热带假丝酵母肉毒碱乙酰基转移酶基因的删除及功能鉴定  

Carnitine Acetyltransferase Gene Disruption and Function Analysis in Candida Tropicalis

作　　者：张利华[1,2] 陈献忠[1,2] 陈振[1,2] 沈微 樊游[1,2] ZHANG Lihua;CHEN Xianzhong;CHEN Zhen;SHEN Wei;FAN You(Key Laboratory of Industrial Biotechnology,Ministry of Education,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2018年第8期880-887,共8页Journal of Food Science and Biotechnology

基　　金：教育部留学回国人员科研启动项目(1014130201150030)

摘　　要：缺乏高效基因删除技术是限制热带假丝酵母菌株代谢工程育种的重要因素。论文发展了一种新型的基因删除技术并利用该技术成功删除了肉毒碱乙酰基转移酶的两个等位基因。首先PCR扩增标记基因(URA3)的3'端324 bp序列作为基因删除辅助序列(gda序列),同向插入到URA3基因的5'端,在此基础上构建两端含有CAT基因同源臂序列的基因删除突变盒,转化宿主菌XZX,获得CAT基因单拷贝和双拷贝敲除的突变株。PCR鉴定和DNA测序结果表明,获得的URA3基因弹出后的突变株,仅在基因组重组位点上残留一个gda序列。进一步鉴定了突变株的生理性能,单拷贝敲除菌株CAT活性较亲本下降80%,但在葡萄糖或烷烃上的生长性能并不受显著影响。双拷贝缺失菌株CAT活性完全丧失,不能利用烷烃生长,同时在葡萄糖培养基中的生长受到显著影响,最终生物量达到7.56(OD600),仅为出发菌株的57.8%。Development of gene deletion method is critical for metabolic engineering of diploid yeast Candida tropicalis.In this study,an efficient genetic manipulation method was developed and using this method two allelic CAT genes were sequentially deleted successfully.The CAT gene deletion cassette arm-gda-URA3-arm which contains a functional URA3 gene flanked by a 324 bp gene disruption auxiliary(gda)sequence direct repeat derived from downstream of the URA3 gene,and homologous arms of CAT gene,was constructed.The growth properties and enzymatic activity of various mutants were characterized.Transformants were isolated from minimal medium plates and sprayed on the 5-FOA selection medium plates again.The resulting mutant strains,in which URA3 marker was pop-out via recombination of gda sequence,were confirmed by PCR and DNA sequencing.After excision,only one copy of the gda sequence remains behind at the recombinant locus.Single CAT mutant and double CAT mutant strains were obtained and characterized.Deletion of one CAT genes could decrease enzyme activity significantly however growth profile was similar with parent strain on either glucose or alkane medium.Moreover,the growth of double CAT genes mutant strain was completely deficient compared to the parent strain using glucose as substrate.In summary,An efficient gene deletion strategy was developed using gda fragment derived from URA3 gene as gene disruption auxiliary sequence.Using this strategy,CAT gene knockout mutants were constructed and its function was investigated.

关 键 词：热带假丝酵母 肉毒碱乙酰基转移酶 基因敲除 URA3基因 基因删除辅助序列 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]