17α-羟基黄体酮C11α-羟化菌株的筛选及工艺优化<sup>*</sup>

doi:10.13523/j.cb.2303053

中国生物工程杂志

2023, Vol. 43

Issue (9): 19-32 DOI: 10.13523/j.cb.2303053

研究报告

17α-羟基黄体酮C11α-羟化菌株的筛选及工艺优化^*

伏家强¹,李会^1,^**(

),王维龙¹,王淑丽²,武胜³,邓庆博⁴,史劲松¹,许正宏⁴

1 江南大学生命科学与健康工程学院无锡 214122
2 天津药业研究院股份有限公司天津 300301
3 津药药业股份有限公司天津 300301
4 江南大学生物工程学院工业生物技术教育部重点实验室无锡 214122

Screening and Fermentation Optimization of 17α-hydroxyprogesterone C11α-hydroxylation Strain

FU Jia-qiang¹,LI Hui^1,^**(

),WANG Wei-long¹,WANG Shu-li²,WU Sheng³,DENG Qing-bo⁴,SHI Jin-song¹,XU Zheng-hong⁴

1 College of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China
2 Tianjin Pharmaceutical Research Institute Limited Company, Tianjin 300301, China
3 Jinyao Pharmaceutical Limited Company, Tianjin 300301, China
4 Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Bio-Engineering, Jiangnan University, Wuxi 214122, China

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摘要：

11α,17α-二羟基黄体酮(11α,17α-dihydroxy progesterone)作为甾体激素类药物的重要中间体,其生物合成主要以17α-羟基黄体酮作为底物转化生成。为探究不同微生物对17α-羟基黄体酮的转化能力,以11株具有甾体羟化能力的菌株为研究对象,通过全细胞生物转化实验,筛选得到了一株转化能力最强的菌株亚麻刺盘孢SF-307。通过单因素实验和正交设计进行菌株发酵培养基组分优化,确定了最适发酵培养基:15 g/L可溶性淀粉、1.8 g/L 氯化铵、0.6 g/L 氯化镁、3 g/L玉米浆。优化培养基后的亚麻刺盘孢SF-307转化产物唯一,当底物投料量为0.5 g/L,添加1%(V/V)乙醇进行助溶,转化56 h时,底物转化率为93.2%,11α,17α-二羟基黄体酮的最高浓度为224.1 mg/L,较优化前提高61.1%。上述结果表明:亚麻刺盘孢SF-307作为一株全新的17α-羟基黄体酮羟化菌株,发酵优化可显著增强17α-羟基黄体酮转化产物的选择性,缩短转化周期,对11α,17α-二羟基黄体酮的工业生产意义重大。

关键词： 11α,17α-二羟基黄体酮; 17α-羟基黄体酮; 生物转化; 亚麻刺盘孢SF-307; 发酵优化

Abstract:

11α,17α-dihydroxy progesterone is an important intermediate of steroid hormone drugs and its biosynthesis is mainly produced by microbial transformation of 17α-hydroxyprogesterone. In order to explore the transformation ability of different microorganisms to 17α-hydroxyprogesterone, 11 strains with steroid hydroxylation ability were selected. Through the whole-cell biotransformation experiment, Colletotrichum lini SF-307 with the strongest transformation ability was obtained. Then, the optimal composition of the fermentation medium was determined by a single-factor experiment and orthogonal design. The most suitable fermentation medium was determined: 15 g/L soluble starch, 1.8 g/L ammonium chloride, 0.6 g/L magnesium chloride, and 3 g/L corn pulp. After optimization, the only product 11α, 17α-dihydroxy progesterone was produced by C. lini SF-307. When the substrate was fed at 0.5 g/L with the addition of 1% (V/V) ethanol for co-solubilization, the substrate conversion was 93.2% and the highest concentration was 224.1 mg/L at 56 h, which was increased by 61.1% compared to the original. The results showed that C. lini SF-307 was a new strain of 17α-hydroxyprogesterone hydroxylation. The fermentation optimization can significantly enhance selectivity of 17α-hydroxyprogesterone conversion products and shorten the transformation period. This study is of great significance to the industrial production of 11α,17α-dihydroxy progesterone.

