Identification of multiple interacting alleles conferring low glycerol and high ethanol yield in Saccharomyces cerevisiae ethanolic fermentation

Hubmann, Georg; Mathé, Lotte; Foulquié-Moreno, María R.; Duitama, Jorge; Nevoigt, Elke; Thevelein, Johan M.

doi:10.1186/1754-6834-6-87

Cited by 44 publications

(33 citation statements)

References 45 publications

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“…However, this scheme requires specific knowledge of which biological parts are responsible for the traits of interest. Quantitative trait locus (QTL) mapping [67], comparative genomics [21, 68], chemical genomics [69], and several other methods have been successfully used to identify such parts, but each approach is labor intensive and costly.…”

Section: Discussionmentioning

confidence: 99%

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

Peris

Moriarty

Alexander

et al. 2017

Biotechnol Biofuels

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BackgroundLignocellulosic biomass is a common resource across the globe, and its fermentation offers a promising option for generating renewable liquid transportation fuels. The deconstruction of lignocellulosic biomass releases sugars that can be fermented by microbes, but these processes also produce fermentation inhibitors, such as aromatic acids and aldehydes. Several research projects have investigated lignocellulosic biomass fermentation by the baker’s yeast Saccharomyces cerevisiae. Most projects have taken synthetic biological approaches or have explored naturally occurring diversity in S. cerevisiae to enhance stress tolerance, xylose consumption, or ethanol production. Despite these efforts, improved strains with new properties are needed. In other industrial processes, such as wine and beer fermentation, interspecies hybrids have combined important traits from multiple species, suggesting that interspecies hybridization may also offer potential for biofuel research.ResultsTo investigate the efficacy of this approach for traits relevant to lignocellulosic biofuel production, we generated synthetic hybrids by crossing engineered xylose-fermenting strains of S. cerevisiae with wild strains from various Saccharomyces species. These interspecies hybrids retained important parental traits, such as xylose consumption and stress tolerance, while displaying intermediate kinetic parameters and, in some cases, heterosis (hybrid vigor). Next, we exposed them to adaptive evolution in ammonia fiber expansion-pretreated corn stover hydrolysate and recovered strains with improved fermentative traits. Genome sequencing showed that the genomes of these evolved synthetic hybrids underwent rearrangements, duplications, and deletions. To determine whether the genus Saccharomyces contains additional untapped potential, we screened a genetically diverse collection of more than 500 wild, non-engineered Saccharomyces isolates and uncovered a wide range of capabilities for traits relevant to cellulosic biofuel production. Notably, Saccharomyces mikatae strains have high innate tolerance to hydrolysate toxins, while some Saccharomyces species have a robust native capacity to consume xylose.ConclusionsThis research demonstrates that hybridization is a viable method to combine industrially relevant traits from diverse yeast species and that members of the genus Saccharomyces beyond S. cerevisiae may offer advantageous genes and traits of interest to the lignocellulosic biofuel industry.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0763-7) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

Peris

Moriarty

Alexander

et al. 2017

Biotechnol Biofuels

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“…The most widely used molecular markers in yeast are natural variations detected as hybridization differences on high-density oligonucleotide arrays (38) or determined by whole-genome sequencing (39,40). Several polygenic traits have been investigated by using these approaches, which led to the identification of loci, genes, and single nucleotide polymorphisms involved in hightemperature growth (39,40), sporulation efficiency (41), mRNA expression profiles (42), acetic acid production (43), resistance to chemical agents (44), high ethanol tolerance (34), maximal ethanol accumulation (45), low glycerol production (46,47), and thermotolerance (48).…”

Section: Discussionmentioning

confidence: 99%

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

et al. 2015

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Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3⌬0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.

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“…QTLs can be mapped at high resolution using high-throughput, whole-genome sequencing of pooled individuals from extremely large inbred populations 16,17 . Using QTL analysis to inform strain engineering has been proposed 3,[18][19][20] , and QTL mapping was previously used with marker assisted breeding for strain development 21 . The value for QTL analysis in strain engineering is sometimes evident in routine follow-up experiments using deletions, allele † replacements, or allele complementation to confirm QTLs; the resulting strains occasionally display slightly improved phenotypes [18][19][20][21][22][23][24] .…”

Section: Introductionmentioning

confidence: 99%

“…Engineering of complex traits can be hindered by unanticipated epistasis among otherwise beneficial alleles 19,23,25 . Epistasis in which the phenotypic effect of one or both alleles is inverted by genetic interaction 26,27 , when unanticipated, is problematic for strain development (e.g.…”

Section: Introductionmentioning

confidence: 99%

QTL-guided metabolic engineering of a complex trait

Maurer

Sutardja

Pinel

et al. 2016

Preprint

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Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic Bulk Reciprocal Hemizygosity analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this "genetic blueprint", and using a dual-guide Cas9-based method to efficiently perform multi-kilobase locus replacements, we engineered an S288C strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait in S. cerevisiae, as well as other organisms.

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Identification of multiple interacting alleles conferring low glycerol and high ethanol yield in Saccharomyces cerevisiae ethanolic fermentation

Cited by 44 publications

References 45 publications

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

QTL-guided metabolic engineering of a complex trait

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