCl 2 ⁇ 4H 2 O 5 ⁇ 10 -4 g/L, hydroxypropyl- ⁇ -cyclodextrin 60 g/L, pH 7.5.
  • the fermentation fermentation production is fermentation for 168 h at 30 ° C, 160 rpm.
  • a fourth object of the present invention is to provide an ADD produced by the recombinant bacteria.
  • a fifth object of the present invention is to provide the use of the recombinant strain or the anthrone C27-monooxygenase gene for the preparation of a medicament.
  • the drug refers to a drug that is anti-tumor, anti-inflammatory, anti-bacterial, anti-viral, anti-hormonal or anti-allergic.
  • the drug refers to a therapeutic organ degenerative drug, a gynecological disease drug, an anti-obesity drug, a drug for preventing coronary heart disease, or an inhibitor of HIV integrase.
  • the present invention screens three isozyme genes of anthrone C27-monooxygenase in Mycobacterium marinum by genetic engineering means, and identifies anthrone by gene knockout and knockout gene replenishment.
  • SMO C27-monooxygenase
  • the present invention demonstrates the function of these three isoenzymes in the metabolism of sterols, and determines its key role in the conversion of sterols, and verifies that it is a key enzyme in the process of synthesis of AD/ADD by mycobacteria to sterols.
  • the present invention is applied in the process of converting sterol to ADD to increase the yield of ADD, and it is found that SMO2 has the most obvious effect on increasing ADD yield. Eventual overexpression of SMO2 increased the yield of ADD from 5.2 g/L to 7.3 g/L, which was 40.4% higher than the original bacteria.
  • the present invention provides useful guidance for the industrialization of ADD production by microbial fermentation.
  • Phytosterol (soy sterol ⁇ 95%) was purchased from Lilly Biotechnology (Huzhou) Co., Ltd., and ADD was purchased from SIGMA, USA.
  • HPLC analysis of ADD ADD has characteristic absorption peaks at 254 nm ultraviolet wavelength, so the product concentration was determined by HPLC.
  • Example 1 Construction of SMO knockout strains and corresponding replenishing strains
  • Three SMO isozymes were screened by querying the genome-wide information of New Mycobacterium.
  • the new strain of M. aeruginosa with SMO enzyme activity in the laboratory was used as the starting strain, and the genes of these three enzymes were obtained by PCR using the chromosome as a template.
  • the knockout gene was obtained by PCR, and the mycobacterial knockout plasmid p2NIL was ligated to construct a knockout plasmid, which was successfully transformed by PCR and transformed into New Mycobacterium.
  • cholest-4-en-3-one as the base , detecting the degradation of the substrate by the knockout strain.
  • a knockout gene replenishing strain was constructed.
  • the complete SMO gene was amplified by PCR, and the integration vector pMV306 was ligated to construct an integrated plasmid. After successful construction by PCR, the corresponding transformation was carried out.
  • a knockout gene replenishing strain was constructed. Under the same conditions, the degradation of the substrate by the supplemented strain was detected by using cholest-4-en-3-one as a substrate. The results showed that the ability of the knockout strain to degrade the cholest-4-en-3-one was hindered, and the knockout gene replenishing strain reduced the hindrance to some extent.
  • the present invention finally demonstrates that the three enzymes of SMO are key enzymes in the degradation of sterols.
  • PCR reaction system 10 ⁇ ExTaq Buffer 5 ⁇ L, dNTP 4 ⁇ L, template DNA 1 ⁇ L, 0.5 ⁇ L of each of the upstream and downstream primers, 1 ⁇ L of ExTaq enzyme, and ddH 2 O were added to a total volume of 50 ⁇ L.
  • PCR reaction conditions 94 ° C for 5 min, 94 ° C for 30 s, 65 ° C for 45 s, 72 ° C for 90 s, 35 cycles, 72 ° C for 10 min, 12 ° C for 10 min.
  • the PCR product was recovered according to the instructions of Shanghai Jierui Company's gel recovery kit.
