三乙酸内酯(triacetic acid lactone, TAL)是一种具有广泛应用前景的聚酮,可用作各种有机化合物的前体。为了提高酿酒酵母(Saccharomyces cerevisiae)中TAL的产量,该研究利用基因组重复序列整合的方法构建了S.cerevisiae多拷贝整合系统,应用该系统提高TAL的生产。首先以绿色荧光蛋白表征多拷贝Delta1整合,通过截短遗传霉素和潮霉素B两种抗生素基因的启动子以及增加抗生素浓度的方法增加筛选压力,进而提高整合效率和拷贝数,最优参数为将抗生素基因启动子截短至15 bp的同时采用160 μg/mL的抗生素。将该优化系统随后用于新的多拷贝整合位点Delta2的表征。最后应用该系统合成TAL,通过HPLC分析TAL产量。结果表明,利用Delta1和Delta2序列表征绿色荧光蛋白的最高拷贝数分别为10和7,Delta1略优于Delta2。应用该系统在S.cerevisiae中合成TAL,Delta1和Delta2的产量分别为1.50、1.17 mmol/L,比单拷贝菌株产量分别提高460%和337%。该多拷贝系统有效提高了TAL的产量,为S.cerevisiae异源途径的表达提供了一种高效、模块化的多拷贝整合方法。
Triacetic acid lactone (TAL) is a promising platform polyketide with broad applications, especially it can be used as a precursor for the synthesis of various organic compounds.This study characterized the repeating sequences on the yeast genome to integrate the TAL biosynthesis pathway into these sites for enhanced gene expression and TAL production by Saccharomyces cerevisiae.Firstly, this study used the green fluorescent protein as the reporter to characterize multi-copy integration by the Delta1 site.It showed that truncating the promoter of a selection marker gene (for geneticin or hygromycin B) or increasing the antibiotic concentration, were effective in improving the integration efficiency and copy numbers.The highest copy number of multi-copy integration was obtained when truncating the antibiotic gene promoter to 15 bp, and using an antibiotic at 160 μg/mL.The optimization system was subsequently used to characterize the second multi-copy integration site, Delta2, with similar results.Then, the optimized multi-copy integration system was applied to introduce the TAL biosynthesis pathway into S.cerevisiae.The highest pathway copies using Delta1 and Delta2 sequences were 10 and 7, respectively, with Delta1 slightly better than Delta2.After 48 h fermentation in YTD medium, TAL titers of the Delta1-integration strain and the Delta2-integration strain were 1.50 mmol/L and 1.17 mmol/L, respectively, 460% and 337% higher than the single-copy integration strain.This study demonstrates that the multi-copy integration system is an efficient approach to introducing heterologous pathways into S.cerevisiae to improve biocatalytic efficiency.
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