|
|
|
| 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 |
|
|
|
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.
|
|
Received: 12 May 2016
Published: 25 November 2016
|
|
|
|
|
| [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. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
