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 inCandida tropicalis

DOI：10.3969/j.issn.1673-1689.2018.08.015

中文关键词: 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

英文关键词: Candida tropicalis,carnitine acetyltransferase,gene disruption,URA3gene,gene disruption auxiliary sequence

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中文摘要:

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

英文摘要:

Development of gene deletion method is critical for metabolic engineering of diploid yeastCandida tropicalis. In this study，an efficient genetic manipulation method was developed and using this method two allelicCATgenes were sequentially deleted successfully. TheCAT 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 theURA3gene，and homologous arms ofCATgene，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 doubleCATmutant strains were obtained and characterized. Deletion of oneCATgenes could decrease enzyme activity significantly however growth profile was similar with parent strain on either glucose or alkane medium. Moreover，the growth of doubleCATgenes 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 fromURA3gene as gene disruption auxiliary sequence. Using this strategy，CATgene knockout mutants were constructed and its function was investigated.

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