  • the recovered product of the gel was connected with pMD18-T vector overnight, transformed into E.coli JM109 competent cells, and the recombinant plasmid was screened using ampicillin resistant plates.
  • the gene bands of about 2.7 kb and 1.3 kb were cleavage, indicating that the recombinant plasmid was successfully constructed.
  • the recombinant plasmids were named pMD18-T-Smo1, pMD18-T-Smo2 and pMD18-T-Smo3.
  • a specific primer was designed to amplify a 300 bp sequence upstream from Smo1, Smo2 and Smo3 by PCR, and a 450 bp sequence was amplified downstream.
  • the upstream and downstream amplified gene sequences were mixed as a template, and a 750 bp sequence was amplified using an upstream primer of the upstream 300 bp sequence and a downstream primer of the 450 bp sequence.
  • the amplified sequence and the knockout plasmid p2NIL were digested with Kpn I and Hind III, and recovered by gel recovery. T 4 DNA ligase was ligated overnight to connect the two fragments. After overnight, the conjugate was transformed into E. coli JM109 competent cells by heat shock.
  • Positive transformants were screened using kanamycin resistant plates.
  • the transformant plasmid was extracted, and the recombinant plasmid was successfully constructed by enzyme digestion.
  • the selectable marker gene cassette in plasmid pGOAL19 was excised by Pac I and inserted into the Pac I restriction site of the constructed recombinant plasmid to construct knockout plasmids p2N- ⁇ Smo1, p2N- ⁇ Smo2 and p2N- ⁇ Smo3.
  • the knockout plasmid was successfully constructed by PCR.
  • Plasmid pMV306 was used to knock out the gene's complement.
  • the corresponding gene fragments in the recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 were digested with the heat shock promoter hsp60 and ligated into the phase by double digestion with Xba I/Cla I.
  • Knockout gene replenishment plasmids p306-Smo1, p306-Smo2 and p306-Smo3 were constructed at the pMV306 restriction site.
  • A will construct a successful recombinant plasmid for transformation into New Mycobacterium phlei by electroporation.
  • the conversion method uses the improved Gordhan and Parish method.
  • Plasmid pMV261 was used for overexpression of genes in New Mycobacterium.
  • pMD18-T-Smo1 was digested with Sac I and Hind III
  • pMD18-T-Smo2 and pMD18-T-Smo3 were digested with BamH I and EcoR I, respectively
  • plasmid pMV261 was digested with the corresponding restriction enzyme site.
  • the corresponding gene fragment and the plasmid pMV261 fragment were purified by gel extraction, and T 4 DNA ligase was ligated overnight to link the two fragments. After overnight, the conjugate was heat-shocked into E. coli JM109 competent cells, and positive transformants were screened using kanamycin resistance plates. .
  • the transformant plasmid was extracted, and the recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 were successfully constructed by restriction enzyme digestion.
  • the recombinant plasmids were successfully constructed and transformed into the high-yield ADD of Mycobacterium neocorum.
  • JC-12 is already in the literature Shao ML, Zhang X, Rao ZM, Xu MJ, Yang TW, Li H, Xu ZH (2015) Enhanced production of androst-1,4-diene-3, 17-dione by Mycobacterium neoaurum JC- In the 12using three-stage fermentation strategy.
  • Example 3 SMO enhances expression of recombinant strains to increase ADD yield
  • the fermentation medium was added according to the inoculation amount of 5%, and 20 g/L of cholesterol was used as a substrate at 30 ° C. Fermentation transformation experiment was carried out by fermentation at 160 rpm for 168 hours.
  • the seed culture medium glucose 10g / L, peptone 10g / l, beef extract 6g / L, NaCl 10g / L, pH 7.5; fermentation medium: cholesterol 20g / L, glucose 20g / L, peptone 10g / L, beef Paste 6g/L, K 2 HPO 4 3g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, MnCl 2 ⁇ 4H 2 O 5 ⁇ 10 -4 g/L, hydroxypropyl- ⁇ -cyclodextrin 60g/ L, pH 7.5.
