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IntroductionFurfural, a main inhibitor produced during pretreatment of lignocellulose, has shown inhibitory effects on S. cerevisiae.MethodIn the present study, new strains named 12–1 with enhanced resistance to furfural were obtained through adaptive laboratory evolution, which exhibited a shortened lag phase by 36 h, and an increased ethanol conversion rate by 6.67% under 4 g/L furfural.Results and DiscussionTo further explore the mechanism of enhanced furfural tolerance, ADR1_1802 mutant was constructed by CRISPR/Cas9 technology, based on whole genome re-sequencing data. The results indicated that the time when ADR1_1802 begin to grow was shortened by 20 h compared with reference strain (S. cerevisiae CEN.PK113-5D) when furfural was 4 g/L. Additionally, the transcription levels of GRE2 and ADH6 in ADR1_ 1802 mutant were increased by 53.69 and 44.95%, respectively, according to real-time fluorescence quantitative PCR analysis. These findings suggest that the enhanced furfural tolerance of mutant is due to accelerated furfural degradation. Importance: Renewable carbon worldwide is vital to achieve “zero carbon” target. Bioethanol obtained from biomass is one of them. To make bioethanol price competitive to fossil fuel, higher ethanol yield is necessary, therefore, monosaccharide produced during biomass pretreatment should be effectively converted to ethanol by Saccharomyces cerevisiae. However, inhibitors formed by glucose or xylose oxidation could make ethanol yield lower. Thus, inhibitor tolerant Saccharomyces cerevisiae is important to this process. As one of the main component of pretreatment hydrolysate, furfural shows obvious impact on growth and ethanol production of Saccharomyces cerevisiae. To get furfural tolerant Saccharomyces cerevisiae and find the underlying mechanism, adaptive laboratory evolution and CRISPR/Cas9 technology were applied in the present study

The salt-tolerant flavor yeast Zygosaccharomyces rouxii is an important food flavor microorganism, but its intracellular stress-resistant trehalose synthesis efficiency has been shown to be low, resulting in its weak high-temperature resistance. The intracellular and extracellular levels of carbohydrates, organic acids, and amino acids of Z. rouxii in a 20-L mechanically stirred ventilated fermenter were analyzed using metabolomics research methods. Our results showed that glucose supplementation could promote the growth of yeast cells, but high temperatures (> 35°C) significantly prevented cell growth. Under three different growth strategies, extracellular glucose was continuously utilized and intracellular glucose was continuously metabolized, but glucose overflow metabolism was inhibited by high temperature, which showed that the level of intracellular/extracellular ethanol was stable. High temperature stimulated significant intracellular trehalose accumulation (c20.5h = 80.78 mg/g Dry Cell Weight (DCW)) but not efflux, as well as xylitol accumulation (c20.5h = 185.97 mg/g DCW) but with efflux (c20.5h = 29.78 g/L). Moreover, heat resistance evaluation showed that xylitol and trehalose had heat-protective effects on Z. rouxii. In addition, a large amount of propionic acid and butyric acid accumulated inside and outside these cells, showing that the conversion of glucose to acid in yeast cells becomes the main pathway of glucose overflow metabolism in high temperatures. In addition, the increased demand of yeast cells for phenylalanine, threonine, and glycine at high temperatures suggested that these metabolites participated in the temperature adaptation of Z. rouxii in different ways. Valine and leucine/isoleucine [branched-chain amino acids (BCAAs)] were mainly affected by the addition of glucose, while glucose, sucrose, aspartic acid/asparagine, and glutamate/glutamine were not affected by this temperature regulation as a whole. This study could help deepen our understanding of the high-temperature adaptation mechanism of salt-tolerant Z. rouxii, and has theoretical significance for the application of highly tolerant yeast to food brewing.

2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for “natural” 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.

We isolated an aromatic strain of yeast (M2013310) from chili sauce. Assembly, annotation, and phylogenetic analysis based on genome sequencing, identified M2013310 as an allodiploid yeast that was closely related to Zygosaccharomyces rouxii. During fermentation, M2013310, produced an aromatic alcohol with a rose-honey scent; gas chromatography tandem mass spectrometry identified this alcohol as 2-phenylethanol. The concentration of 2-phenylethanol reached 3.8 mg/L, 1.79 g/L, and 3.58 g/L, in M3 (NH4+), M3 (NH4+ + Phe), and M3 (Phe) culture media, after 72 h of fermentation, respectively. The mRNA expression levels of ARO8 encoding aromatic aminotransferases I and ARO10 encoding phenylpyruvate decarboxylase by M2013310 in M3 (Phe) were the lowest of the three different forms of media tested. These results indicated that M2013310 can synthesize 2-phenylethanol via the Shikimate or Ehrlich pathways and the production of 2-phenylethanol may be significantly improved by the over-expression of these two genes. Our research identified a promising strain of yeast (M2013310) that could be used to improve the production of 2-phenylethanol.

Cordyceps militaris is a filamentous fungus used for both medicinal and culinary purposes. It exhibits a wide range of pharmacological activities due to its valuable contents of cordycepin, polysaccharides, carotenoids, terpenoids and other metabolites. However, C. militaris strains are highly susceptible to irreversible degradation in agricultural production, which is often manifested as a prolonged color change period and a significant decrease in the production of secondary metabolites. UDP-glycosyltransferases are an important enzyme family that participates in the synthesis of terpenoids by performing the glycosylation of key residues of enzymes or molecules. However, few studies have focused on its effect on the regulation of metabolite production in C. militaris. Therefore, in this study, we performed transcriptome analysis across four different developmental stages of C. militaris to target the putative glycosyltransferase gene CmUGT1, which plays important roles in metabolite production. We further constructed and screened a CmUGT1-overexpressing strain by Agrobacterium tumefaciens-mediated infestation of C. militaris spores. The major metabolite production of the wild-type and CmUGT1-overexpressing C. militaris strains was determined after short-term shake-flask cultivation of mycelia. The results showed that the yields of carotenoids and polysaccharides in the mycelia of the CmUGT1-overexpressing strains were 3.8 and 3.4 times greater than those in the mycelia of the wild type, respectively (p