Key words: 11α,17α-dihydroxy progesterone 17α-hydroxyprogesterone Biotransformation Colletotrichum lini SF-307 Fermentation optimization

收稿日期: 2023-03-20 出版日期: 2023-10-08

ZTFLH:

Q819

基金资助: * 国家重点研发计划(2019YFA0905300);国家自然科学基金(22078126);天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-001-14)

通讯作者: ** 电子信箱:lihui@jiangnan.edu.cn

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	伏家强
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	王淑丽
	武胜
	邓庆博
	史劲松
	许正宏

引用本文:

伏家强, 李会, 王维龙, 王淑丽, 武胜, 邓庆博, 史劲松, 许正宏. 17α-羟基黄体酮C11α-羟化菌株的筛选及工艺优化^*[J]. 中国生物工程杂志, 2023, 43(9): 19-32.

FU Jia-qiang, LI Hui, WANG Wei-long, WANG Shu-li, WU Sheng, DENG Qing-bo, SHI Jin-song, XU Zheng-hong. Screening and Fermentation Optimization of 17α-hydroxyprogesterone C11α-hydroxylation Strain. China Biotechnology, 2023, 43(9): 19-32.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2303053 或 https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I9/19

图1 17α-羟基黄体酮和11α,17α-二羟基黄体酮标准品HPLC图谱

图2 11株菌株转化17α-羟基黄体酮发酵产物的HPLC图谱

图3 17α-羟基黄体酮转化产物的质谱测定

图4 17α-羟基黄体酮转化产物的核磁共振氢谱

图5 17α-羟基黄体酮转化产物的核磁共振碳谱

图6 5株真菌转化17α-羟基黄体酮的结果验证

图7 碳源种类对亚麻刺盘孢转化的影响

图8 可溶性淀粉浓度对亚麻刺盘孢转化的影响

图9 氮源种类对亚麻刺盘孢转化的影响

图10 氯化铵浓度对亚麻刺盘孢转化的影响

图11 无机盐种类对亚麻刺盘孢转化的影响

图12 氯化镁浓度对亚麻刺盘孢转化的影响

表1 正交试验因素及水平 (g/L)

