增加雷斯青霉15α–羟化酶基因拷贝数提高甾体转化效率  

Increasing 15α-Hydroxylase Gene Copy Number in Penicillium raistrickii to Enhance the Transformation Efficiency of Steroid

作　　者：李雪龙 李改 谢东奇 金鹏 毛淑红[1] 王正祥 刘晓光 LI Xuelong;LI Gai;XIE Dongqi;JIN Peng;MAO Shuhong;WANG Zhengxiang;LIU Xiaoguang(College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457,China;College of Chemical Engineering and Materials Science,Tianjin University of Science & Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津科技大学化工与材料学院,天津300457

出　　处：《天津科技大学学报》2019年第4期16-21,29,共7页Journal of Tianjin University of Science & Technology

基　　金：国家高技术研究发展计划(863计划)资助项目(2011AA02A211)

摘　　要：孕二烯酮是第三代高效避孕药的主要成分.雷斯青霉(Penicillium raistrickii)ATCC10490催化甾体左旋乙基甾烯双酮的C15α–羟基化反应合成孕二烯酮的重要中间体C15α–羟基左旋乙基甾烯双酮.为了提高现有生产菌种的转化效率,本文研究了在雷斯青霉ATCC10490中增加甾体C15α–羟化酶基因(PRH)拷贝数对转化效率的影响.分别构建了PRH基因的诱导型和组成型过表达载体pPZP-p800-PRH和pPZP-TrpC-PRH,并通过同源重组的方法定点整合到雷斯青霉基因组.转化实验表明,在出发菌株中增加一个PRH基因拷贝显著提高了甾体左旋乙基甾烯双酮转化效率,重组菌PRH-P800和PRH-TrpC的摩尔转化率分别提高了13%和20%,同时转化时间均缩短了12h.Gestodene is the main component of the third generation of high-performance contraceptives.Penicillium raistrickii ATCC10490 can catalyze 15α-hydroxylation of steroid 13β-ethyl-4-gonene-3,17-dione to synthesize 15α-hydroxy- 13β-ethyl-4-gonene-3,17-dione,a key intermediate of gestodene.To improve the transformation efficiency of P.raistrickii ATCC10490,the effect of copy number of 15α-hydroxylase gene(PRH)in ATCC10490 on steroid transformation efficiency was studied.Vectors pPZP-p800-PRH and pPZP-TrpC-PRH for inducible and constitutive expression of PRH were constructed and integrated into the targeted genome region of ATCC10490 by homologous recombination.Transformation experiments showed that the addition of extra copy of PRH in ATCC10490 significantly increased the conversion of 13 β-ethyl- 4-gonene-3,17-dione.The molar transformation rates of the two engineered strains PRH-P800 and PRH-TrpC,compared with the starting strain,increased by 13% and 20%,respectively,while the transformation time was shortened by 12 h.

关 键 词：雷斯青霉 孕二烯酮 15α–羟化酶 左旋乙基甾烯双酮 

分 类 号：Q781[生物学—分子生物学]