  • the change in yield of ADD was examined.
  • the results showed that the yield of ADD of the recombinant strain JC-12 S2 overexpressing SMO2 was the most obvious after fermentation for 168 h, and increased from 5.2 g/L of the original strain Mycobacterium tuberculosis JC-12 to 7.3 g/L.
  • the yields of ADD of the recombinant bacteria JC-12 S1 and JC-12 S3 were increased to 6.5 g/L and 6.1 g/L, respectively.

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Abstract

提供了一种新金色分枝杆菌，其来源的甾酮C27单加氧酶，及其应用。在高产雄甾二烯二酮（ADD）的新金色分枝杆菌中分别增加SMO同工酶SMO1、SMO2和/或SMO3的表达，可以明显增加ADD的终产量。

Description

一种新金色分枝杆菌来源的甾酮C27-单加氧酶及其应用 技术领域
本发明涉及一种新金色分枝杆菌来源的甾酮C27-单加氧酶及其应用，属于基因工程和酶工程技术领域。
背景技术
甾体激素类药物因其多种生理功能，对机体起着非常重要的调节作用，故在临床上具有广泛的应用。甾体激素类药物被广泛用于抗肿瘤、抗炎症、抗菌、抗病毒、抗激素和抗过敏等。此外，各类性激素类药物是治疗性器官退化和妇科疾病的主要药物，是口服避孕药的主要成分。同时甾体激素类药物还可以作为抗肥胖药物的活性成分，用于预防冠心病以及作为HIV整合酶的抑制剂，预防HIV的传染和用于艾滋病的治疗等。雄甾-4-烯-3,17-二酮(AD)和雄甾-1,4-二烯-3,17-二酮(ADD)作为甾体激素类药物的重要中间体，几乎可以合成所有的甾体激素类药物，在甾体的工业化生产中占有重要的地位。
目前制备AD和ADD主要有两种途径：一是从野生中草药黄姜、穿地龙等植物中提取薯蓣皂苷元，然后进行化学合成。但该途径存在生产成本高、过程复杂、环境污染严重等问题；二是以自然界中丰富存在的动植物甾醇为原料，通过微生物发酵技术对甾醇侧链进行选择性降解而得到目的产物。微生物法具有成本低、对环境温和等优点，越来越受到人们的重视。世界先进国家目前进行甾体药物制备，大多以动植物甾醇为起始原料进行微生物转化，得到甾体药物中间体后，再进一步制备。
众多学者对微生物转化甾醇合成AD/ADD进行了大量的研究，其中分枝杆菌是生产AD/ADD的优良菌株之一。已有报道微生物转化甾醇合成AD/ADD的代谢途径需要很多的酶，但是具体的是那些酶参与还没有完全研究明白。目前研究的进展是仅仅鉴定和证明了甾醇转化的第一步酶胆固醇氧化酶和最后一步酶3-甾酮-Δ1-脱氢酶。其他途径中的酶还未鉴定和应用。
因此，有必要对分枝杆菌转化甾醇合成AD/ADD过程中的关键酶及其功能鉴定进行深入研究，以期在此基础上开发可促进分枝杆菌转化甾醇合成AD/ADD的方法。
发明内容
为了解决上述问题，本发明通过基因敲除及回补的方法，首次在新金色分枝杆菌中筛选到甾醇边链降解过程中关键酶甾酮C27-单加氧酶(SMO)的三个同工酶(SMO1，SMO2，SMO3)。分别将这三个同工酶在高产雄甾-1,4-二烯-3,17-二酮(ADD)的新金色分枝杆菌中加强表达， ADD产量都得到明显提高，其中SMO2的加强表达对提高ADD的产量效果更明显。新金色分枝杆菌中甾酮C27-单加氧酶的功能鉴定和应用均为首次报道，且重组菌对提高ADD的产量较明显。加强表达SMO2基因的菌株的ADD最终产量从5.2g/L提高到7.3g/L。
本发明的第一个目的是提供一种甾酮C27-单加氧酶，所述甾酮C27-单加氧酶的氨基酸序列如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示。
本发明的第二个目的一种ADD产量提高的新金色分枝杆菌重组菌，所述重组菌是过表达了甾酮C27-单加氧酶基因的新金色分枝杆菌；所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