表2 正交试验方案及结果

图13 亚麻刺盘孢转化17α-羟基黄体酮的过程分析

图14 优化前后亚麻刺盘孢转化17α-羟基黄体酮的产物HPLC图谱

[1]	Szaleniec M, Wojtkiewicz A M, Bernhardt R, et al. Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms. Applied Microbiology and Biotechnology, 2018, 102(19): 8153-8171. doi: 10.1007/s00253-018-9239-3 pmid: 30032434
[2]	Sultana N. Microbial biotransformation of bioactive and clinically useful steroids and some salient features of steroids and biotransformation. Steroids, 2018, 136: 76-92. doi: S0039-128X(18)30015-1 pmid: 29360535
[3]	贺颖, 董得时. 高效液相色谱法测定醋酸泼尼松片中相关物质含量. 中国医院用药评价与分析, 2012, 12(6):531-533.
	He Y, Dong D S. Determination of the contents of the related substances in prednisone acetate tablets by HPLC. Evaluation and Analysis of Drug-Use in Hospitals of China, 2012, 12(6):531-533.
[4]	Xia J L, Liu Y M, Chen X Q, et al. Effects of methotrexate combined with hydroxychloroquine sulfate and prednisone acetate on inflammatory response, immune function and liver and renal function in patients with systemic lupus erythematosus. Journal of Hainan Medical University, 2017, 23(22): 41-44.
[5]	Auchus R J, Yu M K, Nguyen S, et al. Use of prednisone with abiraterone acetate in metastatic castration-resistant prostate cancer. The Oncologist, 2014, 19(12): 1231-1240. doi: 10.1634/theoncologist.2014-0167
[6]	Tang C, Wang W, Xue Y X, et al. Effect of MMF immunosuppression based on CNI reduction on CNI-related renal damage after lung transplantation. Journal of Healthcare Engineering, 2022, 2022: 1-7.
[7]	Posadas E, Chi K, de Wit R, et al. Pharmacokinetics, safety, and antitumor effect of apalutamide with abiraterone acetate plus prednisone in metastatic castration-resistant prostate cancer: phase ib study. Clinical Cancer Research, 2020, 26: 3517-3524. doi: 10.1158/1078-0432.CCR-19-3402 pmid: 32366670
[8]	冯进辉, 张汝金, 张峥斌, 等. 系列甾体药物关键中间体转化菌种构建及智能化生产应用. 生物工程学报, 2022, 38(11): 4335-4342.
	Feng J H, Zhang R J, Zhang Z B, et al. Construction of strains for bioconversion of steroid key intermediates and intelligent industrial production. Chinese Journal of Biotechnology, 2022, 38(11): 4335-4342.
[9]	雷灿. 薯蓣皂素清洁生产技术研究. 恩施: 湖北民族学院, 2016.
	Lei C. Environment-friendly extraction of diosgenin from Dioscorea zingiberensis rhizome. Enshi: Hubei University for Nationalities, 2016.
[10]	陶琳, 系祖斌, 艾文, 等. 一种制备11α,17α-羟基黄体酮的方法与流程: 中国, CN115466774A. 2022-12-13[2023-02-21]. https://cprs.patentstar.com.cn/Search/Detail?ANE=8CEA9FEA9FFD9DEA9EFB9BHA4BCA3AAACIFA9FFDADHA9BGE.
	Tao L, Xi Z B, Ai W, et al. A method and process for the preparation of 11α,17α-hydroxyprogesterone: Chinese, CN115466774A. 2022-12-13[2023-02-21]. https://cprs.patentstar.com.cn/Search/Detail?ANE=8CEA9FEA9FFD9DEA9EFB9BHA4BCA3AAACIFA9FFDADHA9BGE.
[11]	Alejandro Z, Rubin B A. Process for the introduction of an 11 alpha-OH group into cyclopentanophenanthrene derivatives by Cunninghamella echinulate: USA, US37867853A. 1957-11-05[2023-02-21]. https://www.freepatentsonline.com/2812286.html.
[12]	乔玉茜. 17α羟基黄体酮11α羟化菌株筛选及其转化工艺研究. 天津: 天津科技大学, 2017.
	Qiao Y Q. Screening of 17α hydroxyprogesterone 11α hydroxylated strain and its transformation technology. Tianjin: Tianjin University of Science and Technology, 2017.
[13]	李茜茜, 时丽荣, 荣绍丰, 等. 赭曲霉毒素A基因敲除的赭曲霉突变菌株及其构建和应用: 中国, CN110564630A, 2019-12-13[2023-02-21]. https://cprs.patentstar.com.cn/Search/Detail?ANE=AIHA5EBA9FDBAIHA9HHG9BHA7CEA9FHG9GAD5BCAADHA9CHG.
	Li Q Q, Shi L R, Rong S H, et al. A mutant strain of Aspergillus ochraceus with ochratoxin a gene knockout and its construction and application: Chinese, CN110564630A. 2019-12-13[2023-02-21]. https://cprs.patentstar.com.cn/Search/Detail?ANE=AIHA5EBA9FDBAIHA9HHG9BHA7CEA9FHG9GAD5BCAADHA9CHG.
