分枝杆菌细胞裂解液催化甾体激素C<sub>1,2</sub>位脱氢反应的研究

doi:10.13523/j.cb.20170804

中国生物工程杂志

2017, Vol. 37

Issue (8): 23-30 DOI: 10.13523/j.cb.20170804

研究报告

分枝杆菌细胞裂解液催化甾体激素C_1,2位脱氢反应的研究

秦梦菲, 孙鸿, 宋浩

天津大学化工学院系统生物工程教育部重点实验室天津化学化工协同创新中心合成生物学平台天津 300072

Studies on the 3-Ketosteriod-1-Dehydrogenation of Steroid Hormone by Cellular lysates of Mycobacterium

QIN Meng-fei, SUN Hong, SONG Hao

School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering, Ministry of Education, SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China

全文: PDF(826 KB) HTML

摘要：

9β，11β-环氧-17α，21-二羟基-16β-甲基孕-1，4-二烯-3，20-二酮（Ⅳ）是生产9-氟甾体激素的关键前体，以9β，11β-环氧-17α，21-二羟基-16β-甲基孕-4-烯-3，20-二酮-21-醋酸酯（Ⅰ）为底物合成Ⅳ是工业化生产Ⅳ的重要方法。通过比较分枝杆菌全细胞转化法与细胞裂解液转化法，发现分枝杆菌全细胞只能将Ⅰ转化为9β，11β-环氧-17α，21-二羟基-16β-甲基孕-4-烯-3，20-二酮（Ⅱ），而细胞裂解液可以有效地将Ⅰ转化为Ⅳ，其反应机制为底物Ⅰ自发水解为中间体Ⅱ，Ⅱ在C_1，2位脱氢酶（KSTD）的催化作用下发生C_1，2位脱氢反应生成产物Ⅳ。为进一步提高产物Ⅳ的转化率，利用基因工程手段在分枝杆菌中分别过表达编码KSTD的关键基因：kstD、kstD3和kstD_M，提高脱氢反应效率，结果表明1 g/L底物Ⅰ在pH7.0的重组菌株MS136-kstD_M细胞裂解液中反应45 h，Ⅳ的转化率为78.4%，比出发菌株提高了38.9%；并优化缓冲液pH，提高反应速率，结果表明1 g/L底物Ⅰ在pH7.5的重组菌株MS136-kstD_M细胞裂解液中反应45 h，Ⅳ的转化率为92.8%，比出发菌株提高了63.4%。

关键词： 分枝杆菌细胞裂解液C_{1, 2}位脱氢酶; 9β, 11β-环氧-17α, 21-二羟基-16β-甲基孕-1, 4-二烯-3, 20-二酮

Abstract:

9β, 11β-Epoxypregn-4-ene-17α, 21-diol-3, 20-dione 21-acetate (Ⅰ) is a substrate for the production of 9β, 11β-Epoxypregn-1, 4-diene-17α, 21-diol-3, 20-dione (Ⅳ), which is a key precursor for the production of many 9-fluoro-substituted corticosteroid hormones. By comparing whole cells catalysis and cellular lysates conversion, it was found that whole cells of Mycobacterium sp. MS136 could only convertⅠto 9β, 11β-Epoxypregn-4-ene-17α, 21-diol-3, 20-dione (Ⅱ), and Ⅰ can be effectively converted toⅣ by cellular lysates.The reaction order is that Ⅰ is spontaneously hydrolyzed to Ⅱ and Ⅱundergoes C_{1, 2}-dehydrogenation reaction toⅣ.In order to improve the productivity of Ⅳ, the key genes kstD, kstD3 and kstD_M encoding C_{1, 2}-dehydrogenase (KSTD)were overexpressed in Mycobacterium sp. MS136 to enhance the C_{1, 2}-dehydrogenation reaction rate, and the results showed that 1 g/L substrate Ⅰ can be converted by recombinant strain MS136-kstD_M cellular lysates at pH 7.0, the productivity of Ⅳreached 78.4% after 45 h, which is 38.9% higher than original strain.The reaction rate is enhanced by optimizing the pH, and the results showed that 1 g/L substrate (Ⅰ) can be converted by recombinant strain MS136-kstD_M cellular lysates at pH 7.5, the productivity of Ⅳreached 92.8% after 45 h, which was 63.4% higher than original strain.

Key words: 9β, 11β-Epoxypregn-1, 4-diene-17α, 21-diol-3, 20-dionecellular Lysates of Mycobacterium 3-ketosteriod-1-dehydrogenase

收稿日期: 2017-02-23 出版日期: 2017-08-25

ZTFLH:

G819

基金资助:

国家青年千人基金资助项目（SQK13001）

通讯作者: 宋浩 E-mail: hsong@tju.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	孙鸿
	秦梦菲
	宋浩

引用本文:

秦梦菲, 孙鸿, 宋浩. 分枝杆菌细胞裂解液催化甾体激素C_1,2位脱氢反应的研究[J]. 中国生物工程杂志, 2017, 37(8): 23-30.

