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

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
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< i> 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