在本发明的一种实施方式中，所述甾酮C27-单加氧酶基因Smo1的氨基酸序列如SEQ ID NO.4所示，核苷酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，所述甾酮C27-单加氧酶基因Smo2的氨基酸序列如SEQ ID NO.5所示，核苷酸序列如SEQ ID NO.2所示。
在本发明的一种实施方式中，所述甾酮C27-单加氧酶基因Smo3的氨基酸序列如SEQ ID NO.6所示，核苷酸序列如SEQ ID NO.3所示。
在本发明的一种实施方式中，所述新金色分枝杆菌重组菌是以新金色分枝杆菌出发菌过表达了甾酮C27-单加氧酶基因。
在本发明的一种实施方式中，所述出发菌可以是以下任意一种：Mycobacterium neoaurum ATCC 25795、Mycobacterium neoaurum ZJUVN(CGMCC 5477)、Mycobacterium neoaurum NwIB-01(CCTCC M 209094)、Gordonia neofelifaecis NRRL B-59395(CCTCCAB-209144)、Arthrobacter simplex(TCCC 11037)、Mycobacterium fortuitum subsp.fortuitum(MTCC 929)或Mycobacterium neoaurum JC-12。
其中，Mycobacterium neoaurum ATCC 25795已经在文献Yao K,Xu L-Q,Wang F-Q,Wei D-Z(2014)Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795to produce9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols.Metab Eng 24(0):181-191doi:http://dx.doi.org/10.1016/j.ymben.2014.05.005中公开了。
Mycobacterium neoaurum ZJUVN(CGMCC 5477)已经在文献Zhang X-y,Peng Y,Su Z-r,Chen Q-h,Ruan H,He G-q(2013)Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08.Journal of Zhejiang University SCIENCE B 14(2):132-143doi:10.1631/jzus.B1200067中公开了。
Mycobacterium neoaurum NwIB-01(CCTCC M 209094)已经在文献Wei W,Fan S-Y,Wang F-Q,Wei D-Z(2014)Accumulation of androstadiene-dione by overexpression of heterologous3-ketosteroidΔ1-dehydrogenase in Mycobacterium neoaurum NwIB-01.World J Microbiol Biotechnol 30(7):1947-1954doi:10.1007/s11274-014-1614-3中公开了。
Gordonia neofelifaecis NRRL B-59395(CCTCC AB-209144)已经在文献Liu Y,Chen G,Ge F,Li W,Zeng L,Cao W(2011)Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete Gordonia neofelifaecis.World J Microbiol Biotechnol 27(4):759-765doi:10.1007/s11274-010-0513-5中公开了。
Arthrobacter simplex(TCCC 11037)已经在文献Wang M,Zhang LT,Shen YB,Ma YH,Zheng Y,Luo JM(2009)Effects of hydroxypropyl-β-cyclodextrin on steroids 1-en-dehydrogenation biotransformation by Arthrobacter simplex TCCC 11037.J Mol Catal,B Enzym 59(1–3):58-63doi:http://dx.doi.org/10.1016/j.molcatb.2008.12.017中公开了。
