Abstract
BackgroundStress 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.ResultsThree 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 ℃.ConclusionsIrrE 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.
Highlights
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
Expressing IrrE to enhance yeast tolerance to acetic acid To study the effect of IrrE on the inhibitor tolerance of S. cerevisiae, we first evaluated the IrrE role on strain tolerance to acetic acid, a primary inhibitor in lignocellulose hydrolysates
The results showed that the heterologous expression of IrrE slightly enhanced the ability of S. cerevisiae cells to resist acetic acid shock, but it did not confer to strains the desired acid tolerance
Summary
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. IrrE, a global regula‐ tory protein from the prokaryotic organism Deinococcus radiodurans, was engineered to confer yeast improved toler‐ ance to the inhibitors in lignocellulose hydrolysates or high temperatures. Furan aldehydes, and carboxylic acids are three main groups of inhibitory molecules present in the lignocellulosic pretreated hydrolysates [12]. Our group has conducted studies on the tolerance mechanism against multiple inhibitors of FAP and has found increasing proline and myoinositol as the new determinants for improving strain tolerance to FAP [13, 14]. Simultaneously regulating multiple genes may be a feasible route to enhance strain tolerance to complex inhibitors
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