Combination of mutagenesis and adaptive evolution to engineer salt-tolerant and aroma-producing yeast for soy sauce fermentation.

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The moromi fermentation of high-salt liquid-state fermentation (HLF) soy sauce is usually performed in high-brine solution (17-20%, w/w), which decreases the metabolic activity of aroma-producing yeast. To enhance the soy sauce flavors, increasing the salt tolerance of aroma-producing yeasts is very important for HLF soy sauce fermentation. In the present study, atmospheric and room-temperature plasma (ARTP) was first used to mutate the aroma-producing yeast Wickerhamomyces anomalus, and the salt tolerant strains were obtained by selection of synthetic medium with a sodium chloride concentration of 18% (w/w). Furthermore, adaptive laboratory evolution (ALE) was used to improve the salt tolerance of the mutant strains. The results obtained indicated that the combination use of ARTP and ALE markedly increased the NaCl tolerance of the yeast by increasing the cellular accumulation of K+ and removal of cytosolic Na+ , in addition to promoting the production of glycerin and strengthening the integrity of the cell membrane and cell wall. In soy sauce fermentation, the engineered strains improved the physicochemical parameters of HLF soy sauce compared to those produced by the wild-type strain, and the engineered strains also increased the alcohol, acid and aldehyde production, and enriched the types of esters in the soy sauce. The results of the present study indicated that the combination of ARTP mutagenesis and ALE significantly improved the salt tolerance of the aroma-producing yeast, and also enhanced the production of volatiles of HLF soy sauce. © 2021 Society of Chemical Industry.