Department of Biotechnology, University of the Western Cape, Bellville 7530, South Africa.
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
FEMS Yeast Res. 2019 Jun 1;19(4). doi: 10.1093/femsyr/foz035.
To enable Saccharomyces cerevisiae to produce renewable fuels from lignocellulose in a consolidated bioprocess, a heterologous cellulase system must be engineered into this yeast. In addition, inherently low secretion titers and sensitivity to adverse environmental conditions must be overcome. Here, two native S. cerevisiae genes related to yeast stress tolerance, YHB1 and SET5, were overexpressed under transcriptional control of the constitutive PGK1 promoter and their effects on heterologous secretion of Talaromyces emersonii cel7A cellobiohydrolase was investigated. Transformants showed increased secreted enzyme activity that ranged from 22% to 55% higher compared to the parental strains and this did not lead to deleterious growth effects. The recombinant strains overexpressing either YHB1 or SET5 also demonstrated multi-tolerant characteristics desirable in bioethanol production, i.e. improved tolerance to osmotic and heat stress. Quantitative reverse transcriptase PCR analysis in these strains showed decreased transcription of secretion pathway genes. However, decreased unfolded protein response was also observed, suggesting novel mechanisms for enhancing enzyme production through stress modulation. Overexpression of YHB1 in an unrelated diploid strain also enhanced stress tolerance and improved ethanol productivity in medium containing acetic acid. To our knowledge, this is the first demonstration that improved heterologous secretion and environmental stress tolerance could be engineered into yeast simultaneously.
为了使酿酒酵母能够在一个整合生物加工过程中从木质纤维素生产可再生燃料，必须将异源纤维素酶系统工程化到这种酵母中。此外，必须克服固有低分泌效价和对不利环境条件的敏感性。在这里，两个与酵母应激耐受相关的天然酿酒酵母基因 YHB1 和 SET5 在组成型 PGK1 启动子的转录控制下过表达，并研究了它们对塔宾曲霉 cel7A 纤维二糖水解酶异源分泌的影响。转化体显示出增加的分泌酶活性，与亲本菌株相比，活性提高了 22%至 55%，而这并没有导致有害的生长效应。过表达 YHB1 或 SET5 的重组菌株还表现出在生物乙醇生产中所需的多重耐受特性，即提高了对渗透压和热应激的耐受性。在这些菌株中进行的定量逆转录 PCR 分析显示，分泌途径基因的转录减少。然而，也观察到未折叠蛋白反应的减少，这表明通过应激调节增强酶生产的新机制。在一个不相关的二倍体菌株中过表达 YHB1 也增强了应激耐受性，并提高了含有乙酸的培养基中的乙醇生产率。据我们所知，这是首次证明可以同时将改进的异源分泌和环境应激耐受工程化到酵母中。
