基于筛选标记整合拷贝增加的酿酒酵母外源蛋白高效表达

High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae

摘要:

摘要:【目的】利用酿酒酵母内部核糖体进入位点(IRES)介导构建外源蛋白高效表达系统，构建酿酒酵母蛋白表达工程菌，为酿酒酵母在代谢工程中的应用奠定基础。【方法】首先分别构建含启动子Pilv5，Padh2，Ptdh3 的Promoter-mCherry-TIF4631 IRES-URA3共表达框，利用同源重组的方法将共表达框整合到酿酒酵母W303-1B-A 基因组中，经URA3功能回复筛选转化子。然后比较转化子中mCherry荧光强度的差异，以表征三种启动子在共表达框中的应用效果。利用荧光定量PCR测定并分析转化子中整合DNA片段在基因组中的拷贝数，并在无选择压力的条件下连续传代培养转化子，分析其遗传稳定性。最后以木糖还原酶基因XYL1，β-半乳糖苷酶基因LACZ替换共表达框中的mCherry基因，检测木糖还原酶(xylose reductase)和β-半乳糖苷酶(β-galactosidase)的酶活力、蛋白表达量等，以验证该表达框的应用效果。【结果】整合DNA片段的拷贝数和mCherry的表达量受启动子影响。其中含Padh2的转化子最低，含Ptdh3的转化子居中，含Pilv5的转化子最高。含有Pilv5启动子且mCherry表达量最高的转化子，整合DNA片段在基因组中的拷贝数为47，构建的工程菌株具有较好的遗传稳定性。在含Pilv5启动子和TIF4631 IRES的表达框中，木糖还原酶成功表达，其中活力最高的转化子WIX-10的酶活力为0.209 U/mg粗蛋白;β-半乳糖苷酶也成功表达，其中酶活力最高的转化子WIL-1的酶活力为12.58 U/mg 粗蛋白。【结论】在酿酒酵母中，由Pilv5启动子和TIF4631 IRES介导的外源蛋白表达系统能够高效表达外源蛋白，为外源蛋白在酿酒酵母中表达提供了新的策略，也为该系统在酿酒酵母代谢工程中的应用提供了充分的实验依据。

Abstract:

Abstract:［Objective］ High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed，which could be used for other applications of S. cerevisiae in metabolic engineering．［Method］ We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5，Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A．The URA3 + transformants were selected．By comparing the difference in the mean florescence value of mCherry in transformants，the effect of three promoters was detected in the co-expression cassette．The copy numbers of the interested genes in the genome were determined by Real-Time PCR．We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure． To verify the application of co-expression cassette，the ORF of mCherry was replaced by β-galactosidase (LACZ) and xylose reductase (XYL1)．The enzyme activities and production of β-galactosidase and xylose reductase were detected.［Result］ mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter． The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5．The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES．The highest enzyme activity was 0.209 U/mg crude protein in the transformants WIX-10.β-galactosidase was also expressed successfully．The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein.［Conclusion］ The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae．This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering application of this system in yeast.