一株高效氧化胆固醇的简单节杆菌构建与转化条件优化

doi:10.13523/j.cb.20161110

中国生物工程杂志

2016, Vol. 36

Issue (11): 70-75 DOI: 10.13523/j.cb.20161110

技术与方法

一株高效氧化胆固醇的简单节杆菌构建与转化条件优化

张璟, 张玉富, 秦慧民, 毛淑红, 路福平

天津科技大学生物工程学院工业发酵微生物教育部重点实验室天津 300457

Construction of Artificial Arthrobacter simplex for Oxidizing Cholesterol, and Optimization of Bioconversion Condition

ZHANG Jing, ZHANG Yu fu, QIN Hui min, MAO Shu hong, LU Fu ping

Key Laboratory of Industrial Fermentation Microbiology, National Engineering Laboratory for Industrial Enzymes(NELIE), College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

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摘要：

为实现胆固醇的高效生物氧化，利用基因工程手段将编码简单节杆菌胆固醇氧化酶的DNA片段克隆到质粒pTY2中，构建pTY2-5332插入表达载体。该载体以简单节杆菌基因组中的16S rDNA位点为整合点，提高了胆固醇氧化酶在基因组中的拷贝数，实现了简单节杆菌胆固醇氧化酶的过表达。重组菌的生长实验分析表明，插入到16S rDNA位点的胆固醇氧化酶没有影响简单节杆菌的生长。重组菌可在20h将2g/L的胆固醇完全转化为4-胆甾烯-3酮，比原始菌的转化时间缩短了4h，提高了胆固醇的转化效率。经过转化条件优化确定了该重组菌以2%的接种量后继续培养16h，然后胆固醇经120目过筛后投料量为2g/L，并添加2%（体积比）二甲基甲酰胺作为促溶剂；胆固醇添加18h后可完全转化为4-胆甾烯-3-酮，是优化前的1.11倍。

关键词： 胆固醇氧化酶; 4-胆甾烯-3-酮; 16S rDNA; 胆固醇; 简单节杆菌

Abstract:

To improve the ability of conversion cholesterol to 4-cholesten-3-one, the cholesterol oxidase gene from the Arthrobacter simplex was cloned and the integrative expression vector pTY2-5332 was constructed. The recombinant plasmid pTY2-5332 was transformed into Arthrobacter simplex by eletroporation and inserted into the 16S rDNA site to increase the copy number of the cholesterol oxidase in genome. Result of the growth data indicates that a small amount of the 16S rDNA site disrupted has little effect on the growth of the Arthrobacter simplex. The recombinant strain can convert cholesterol completely to 4-cholesten-3-one in 20 hours when the cholesterol concentration is 2g/L, reduce 4 hours compared with the wild-type Arthrobacter simplex, and are capable of increasing cholesterol conversion efficiency. The optimal conversion condition of cholesterol was 2% inoculum size and continuous culture of 16h, then 2g/L substrate passed through a 120 mesh screen and 2% DMF were added to the medium. Under this contions, the cholesterol converted into 4-cholesten-3-one in 18 hours completely, which was 1.11 times higher than the recombinant strain.

Key words: 4-cholesten-3-one Cholesterol oxidase Arthrobacter simplex 16S rDNA Cholesterol

收稿日期: 2016-05-12 出版日期: 2016-11-25

ZTFLH:

Q78

基金资助:

国家高技术研究发展计划资助项目（2011AA02A211-06）

通讯作者: 路福平,lfp@tust.edu.cn E-mail: lfp@tust.edu.cn

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引用本文:

张璟, 张玉富, 秦慧民, 毛淑红, 路福平. 一株高效氧化胆固醇的简单节杆菌构建与转化条件优化[J]. 中国生物工程杂志, 2016, 36(11): 70-75.

ZHANG Jing, ZHANG Yu fu, QIN Hui min, MAO Shu hong, LU Fu ping. Construction of Artificial Arthrobacter simplex for Oxidizing Cholesterol, and Optimization of Bioconversion Condition. China Biotechnology, 2016, 36(11): 70-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20161110 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I11/70

[1] Suzuki K.Anti-obesity effect of cholest-4-en-3-one, an intestinal catabolite of cholesterol, on mice. Journal of Nutritional Science and Vitaminology, 1993, 35(5):537-543.
[2] Barton D H R.The invention of chemical reactions:the last five years. Tetrahedron, 1992, 48(13):2529-2544.
[3] Santhanam H K, Shreve G S. Solvent selection and productivity in multiphase biotransformation systems. Biotechnology Progress, 1994, 10(2):187-192.
[4] Phase N, Patil S. Natural oils are better than organic solvents for the conversion of soybean sterols to 17-ketosteroids by mycobacterium fortuiurn. World J Microbial Biotechnol, 1994, 10(2):228-229.
[5] Imai Y, Sato R. Studies on the substrate interactions with P450 in drug hydroxylation by liver microsomes. J Biochem, 1967, 62(2):239-249.
[6] Overhage J, Priefert H, Rabenhorst J, et al. Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase(vdh) gene. Appl Microbiol Biotechnol, 1999, 52(6):820-828.
[7] Saxena S. Fungal biotransformation of cannabinoids:potential for new effective drugs. CURR Opin Drug Disc, 2009, 12(2):305-312.
[8] Liu W H, Horng W C, Tsai M S. Bioconversion of cholesterol to cholest-4-en-3-one in aqueous/organic solvent two-phase reactors. Enzyme and Microbial Technology, 1996, 18(3):184-189.
[9] 吕陈锋, 陈毅力, 王龙刚, 等. 利用胆固醇氧化酶转化胆固醇制备胆甾-4-烯-3-酮. 无锡轻工大学学报, 2001, 20(5):485-488. Lu C F, Chen Y L, Wang L G, et al. Bioconversion of cholesterol by cholesterol oxidase from Brevibacterium sp.. Journal of Wuxi University of Light Industry, 2001, 20(5):485-488.
[10] Shtratnikova V Y, Schelkunov M I, Pekov Y A, et al. Complete genome sequence of steroid-transforming Nocardioides simplex VKM Ac-2033D. Genome Announc, 2015, 3(1):e01406.
[11] Zhang H T, Tian Y, Wang J L, et al. Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation. Appl Microbiol Biotechnol, 1993, 97(21):9503-9514.
[12] Vrielink A, Ghisla S. Cholesterol oxidase:biochemistry and structural features. Federation of European Biochemical Societies Journal, 2009, 276(23):6826-6843.
[13] Pollegioni L. Cholesterol oxidase:a model flavoprotein oxidase and a biotechnological tool. Federation of European Biochemical Societies Journal, 2009, 276(23):6825.
[14] Noriyuki Doukyu. Characteristics and biotechnological applications of microbial cholesterol oxidases. Applied Microbiology and Biotechnology, 2009, 83(5):825-837.
[15] 李晓丹.大肠杆菌单拷贝rRNA操纵元菌株的构建及16S rRNA基因敲除中的质粒重排.南京:南京农业大学,2007. Li X D. Construction of An Escherichia coli Strain with Single Copy of rRNA Operon and the Plasmid Rearrangement During Homologous Recombination. Nanjing:Nanjing Agricultural University, 2007.
[16] Rossolini G M, Riccio M L, Gallo E, et al. Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination. Gene, 1992, 119(1):75-81.
[17] Condon Y C, Liveris D, Squires C, et al. rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation. Journal of Bacteriology, 1995, 177(14):4152-4156.

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