[14]	Restaino O F, Barbuto Ferraiuolo S, Perna A, et al. Biotechnological transformation of hydrocortisone into 16α-hydroxyprednisolone by coupling Arthrobacter simplex and Streptomyces roseochromogenes. Molecules, 2020, 25(21): 4912. doi: 10.3390/molecules25214912
[15]	Subedi P, Kim K H, Hong Y S, et al. Enzymatic characterization and comparison of two steroid hydroxylases CYP154C3-1 and CYP154C3- 2 from Streptomyces Species. Journal of Microbiology and Biotechnology, 2021, 31(3): 464-474. doi: 10.4014/jmb.2010.10020
[16]	孙锦. 三羟基雄甾烯酮高效转化菌株的选育及其羟化酶研究. 无锡: 江南大学, 2017.
	Sun J. Breeding of trihydroxy androstenone efficient transformation strain and study on its hydroxylase. Wuxi: Jiangnan University, 2017.
[17]	付珍珍, 李恒, 李会, 等. Gibberella intermedia CA3-1羟基化去氢表雄酮的工艺. 生物加工过程, 2015(1): 1-5.
	Fu Z Z, Li H, Li H, et al. C7α-hydroxylation of dehydroepiandrosterone by Gibberella intermedia CA3-1. Chinese Journal of Bioprocess Engineering, 2015(1): 1-5.
[18]	Chen X L, Luo X R, Cao F F, et al. Molecular cloning, expression of CPR gene from Rhizopus oryzae into Rhizopus nigericans and its application in the 11α-hydroxylation of 16α, 17-epoxy-progesterone. Enzyme and Microbial Technology, 2014, 66: 28-34. doi: 10.1016/j.enzmictec.2014.08.002
[19]	艾露, 陈文慧, 史京辉, 等. 赭曲霉11α羟化酶的克隆表达及关键氨基酸位点分析. 生物技术通报, 2023(4): 114-123. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0572
	Ai L, Chen W H, Shi J H, et al. Cloning and expression of 11αhydroxylase from Aspergillus ochraceus and analysis of key amino acid sites. Biotechnology Bulletin, 2023(4): 114-123. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0572
[20]	Fan Y X, Lu Y L, Zhang L W, et al. Enhancing NADPH regeneration and increasing hydroxylation efficiency with P450 monooxygenase through strengthening expression of glucose-6-phosphate dehydrogenase in industrial filamentous fungi. Biocatalysis and Agricultural Biotechnology, 2017, 11: 307-311. doi: 10.1016/j.bcab.2017.08.004
[21]	尹思淇, 李聪, 吴燕, 等. Colletotrichum lini ST-1静息细胞转化DHEA制备7α, 15α-diOH-DHEA的工艺. 食品与生物技术学报, 2016, 35(8): 801-805.
	Yin S Q, Li C, Wu Y, et al. Bioconversion of dehydroepiandrosterone to 3β, 7α, 15α-trihydroxy-5-androsten-17-one by Colletotrichum lini ST-1 resting cells. Journal of Food Science and Biotechnology, 2016, 35(8): 801-805.
[22]	赵有玺, 龚平. 雄甾烯酮转化菌的诱变育种和发酵条件优化. 食品与生物技术学报, 2010, 29(1):150-154.
	Zhao Y X, Gong P. Mutant and optimization of androstenone translation strain Mycobacterium sp. SH5. Journal of Food Science and Biotechnology, 2010, 29(1):150-154.
[23]	樊金华, 薛皎亮, 谢映平, 等. 布氏白僵菌液体培养条件的优化研究. 山西大学学报, 2013, 36(2): 271-274.
	Fan J H, Xue J L, Xie Y P, et al. Optimization of fermentation condition of Beauveria brongniartii. Journal of Shanxi University, 2013, 36(2): 271-274.
[24]	庞光武, 梁智群. 海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化. 中国生物工程杂志, 2022, 42(12): 27-36.
	Pang G W, Liang Z Q. Mutagenic breeding and optimization of fermentation conditions of fibrinolytic enzyme from marine Bacillus subtilis. China Biotechnology, 2022, 42(12): 27-36.
[25]	Tomita A, Zhang M F, Jin F, et al. ATP-dependent modulation of MgtE in Mg2+ homeostasis. Nature Communications, 2017, 8(1): 1-11. doi: 10.1038/s41467-016-0009-6
[26]	付珍珍. 生物转化制备三羟基雄甾烯酮赤霉菌的筛选及工艺优化. 无锡: 江南大学, 2014.
	Fu Z Z. Screening and process optimization of Monascus for preparing trihydroxyandrostenone by biotransformation. Wuxi: Jiangnan University, 2014.
[27]	艾正文. 基于转录组学的Streptococcus thermophilus TF96中心碳代谢机制的研究. 哈尔滨: 东北农业大学, 2017.
	Ai Z W. Study on carbon metabolism mechanism of Streptococcus thermophilus TF96 center based on transcriptomics. Harbin: Northeast Agricultural University, 2017.
[28]	陈海立. 基于“OSMAC”策略挖掘两株三七内生真菌次级代谢产物的合成潜能. 重庆: 重庆大学, 2020.
	Chen H L. Mining the synthetic potential of secondary metabolites of two endophytic fungi from Panax notoginseng based on “OSMAC” strategy. Chongqing: Chongqing University, 2020.

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