Mycobacterium fortuitum subsp.fortuitum(MTCC 929)已经在文献GullaV,Banerjee T,Patil S(2010)Bioconversion of soysterols to androstenedione by Mycobacterium fortuitum subsp.fortuitum NCIM 5239,a mutant derived from total sterol degrader strain.J Chem Technol Biotechnol 85(8):1135-1141doi:10.1002/jctb.2410中公开了。
Mycobacterium neoaurum JC-12已经在文献Shao ML,Zhang X,Rao ZM,Xu MJ,Yang TW,Li H,Xu ZH(2015)Enhanced production of androst-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12using three-stage fermentation strategy.PLoS ONE 10(9):e0137658中公开了。
在本发明的一种实施方式中，所述过表达，具体是将甾酮C27-单加氧酶基因连接到质粒pMV261上得到重组质粒，然后将重组质粒转化到出发菌中。
本发明的第三个目的是提供一种通过过表达甾酮C27-单加氧酶基因提高ADD产量的方法，所述方法是利用本发明的所述的新金色分枝杆菌重组菌为生产菌株发酵生产ADD；所述重组菌过表达甾酮C27-单加氧酶基因；所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
在本发明的一种实施方式中，所述甾酮C27-单加氧酶基因的氨基酸序列如SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.6所示。
在本发明的一种实施方式中，所述发酵生产是以植物甾醇或/和胆固醇为底物。
在本发明的一种实施方式中，用于发酵的发酵培养基成分：胆固醇20g/L，葡萄糖20g/L，蛋白胨10g/L，牛肉膏6g/L，K2HPO4 3g/L，MgSO4·7H2O 0.5g/L，MnCl2·4H2O 5×10-4 g/L，羟丙基-β-环糊精60g/L，pH 7.5。
在本发明的一种实施方式中，所述发酵发酵生产是在30℃，160rpm条件下发酵168h。
本发明的第四个目的是提供所述重组菌生产得到的ADD。
本发明的第五个目的是提供所述重组菌或者所述甾酮C27-单加氧酶基因在制备药物方面的应用。
在本发明的一种实施方式中，所述药物是指抗肿瘤、抗炎症、抗菌、抗病毒、抗激素或者抗过敏的药物。
在本发明的一种实施方式中，所述药物是指治疗性器官退化药物、妇科疾病药物、抗肥胖药物、预防冠心病的药物或者HIV整合酶的抑制剂等。
本发明的有益效果：
(1)本发明通过基因工程手段筛选到新金色分枝杆菌中甾酮C27-单加氧酶的3个同工酶基因，借助基因敲除和敲除基因回补的方法，鉴定了甾酮C27-单加氧酶(SMO)甾醇转化过程中的第二步酶。本发明证明了这三个同工酶在甾醇代谢过程中的功能，确定了其在甾醇转化过程中的关键作用，验证了其是分枝杆菌转化甾醇合成AD/ADD过程中的关键酶。
(2)本发明在确定关键酶的基础上，对其在甾醇转化合成ADD的过程中进行应用以期提高ADD的产量，发现SMO2对提高ADD产量效果最为明显。最终过量表达SMO2使得ADD的产量从5.2g/L提高到7.3g/L，比原始菌提高了40.4％。本发明为微生物发酵法提高ADD产量的工业化提供了有益的指导。
具体实施方式
主要试剂：植物甾醇(大豆甾醇≥95％)购自礼来生物技术(湖州)有限公司，ADD购自美国SIGMA公司。
ADD的HPLC分析：ADD在254nm紫外波长下均有特征吸收峰，所以采用HPLC法测定产物浓度。色谱条件：色谱柱：DimosoilC18(5μl，250mm×4.6mm)，流动相：甲醇-水(V/V＝70:30)，检测器：UV Detector，检测波长：254nm，柱温：30℃，进样量：10μL，流速：1.0ml/min。
实施例1：SMO敲除菌株及相应回补菌株的构建
