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Saccharomyces cerevisiae FLO1 Gene Demonstrates Genetic Linkage to Increased Fermentation Rate at Low Temperatures.

ABSTRACT: Low fermentation temperatures are of importance to food and beverage industries working with Saccharomyces cerevisiae Therefore, the identification of genes demonstrating a positive impact on fermentation kinetics is of significant interest. A set of 121 mapped F₁ progeny, derived from a cross between haploid strains BY4716 (a derivative of the laboratory yeast S288C) and wine yeast RM11-1a, were fermented in New Zealand Sauvignon Blanc grape juice at 12.5°. Analyses of five key fermentation kinetic parameters among the F₁ progeny identified a quantitative trait locus (QTL) on chromosome I with a significant degree of linkage to maximal fermentation rate (V_max) at low temperature. Independent deletions of two candidate genes within the region, FLO1 and SWH1, were constructed in the parental strains (with S288C representing BY4716). Fermentation of wild-type and deletion strains at 12.5 and 25° confirmed that the genetic linkage to V_max corresponds to the S288C version of the FLO1 allele, as the absence of this allele reduced V_max by ?50% at 12.5°, but not at 25°. Reciprocal hemizygosity analysis (RHA) between S288C and RM11-1a FLO1 alleles did not confirm the prediction that the S288C version of FLO1 was promoting more rapid fermentation in the opposing strain background, suggesting that the positive effect on V_max derived from S288C FLO1 may only provide an advantage in haploids, or is dependent on strain-specific cis or trans effects. This research adds to the growing body of evidence demonstrating the role of FLO1 in providing stress tolerance to S. cerevisiae during fermentation.

SUBMITTER: Deed RC

PROVIDER: S-EPMC5345705 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Saccharomyces cerevisiae FLO1 Gene Demonstrates Genetic Linkage to Increased Fermentation Rate at Low Temperatures.

Deed Rebecca C RC Fedrizzi Bruno B Gardner Richard C RC

G3 (Bethesda, Md.) 20170310 3

Low fermentation temperatures are of importance to food and beverage industries working with Saccharomyces cerevisiae Therefore, the identification of genes demonstrating a positive impact on fermentation kinetics is of significant interest. A set of 121 mapped F1 progeny, derived from a cross between haploid strains BY4716 (a derivative of the laboratory yeast S288C) and wine yeast RM11-1a, were fermented in New Zealand Sauvignon Blanc grape juice at 12.5°. Analyses of five ke ...[more]

PMID: 28143947

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Project description:Background:High 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. Results:To 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. Conclusions:The 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.

Project description:BACKGROUND:Genetic engineering of industrial microorganisms often suffers from undesirable side effects on essential functions. Reverse engineering is an alternative strategy to improve multifactorial traits like low glycerol/high ethanol yield in yeast fermentation. Previous rational engineering of this trait always affected essential functions like growth and stress tolerance. We have screened Saccharomyces cerevisiae biodiversity for specific alleles causing lower glycerol/higher ethanol yield, assuming higher compatibility with normal cellular functionality. Previous work identified ssk1E330N…K356N as causative allele in strain CBS6412, which displayed the lowest glycerol/ethanol ratio. RESULTS:We have now identified a unique segregant, 26B, that shows similar low glycerol/high ethanol production as the superior parent, but lacks the ssk1E330N…K356N allele. Using segregants from the backcross of 26B with the inferior parent strain, we applied pooled-segregant whole-genome sequence analysis and identified three minor quantitative trait loci (QTLs) linked to low glycerol/high ethanol production. Within these QTLs, we identified three novel alleles of known regulatory and structural genes of glycerol metabolism, smp1R110Q,P269Q, hot1P107S,H274Y and gpd1L164P as causative genes. All three genes separately caused a significant drop in the glycerol/ethanol production ratio, while gpd1L164P appeared to be epistatically suppressed by other alleles in the superior parent. The order of potency in reducing the glycerol/ethanol ratio of the three alleles was: gpd1L164P?>?hot1P107S,H274Y???smp1R110Q,P269Q. CONCLUSIONS:Our results show that natural yeast strains harbor multiple specific alleles of genes controlling essential functions, that are apparently compatible with survival in the natural environment. These newly identified alleles can be used as gene tools for engineering industrial yeast strains with multiple subtle changes, minimizing the risk of negatively affecting other essential functions. The gene tools act at the transcriptional, regulatory or structural gene level, distributing the impact over multiple targets and thus further minimizing possible side-effects. In addition, the results suggest polygenic analysis of complex traits as a promising new avenue to identify novel components involved in cellular functions, including those important in industrial applications.

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Project description:BackgroundSaccharomyces cerevisiae, a key organism used for the manufacture of renewable fuels and chemicals, has been engineered to utilize non-native sugars derived from plant cell walls, such as cellobiose and xylose. However, the rates and efficiencies of these non-native sugar fermentations pale in comparison with those of glucose. Systems biology methods, used to understand biological networks, hold promise for rational microbial strain development in metabolic engineering. Here, we present a systematic strategy for optimizing non-native sugar fermentation by recombinant S. cerevisiae, using cellobiose as a model.ResultsDifferences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induces mitochondrial activation and reduces amino acid biosynthesis under fermentation conditions. Furthermore, glucose-sensing and signaling pathways and their target genes, including the cAMP-dependent protein kinase A pathway controlling the majority of glucose-induced changes, the Snf3-Rgt2-Rgt1 pathway regulating hexose transport, and the Snf1-Mig1 glucose repression pathway, were at most only partially activated under cellobiose conditions. To separate correlations from causative effects, the expression levels of 19 transcription factors perturbed under cellobiose conditions were modulated, and the three strongest promoters under cellobiose conditions were applied to fine-tune expression of the heterologous cellobiose-utilizing pathway. Of the changes in these 19 transcription factors, only overexpression of SUT1 or deletion of HAP4 consistently improved cellobiose fermentation. SUT1 overexpression and HAP4 deletion were not synergistic, suggesting that SUT1 and HAP4 may regulate overlapping genes important for improved cellobiose fermentation. Transcription factor modulation coupled with rational tuning of the cellobiose consumption pathway significantly improved cellobiose fermentation.ConclusionsWe used systems-level input to reveal the regulatory mechanisms underlying suboptimal metabolism of the non-glucose sugar cellobiose. By identifying key transcription factors that cause suboptimal cellobiose fermentation in engineered S. cerevisiae, and by fine-tuning the expression of a heterologous cellobiose consumption pathway, we were able to greatly improve cellobiose fermentation by engineered S. cerevisiae. Our results demonstrate a powerful strategy for applying systems biology methods to rapidly identify metabolic engineering targets and overcome bottlenecks in performance of engineered strains.

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Saccharomyces cerevisiae FLO1 Gene Demonstrates Genetic Linkage to Increased Fermentation Rate at Low Temperatures.

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<i>Saccharomyces cerevisiae FLO1</i> Gene Demonstrates Genetic Linkage to Increased Fermentation Rate at Low Temperatures.

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