QIN Meng-fei, SUN Hong, SONG Hao. Studies on the 3-Ketosteriod-1-Dehydrogenation of Steroid Hormone by Cellular lysates of Mycobacterium. China Biotechnology, 2017, 37(8): 23-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170804 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I8/23

[1] Tong W Y, Dong X. Microbial biotransformation:recent developments on steroid drugs. Recent Pat Biotechnol, 2009, 3(2):141-153.
[2] Croxatto H B. Progestin implants. Steroids, 2000, 65(10-11):681-685.
[3] Hughes D T, Sperandio V. Inter-kingdom signalling:communication between bacteria and their hosts. Nat Rev Microbiol, 2008, 6(2):111-120.
[4] Bragin J, Saowakhon S, Manosroi A. A novel one-step biotransformation of cortexolone-21-acetate to hydrocortisone acetate using Cunninghamella blakesleeana ATCC 8688a. Enzyme Microb Technol, 2007, 41(3):322-325.
[5] Funder J W. Minireview:aldosterone and mineralocorticoid receptors:past, present, and future. Endocrinology, 2010, 151(11):5098-5102.
[6] García J L, Uhía I, Galán B. Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol, 2012, 5(6):679-699.
[7] Bragin E Y, Shtratnikova V Y, Dovbnya D V, et al. Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains. J Steroid Biochem Mol Biol, 2013, 138(10):41-53.
[8] Xie R, Shen Y, Qin N, et al. Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium neoaurum. J Ind Microbiol Biotechnol, 2015, 42(4):507-513.
[9] Cruz A, Angelova B, Fernandes P, et al. Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in bis-(2-ethylhexyl) phthalate. Biocatal Biotransform, 2004, 22(3):189-194.
[10] Fokina V V, Sukhodol'skaya G V, Gulevskaya S A, et al. The 1(2)-dehydrogenation of steroid substrates by Nocardioides simplex VKM Ac-2033D. Microbiology, 2003, 72(1):24-29.
[11] Fokina V V, Donova M V. 21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D. J Steroid Biochem Mol Biol, 2003, 87(4-5):319-325.
[12] Donova M V. Transformation of steroids by actinobacteria:a review. Applied Biochemistry and Microbiology, 2007, 43(1):5-18.
[13] Donova M V, Egorova O V. Microbial steroid transformations:Current state and prospects. Appl Microbiol Biotechnol, 2012, 94(6):1423-1447.
[14] Fernandes P, Cruz A, Angelova B, et al. Microbial conversion of steroid compounds:Recent developments. Enzyme Microb Technol, 2003, 32(6):688-705.
[15] Wang F Q, Yao K, Xu L Q, et al. Characterization and engineering of 3-ketosteroid-△¹-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3, 17-dione through the catabolism of sterols. Metabolic Engineering, 2014, 24:181-191.
[16] Wang F Q, Wei W, Fan S Y, et al. Inactivation and augmentation of the primary 3-ketosteroid-Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01:biotransformation of soybean phytosterols to 4-androstene-3, 17-dione or 1, 4-androstadiene-3, 17-dione. Appl Environ Microbiol, 2010, 76(13):4578-4582.
[17] Zhang W Q, Shao M L, Rao Z M, et al. Bioconversion of 4-androstene-3,17-dione toandrost-1,4-diene-3,17-dione by recombinant Bacillus subtilis expressing ksdd gene encoding 3-ketosteroid-Δ¹-dehydrogenasefrom Mycobacterium neoaurum JC-12. J Steroid Biochem MolBiol, 2013, 135(1):36-42.
[18] Li Y, Lu F, Sun T, et al. Expression of ksdD gene encoding 3-ketosteroid-Δ¹-dehydrogenase from Arthrobacter simplex in Bacillus subtilis. Lett Appl Microbiol, 2007, 44(5):563-568.
[19] Malaviya A, Gomes J. Androstenedione production by biotransformation of phytosterols. Bioresource Technology, 2008, 99(15):6725-6737.
[20] Shao M, Zhang X, Rao Z, et al. Enhanced production of androst-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12 using three-stage fermentation strategy. PLoS One, 2015, 10(9):e0137658.
[21] Flett F, Mersinias V, Smith C P. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol Lett, 1997, 155(2):223-229.
[22] Van D G, Hessels G I, Van G R, et al. Targeted disruption of the kstD geneencoding 3-ketosteroid-Δ¹-dehydrogenase isoenzyme of Rhodococcus erythropolis SQ1. Appl Environ Microbiol, 2000, 66(5):2029-2036.

[1]	王曦光, 王娟, 张琳. 拟南芥蛋白质丰度与基因翻译效率关联分析[J]. 中国生物工程杂志, 2017, 37(2): 40-47.

Viewed

Full text

Abstract

Cited

Shared

Discussed