通过查询新金色分枝杆菌的全基因组信息，筛选到3个SMO同工酶。以实验室具有SMO酶活力的新金色分枝杆菌作为出发菌株，以其染色体为模板，利用PCR手段获得这3个酶的基因。通过基因敲除引物的设计，利用PCR手段获得敲除基因，连接分枝杆菌敲除质粒p2NIL，构建敲除质粒，经PCR验证构建成功后，转化新金色分枝杆菌中。以胆甾-4-烯-3-酮作为底 物，检测敲除菌株对底物的降解情况。在敲除菌株的基础上，构建敲除基因回补菌株，通过设计引物，利用PCR手段扩增出完整的SMO基因，连接整合载体pMV306构建整合型质粒，经PCR验证构建成功后，转化相应的敲除菌株中，构建敲除基因回补菌株，相同条件下，以胆甾-4-烯-3-酮作为底物，检测回补菌株对底物的降解情况。检测结果表明基因敲除菌株对胆甾-4-烯-3-酮的降解能力受到阻碍，而敲除基因回补菌株在一定程度上减少了这种阻碍作用。本发明最终证明SMO这三个酶在甾醇降解过程中是关键酶。
重组菌株的具体构建方法如下：
(1)甾酮C27-单加氧酶(SMO)基因全序列的克隆
根据GENBANK网站公布的Mycobacterium neoaurum VKM Ac-1815D的全基因组序列(NC-023036)查找到3个SMO基因(Smo1，Smo2，Smo3)，根据具体的基因序列设计相应的基因引物。以制备的新金色分枝杆菌染色体DNA为模板，通过PCR扩增出相应的基因全序列。
PCR反应体系：10×ExTaq Buffer 5μL，dNTP 4μL，模板DNA 1μL，上下游引物各0.5μL，ExTaq酶1μL，ddH2O补齐至总体积50μL。PCR反应条件：94℃5min，94℃30s，65℃45s，72℃90s，循环35次，72℃10min，12℃10min。
参照上海捷瑞公司胶回收试剂盒说明书回收PCR产物，胶回收产物按一定比例与pMD18-T vector过夜连接，转化E.coli JM109感受态细胞，使用氨苄青霉素抗性平板筛选重组菌，重组质粒经酶切释放出大小约为2.7kb和1.3kb的基因条带，表明重组质粒构建成功，重组质粒命名为pMD18-T-Smo1，pMD18-T-Smo2和pMD18-T-Smo3。
(2)新金色分枝杆菌基因敲除质粒的构建
设计特定引物通过PCR的方式从Smo1，Smo2和Smo3上游扩增出300bp的序列，从下游扩增出450bp的序列。将上下游扩增出来的基因序列混合作为模版，利用上游300bp序列的上游引物和450bp序列的下游引物扩增出750bp的序列。将扩增出来的序列和敲除质粒p2NIL经Kpn I和Hind III双酶切，胶回收纯化，T4DNA连接酶过夜连接两片段，过夜后将连接物热击转化E.coli JM109感受态细胞，使用卡那霉素抗性平板筛选阳性转化子。提取转化子质粒，重组质粒经酶切验证构建成功。用Pac I单酶切将质粒pGOAL19中的选择标记基因盒切下来插入到构建好的重组质粒的Pac I酶切位点中，构建敲除质粒p2N-ΔSmo1，p2N-ΔSmo2和p2N-ΔSmo3。经PCR验证，敲除质粒构建成功。
(3)新金色分枝杆菌敲除基因回补质粒的构建
质粒pMV306用于敲除基因的回补。用Xba I/Cla I双酶切，将重组质粒p261-Smo1，p261-Smo2和p261-Smo3中的相应的基因片段连同热激启动子hsp60一同酶切下来连接到相 应的pMV306酶切位点上，构建敲除基因回补质粒p306-Smo1，p306-Smo2和p306-Smo3。
(4)重组质粒电转化法转化到新金色分枝杆菌
A将构建成功的重组质粒用电转化法转化新金色分枝杆菌中。转化方法采用改进的Gordhan and Parish法。
B重组菌株M.neoaurum阳性转化子的筛选；
挑取在具有相应抗生素压力平板上长出的菌落，摇瓶发酵，提取质粒进行酶切验证。
实施例2：SMO加强表达重组菌株的构建
质粒pMV261用于新金色分枝杆菌中基因的过量表达。用Sac I和Hind III双酶切pMD18-T-Smo1，用BamH I和EcoR I分别双酶切pMD18-T-Smo2和pMD18-T-Smo3，同时用相应的酶切位点酶切质粒pMV261，胶回收纯化相应的基因片段和质粒pMV261片段，T4DNA连接酶过夜连接两片段，过夜后将连接物热击转化E.coli JM109感受态细胞，使用卡那霉素抗性平板筛选阳性转化子。提取转化子质粒，重组质粒p261-Smo1，p261-Smo2和p261-Smo3经酶切验证构建成功，将构建成功的重组质粒，分别电转化到高产ADD的新金色分枝杆菌JC-12(Mycobacterium neoaurum JC-12已经在文献Shao ML,Zhang X,Rao ZM,Xu MJ,Yang TW,Li H,Xu ZH(2015)Enhanced production of androst-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12using three-stage fermentation strategy.PLoS ONE10(9):e0137658中公开)中，分别获得重组菌株JC-12S1，JC-12S2和JC-12S3。
