Abstract
BackgroundGenetic 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.ResultsWe 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.ConclusionsOur 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.
Highlights
Rational genetic modification of industrial microorganisms using targeted deletion and/or overexpression of structural or regulatory genes very often results in undesirable side effects on other essential functions [1,2,3,4,5,6,7,8]
We present a novel approach to identify minor Quantitative trait locus (QTL), which does not suffer from the drawbacks that the phenotypic difference between the parent strains becomes smaller or that the number of segregants required for the screening increases
A backcross with the single segregant displaying low glycerol yield and lacking the ssk1E330N...K356N allele led to the identification of three new minor QTLs, in which we identified as causative genes specific alleles of known genes in glycerol metabolism and its regulation, each causing a reduction of glycerol yield
Summary
Rational genetic modification of industrial microorganisms using targeted deletion and/or overexpression of structural or regulatory genes very often results in undesirable side effects on other essential functions [1,2,3,4,5,6,7,8]. Rational genetic engineering of glycerol yield by modification of the main structural gene, GPD1, encoding glycerol 3-phosphate dehydrogenase (GPDH), the rate limiting enzyme of the glycerol biosynthesis pathway, has not been successful in obtaining appropriate industrial yeast strains with a modified glycerol/ethanol ratio due to the negative side-effects on other phenotypic traits. Genetic analysis of natural S. cerevisiae strains exhibiting an inherent glycerol yield significantly different from that of the industrial yeast strains to be improved, offers a promising strategy to identify mutant alleles suitable as gene tools for engineering glycerol production to obtain lower or higher yield, without causing negative side-effects on other essential traits. Previous work identified ssk1E330N...K356N as causative allele in strain CBS6412, which displayed the lowest glycerol/ ethanol ratio
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