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

doi:10.13523/j.cb.2303053

China Biotechnology

2023, Vol. 43

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

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

Download:

HTML

PDF(1963KB) HTML
Export: BibTeX | EndNote (RIS)

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

Received: 20 March 2023 Published: 08 October 2023

ZTFLH:

Q819

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Jia-qiang FU
	Hui LI
	Wei-long WANG
	Shu-li WANG
	Sheng WU
	Qing-bo DENG
	Jin-song SHI
	Zheng-hong XU

Cite this article:

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.

URL:

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

Fig.1 HPLC profiles of standard samples of 17α-hydroxyprogesterone and 11α,17α-dihydroxy progesterone

Fig.2 HPLC profiles of fermentation products of 17α-hydroxyprogesterone by 11 strains

Fig.3 MS of transformation products of 17α-hydroxyprogesterone

Fig.4 ¹H-NMR spectrum of transformation products of 17α-hydroxyprogesterone

Fig.5 ¹³C-NMR spectrum of transformation products of 17α-hydroxyprogesterone

Fig.6 Results validation of transformation of 17α-hydroxyprogesterone by 5 fungal strains

Fig.7 Effects of different carbon sources on the conversion of C. lini

Fig.8 Effects of different concentrations of soluble starch on the conversion of C. lini

Fig.9 Effects of different nitrogen sources on the conversion of C. lini

Fig.10 Effects of different concentrations of ammonium chloride on the conversion of C. lini

Fig.11 Effects of different mineral salts on the conversion of C. lini

Fig.12 Effects of different concentrations of magnesium chloride on the conversion of C. lini

Table 1 Factors and levels of orthogonal test (g/L)

Table 2 Orthogonal test and results

Fig.13 Process analysis of 17α-hydroxyprogesterone transformation by C. lini

Fig.14 HPLC profiles of 17α-hydroxyprogesterone transformed by C. lini before and after optimization A:No substrate addition for C. lini before optimization;B:Substrate addition for C. lini before optimization;C:No substrate addition for C. lini after optimization;D:Substrate addition for C. lini after optimization


[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.

[3]	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.

[8]	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.

[9]	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.

[10]	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.

[12]	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.

[13]	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.

[16]	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.

[17]	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

[19]	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.

[21]	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.

[22]	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.

[23]	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.

[24]	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.

[26]	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.

[27]	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.

[28]	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.

[1]	ZHOU Li-ya, NIU Yu-jie, ZHENG Xiao-bing, JIANG Yan-jun, MA Li, BAI Jing, HE Ying. Molecular Modification and Enzymatic Properties of Glutamate Decarboxylase[J]. China Biotechnology, 2023, 43(5): 24-36.

[2]	SHANG Hua-rong, SUN Jian-zhong, ZHU Dao-chen. Research Progress of Microbial Depolymerization of Lignin to Synthesize Polyhydroxyalkanoates[J]. China Biotechnology, 2023, 43(2/3): 152-164.

[3]	PANG Guang-wu,LIANG Zhi-qun. Mutagenic Breeding and Optimization of Fermentation Conditions of Fibrinolytic Enzyme from Marine Bacillus subtilis[J]. China Biotechnology, 2022, 42(12): 27-36.

[4]	Xin-miao WANG,Kang ZHANG,Sheng CHEN,Jing WU. Recombinant Expression and Fermentation Optimization of Dictyoglomus thermophilum Cellobiose 2-Epimerase in Bacillus subtilis[J]. China Biotechnology, 2019, 39(7): 24-31.

[5]	REN Li-qiong,WU Jing,CHEN Sheng. Co-Expression of N-Acetyltransferase Enhances the Expression of Aspergillus nidulans α-Glucosidase in Pichia pastoris[J]. China Biotechnology, 2019, 39(10): 75-81.

[6]	Zheng-san ZUO,Dong-sheng GUO,Xiao-jun JI,Ping SONG,He HUANG. Polyunsaturated Fatty Acids and Their Derivatives in the Intestinal Tract:a Review[J]. China Biotechnology, 2018, 38(11): 66-75.

[7]	YAO Ren-hui, DONG Zhuo, LI Hui. *Biotransformation of Androst-4-en-3,17-dione by Gibberella intermedia* C2**[J]. China Biotechnology, 2017, 37(3): 73-77.

[8]	ZHANG Qi, ZHANG Lu, XU You-qiang, PEI Jiang-sen, CHENG Chi. L-asparagine Production by Biotransformation Method[J]. China Biotechnology, 2016, 36(1): 63-67.

[9]	QIAN Xin, GUO Hong-yan, ZHOU Qing-feng. Construction of 4-Hydroxyphenylacetate-3-hydroxylase A Expression Strain and Its Biotransformation Effect on Hydroxytyrosol[J]. China Biotechnology, 2015, 35(3): 56-60.

[10]	LIU Hui-li, LI Yuan-yuan, JU Rui-cheng, ZHAO Hong-tao, YANG Qing. *Isolation, Identification and Fermentation Optimization of Antagonistic Bacillus subtilis* KC-5**[J]. China Biotechnology, 2014, 34(3): 96-102.

[11]	ZHAO Wei-rui, SHENG Hu, JUN Huang, MEI Le-he. Improve Microorganism Cell Permeability for Whole-Cell Bioprocess：Methods and Strategies[J]. China Biotechnology, 2014, 34(3): 125-131.

[12]	CHEN Xu-sheng, REN Xi-dong, ZENG Xin, DONG Nan, MAO Zhong-gui. *ε-Poly-L-lysine Production from Precursor L-lysine by Streptomyces* sp. M-Z18**[J]. China Biotechnology, 2013, 33(1): 53-59.

[13]	JIAO Jing-yu, WU Mian-bin, ZHAO Jiong-feng, LIN Jian-ping, YANG Li-rong. Research Advances in Improvement of Xylitol Producing Strains by Genetic Engineering Technology[J]. China Biotechnology, 2012, 32(11): 124-131.

[14]	JIAO Jing-yu, WU Mian-bin, ZHAO Jiong-feng, LIN Jian-ping, YANG Li-rong. Research Advances in Improvement of Xylitol Producing Strains by Genetic Engineering Technology[J]. China Biotechnology, 2012, 32(11): 124-131.

[15]	GAO Hui-hui, CHEN Sheng, WU Jing, CHEN Jian. *Fermentation Optimization in Shake Flasks of Corynebacterium nitrilophilus* NHase Applied in Surface Modification of Polyacrylonitrile Fibers**[J]. China Biotechnology, 2011, 31(8): 54-60.

Viewed

Full text

Abstract

Cited

Shared

Discussed