实施例3：SMO加强表达重组菌株提高ADD的产量
将重组菌株JC-12S1，JC-12S2和JC-12S3在种子培养基上活化后，按照5％的接种量接入发酵培养基，以20g/L的胆固醇为底物，在30℃，160rpm条件下发酵168h，进行发酵转化实验。其中，种子培养基：葡萄糖10g/L，蛋白胨10g/l，牛肉膏6g/L，NaCl 10g/L，pH 7.5；发酵培养基：胆固醇20g/L，葡萄糖20g/L，蛋白胨10g/L，牛肉膏6g/L，K2HPO4 3g/L，MgSO4·7H2O 0.5g/L，MnCl2·4H2O 5×10-4g/L，羟丙基-β-环糊精60g/L，pH 7.5。
检测ADD的产量变化。结果表明发酵转化168h，过量表达SMO2的重组菌JC-12S2的ADD的产量提高最明显，从出发菌株新金色分枝杆菌JC-12的5.2g/L提高到7.3g/L。此外，重组菌JC-12S1和JC-12S3的ADD的产量分别提高到6.5g/L和6.1g/L。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2016097110-appb-000001
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Figure PCTCN2016097110-appb-000007
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Figure PCTCN2016097110-appb-000009

Claims (10)

  1. 一种ADD产量提高的新金色分枝杆菌重组菌，所述重组菌是过表达了甾酮C27-单加氧酶基因的新金色分枝杆菌；所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
  2. 根据权利要求1所述的新金色分枝杆菌重组菌，其特征在于，所述甾酮C27-单加氧酶基因Smo1、Smo2、Smo3的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
  3. 根据权利要求1所述的新金色分枝杆菌重组菌，其特征在于，所述过表达，具体是将甾酮C27-单加氧酶基因连接到质粒pMV261上得到重组质粒，然后将重组质粒转化到出发菌中。
  4. 一种通过过表达甾酮C27-单加氧酶基因提高ADD产量的方法，其特征在于，所述方法是将利用权利要求1～3任一所述的新金色分枝杆菌重组菌为生产菌株发酵生产ADD；所述新金色分枝杆菌重组菌过表达甾酮C27-单加氧酶基因；所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
  5. 根据权利要求4所述的方法，其特征在于，所述甾酮C27-单加氧酶基因的氨基酸序列如SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.6所示。
  6. 根据权利要求4所述的方法，其特征在于，所述发酵生产是以植物甾醇或/和胆固醇为底物进行发酵生产。
  7. 一种甾酮C27-单加氧酶基因，其特征在于，所述甾酮C27-单加氧酶基因的氨基酸序列如SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.6所示。
  8. 权利要求1～3任一所述的新金色分枝杆菌重组菌或者权利要求7所述的甾酮C27-单加氧酶基因在制备药物方面的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述药物是指抗肿瘤、抗炎症、抗菌、抗病毒、抗激素或者抗过敏的药物。
  10. 根据权利要求8所述的应用，其特征在于，所述药物是指治疗性器官退化药物、妇科疾病药物、抗肥胖药物、预防冠心病的药物或者HIV整合酶的抑制剂。
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