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BackgroundHigh temperature inhibits cell growth and ethanol fermentation of Saccharomyces cerevisiae. As a complex phenotype, thermotolerance usually involves synergistic actions of many genes, thereby being difficult to engineer. The overexpression of either endogenous or exogenous stress-related transcription factor genes in yeasts was found to be able to improve relevant stress tolerance of the hosts.ResultsTo increase ethanol yield of high-temperature fermentation, we constructed a series of strains of S. cerevisiae by expressing 8 transcription factor genes from S. cerevisiae and 7 transcription factor genes from thermotolerant K. marxianus in S. cerevisiae. The results of growth curve measurements and spotting test show that KmHsf1 and KmMsn2 can enhance cell growth of S. cerevisiae at 40–42 °C. According to the results of batch fermentation at 43 °C with an initial glucose concentration of 104.8 g/l, the fermentation broths of KmHSF1 and KmMSN2-expressing strains could reach final ethanol concentrations of 27.2 ± 1.4 and 27.6 ± 1.2 g/l, respectively, while the control strain just produced 18.9 ± 0.3 g/l ethanol. Transcriptomic analysis found that the expression of KmHSF1 and KmMSN2 resulted in 55 (including 31 up-regulated and 24 down-regulated) and 50 (including 32 up-regulated and 18 down-regulated) genes with different expression levels, respectively (padj < 0.05). The results of transcriptomic analysis also reveal that KmHsf1 might increase ethanol production by regulating genes related to transporter activity to limit excessive ATP consumption and promote the uptake of glucose; while KmMsn2 might promote ethanol fermentation by regulating genes associated with glucose metabolic process and glycolysis/gluconeogenesis. In addition, KmMsn2 might also help to cope with high temperature by regulating genes associated with lipid metabolism to change the membrane fluidity.ConclusionsThe transcription factors KmHsf1 and KmMsn2 of thermotolerant K. marxianus can promote both cell growth and ethanol fermentation of S. cerevisiae at high temperatures. Different mechanisms of KmHsf1 and KmMsn2 in promoting high-temperature ethanol fermentation of S. cerevisiae were revealed by transcriptomic analysis.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0984-9) contains supplementary material, which is available to authorized users.

BackgroundLow ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation. As a complex phenotype, ethanol tolerance involves synergistic actions of many genes that are widely distributed throughout the genome, thereby being difficult to engineer. TATA-binding protein is the most common target of global transcription machinery engineering for improvement of complex phenotypes.ResultsA random mutagenesis library of K. marxianus TATA-binding protein Spt15 was constructed and subjected to screening under ethanol stress. Two mutant strains with improved ethanol tolerance were identified, one of which (denoted as M2) exhibited increased ethanol productivity. The mutant of Spt15 in strain M2 (denoted as Spt15-M2) has a single amino acid substitution at position 31 (Lys → Glu). RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from Spt15-M2. Spt15-M2 caused changes in transcriptional level of most of the genes in the central carbon metabolism network. Compared with control strain, 444 differentially expressed genes (DEGs) were identified in strain M2 (fold change > 2, Padj < 0.05), including 48 up-regulated and 396 down-regulated. The up-regulated DEGs are involved in amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway, while the down-regulated DEGs are related to ribosome biogenesis, translation and protein synthesis. Five candidate genes (GAP1, GNP1, FAR1, STE2 and TEC1), which were found to be up-regulated in M2 strain, were overexpressed for a gain-of-function assay. However, the overexpression of no single gene helped improve ethanol tolerance as SPT15-M2 did.ConclusionsThis work demonstrates that ethanol tolerance of K. marxianus can be improved by engineering its TATA-binding protein. A single amino acid substitution (K31E) of TATA-binding protein Spt15 is able to bring differential expression of hundreds of genes that acted as an interconnected network for the phenotype of ethanol tolerance. Future perspectives of this technique in K. marxianus were discussed.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1206-9) contains supplementary material, which is available to authorized users.

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