Stress tolerance is one of the important desired microbial traits for industrial bioprocesses, and global regulatory protein engineering is an efficient approach to improve strain tolerance. In our study, IrrE, a global regulatory protein from the prokaryotic organism Deinococcus radiodurans, was engineered to confer yeast improved tolerance to the inhibitors in lignocellulose hydrolysates or high temperatures. Three IrrE mutations were developed through directed evolution, and the expression of these mutants could improve the yeast fermentation rate by threefold or more in the presence of multiple inhibitors. Subsequently, the tolerance to multiple inhibitors of single-site mutants based on the mutations from the variants were then evaluated, and 11 mutants, including L65P, I103T, E119V, L160F, P162S, M169V, V204A, R244G, Base 824 Deletion, V299A, and A300V were identified to be critical for the improved representative inhibitors, i.e., furfural, acetic acid and phenol (FAP) tolerance. Further studies indicated that IrrE caused genome-wide transcriptional perturbation in yeast, and the mutant I24 led to the rapid growth of Saccharomyces cerevisiae by primarily regulating the transcription level of transcription activators/factors, protecting the intracellular environment and enhancing the antioxidant capacity under inhibitor environments, which reflected IrrE plasticity. Meanwhile, we observed that the expression of the wild-type or mutant IrrE could also protect Saccharomyces cerevisiae from the damage caused by thermal stress. The recombinant yeast strains were able to grow with glucose at 42 ℃. IrrE from Deinococcus radiodurans can be engineered as a tolerance-enhancer for Saccharomyces cerevisiae. Systematic research on the regulatory model and mechanism of a prokaryotic global regulatory factor IrrE to increase yeast tolerance provided valuable insights for the improvements in microbial tolerance to complex industrial stress conditions.

中文翻译：

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工程原核生物调节剂IrrE可增强发芽酵母的抗逆性

压力耐受性是工业生物过程重要的重要微生物特性之一，而全局调节蛋白工程是提高菌株耐受性的有效方法。在我们的研究中，IrrE是一种原核生物放线菌Deinococcus radiodurans的全球调节蛋白，被设计用于赋予酵母对木质纤维素水解产物或高温中抑制剂更高的耐受性。通过定向进化开发了三个IrrE突变体，在多种抑制剂的存在下，这些突变体的表达可以使酵母发酵速率提高三倍或更多。随后，基于变体的突变，评估了对单一位点突变体的多种抑制剂的耐受性，并评估了11个突变体，包括L65P，I103T，E119V，L160F，P162S，M169V，V204A，R244G，Base 824缺失，V299A，鉴定出A3和A300V对于改善代表性的抑制剂（例如糠醛，乙酸和苯酚（FAP）的耐受性）至关重要。进一步的研究表明，IrrE在酵母中引起全基因组转录扰动，而突变体I24通过主要调节转录激活因子/因子的转录水平，保护细胞内环境并增强抑制剂环境下的抗氧化能力，导致酿酒酵母快速生长。 ，反映出IrrE可塑性。同时，我们观察到野生型或突变IrrE的表达也可以保护酿酒酵母免受热应激造成的损害。重组酵母菌株能够在42℃葡萄糖条件下生长。可以将放射性杜鹃球菌的IrrE工程化为啤酒酵母的耐受性增强剂。对增加酵母耐受性的原核生物全球调节因子IrrE的调节模型和机理的系统研究为改善对复杂工业压力条件的微生物耐受性提供了宝贵的见解。