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Abstract A suitable nutrient supply, especially of vitamins, is very significant for the deep display of the inherent genetic properties of microorganisms. Here, using the chemically defined minimal medium (MM) for yeast, nicotinamide and inositol were confirmed to be more beneficial for the performance of two industrial baker's yeasts, a conventional and a high-sugar-tolerant strain. Increasing nicotinamide or inositol to proper levels could enhance the both strains on cell growth and activity and product performance, including trehalose accumulation and leavening performance. The activity of key enzymes (PCK, TPS) and the content of intermediate metabolites (G6P, UDPG) in the trehalose synthesis pathway were promoted by a moderate supply of nicotinamide and inositol. That were also proved that an appropriate amount of niacinamide promoted the transcription of longevity-related genes (PNC1, SIR2), and the proper concentration of inositol altered the phospholipid composition in cells, namely, phosphatidylinositol and phosphatidyl choline. Furthermore, the cell growth and the leavening performance of the both strains were promoted after adjusting inositol to choline to the proper ratio, resulting directly in content changes of phosphatidylinositol and phosphatidyl choline in the cells. While the two strains responded to the different proper ratio of inositol to choline probably due to their specific physiological characteristics. Such beneficial effects of increased nicotinamide levels were confirmed in natural media, molasses and corn starch hydrolyzed sugar media. Meanwhile, such adjustment of inositol to choline ratio could lessen the inhibition of excess inositol on cell growth of the two tested strains in corn starch hydrolyzed sugar media. However, in molasse, such phenomenon was not observed probably since there was higher Ca2+ in it. The results indicated that the effects of nutrient factors, such as vitamins, on cell growth and other properties found out from the simple chemically defined minimal medium were an effective measure to use in improving the recipe of natural media at least for baker's yeast.

Abstract Improving the resistance of Saccharomyces cerevisiae to vanillin, derived from lignin, will benefit the design of robust cell factories for lignocellulosic biorefining. The transcription factor Yrr1p mediates S. cerevisiae resistance to various compounds. In this study, eleven predicted phosphorylation sites were mutated, among which 4 mutants of Yrr1p, Y134A/E and T185A/E could improve vanillin resistance. Both dephosphorylated and phosphorylated mutations at Yrr1p 134 and 185 gathered in the nucleus regardless of the presence or absence of vanillin. However, the phosphorylated mutant Yrr1p inhibited target gene expression, while dephosphorylated mutants promoted expression. Transcriptomic analysis showed that the dephosphorylated Yrr1p T185 mutant, under vanillin stress, upregulated ribosome biogenesis and rRNA processing. These results demonstrate the mechanism by which Yrr1p phosphorylation regulates the expression of target genes. The identification of key phosphorylation sites in Yrr1p offers novel targets for the rational construction of Yrr1p mutants to improve resistance to other compounds.

Studying the mechanisms of resistance to vanillin in microorganisms, which is derived from lignin and blocks a major pathway of DNA double-strand break repair in yeast, will benefit the design of robust cell factories that produce biofuels and chemicals using lignocellulosic materials. A high vanillin-tolerant Saccharomyces cerevisiae strain EMV-8 carrying site mutations compared to its parent strain NAN-27 was selected for the analyses. The dynamics of the chromatin structure of eukaryotic cells play a critical role in transcription and the regulation of gene expression and thus the phenotype. Consequently, Hi-C and transcriptome analyses were conducted in EMV-8 and NAN-27 in the log phase with or without vanillin stress to determine the effects of mutations and vanillin disturbance on the dynamics of three-dimensional chromosome organization and the influence of the organization on the transcriptome. The outcomes indicated that the chromosome interaction pattern disturbed by vanillin stress or genetic mutations in the log phase was similar to that in mouse cells. The short chromosomes contact the short chromosomes, and the long chromosomes contact the long chromosomes. In response to vanillin stress, the boundaries of the topologically associating domain (TAD) in the vanillin-tolerant strain EMV-8 were more stable than those in its parent strain NAN-27. The motifs of SFL1, STB3, and NHP6A/B were enriched at TAD boundaries in both EMV-8 and NAN-27 with or without vanillin, indicating that these four genes were probably related to TAD formation. The Indel mutation of YRR1, whose absence was confirmed to benefit vanillin tolerance in EMV-8, caused two new interaction sites that contained three genes, WTM2, PUP1, and ALE1, whose overexpression did not affect vanillin resistance in yeast. Overall, our results revealed that in the log phase, genetic mutations and vanillin disturbance have a negligible effect on three-dimensional chromosome organization, and the reformation or disappearance of TAD boundaries did not show an association with gene expression, which provides an example for studying yeast chromatin structure during stress tolerance using Hi-C technology.

As a potential alternative to fossil-based fuels, cellulosic ethanol has attracted much attention due to its great benefit to energy sustainability and environmental friendliness. However, at present, the industrial competitiveness of cellulosic ethanol production is still insufficient compared with fossil-based fuels because of the higher costs. Expanding the range of lignocellulosic biomass may be a promising measure to promote the economical production of cellulosic ethanol. Corn fiber, a byproduct from the corn deep-processing, is an attractive feedstock for cellulosic ethanol production because of its rich carbohydrate content (generally exceeding 65% of dry weight), almost no transportation cost, and low lignin content allow it to be easily handled. This study first optimized the hydrolysis conditions, including the pretreatment and enzymolysis process based on dilute sulfuric acid, to achieve a high sugar yield. Then, the corn fiber hydrolysates obtained under different hydrolysis conditions were suitably fermented by different C5/C6 co-fermentation Saccharomyces cerevisiae, indicating that the hydrolysate at high solid loading (20%) needs to detoxification to a certain extent but not low solid loading (10%) to achieve high ethanol yield. Finally, the fermentation of the 20% solid loading hydrolysates with resin detoxification was performed in a 50 L bioreactor, achieving the sugar (glucose and xylose) metabolic rate of 2.24 g L −1 h −1 and ethanol yield of 92% of the theoretical value, which are the highest reported levels to date. This study provided a potential process route for cellulosic ethanol production from corn fiber from the perspective of the suitability between the upstream hydrolysis process and the downstream fermentation strain.

The efficient conversion of cellulosic sugars is vital for the economically viable production of biofuels/biochemicals from lignocellulosic biomass hydrolysates. Based on comprehensive screening, Saccharomyces cerevisiae RC212 was chosen as the chassis strain for multiple integrations of heterologous β-glucosidase and β-xylosidase genes in the present study. The resulting recombinant BLN26 and LF1 form a binary synthetic consortium, and this co-culture system achieved partial fermentation of four sugars (glucose, xylose, cellobiose, and xylo-oligosaccharides). Then, we developed a ternary S. cerevisiae consortium consisting of LF1, BSGIBX, and 102SB. Almost all four sugars were efficiently fermented to ethanol within 24 h, and the ethanol yield is 0.482 g g−1 based on the consumed sugar. To our knowledge, this study represents the first exploration of the conversion of mixtures of glucose, xylose, cellobiose, and xylo-oligosaccharides by a synthetic consortium of recombinant S. cerevisiae strains. This synthetic consortium and subsequent improved ones have the potential to be used as microbial platforms to produce a wide array of biochemicals from lignocellulosic hydrolysates.

Abstract Background Vanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin. Results In wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae. Conclusions Cells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

Summary Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5′ caps, thus cannot be translated to proteins. To screen high‐nucleic‐acid content yeasts more efficiently, a cap‐independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I‐recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo‐dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap‐dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high‐nucleic‐acid content yeasts.