Abstract
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.
Highlights
Lignocellulosic biomass is a common resource across the globe, and its fermentation offers a prom‐ ising option for generating renewable liquid transportation fuels
Synthetic hybrids have intermediate traits or display heterosis To determine whether interspecies hybridization could introduce traits from other species relevant to biofuel production, we generated eight synthetic hybrids
We crossed haploid strains of S. cerevisiae previously engineered and experimentally evolved for xylose fermentation (Y128 and Y101, a heterothallic haploid derivative of Y73) [24, 25] with previously generated haploid derivatives of the reference strains of S. mikatae, S. kudriavzevii, S. uvarum [28, 50], and our new heterothallic haploid derivative of S. eubayanus (Table 1; Fig. 1), four of the most divergent members of the genus
Summary
Lignocellulosic biomass is a common resource across the globe, and its fermentation offers a prom‐ ising option for generating renewable liquid transportation fuels. Hydrolysates made from lignocellulosic sources contain high levels of pentose sugars, xylose, which native S. cerevisiae consumes poorly or not at all [8] These hydrolysates contain potent fermentation inhibitors that are mainly derived from the deconstruction of biomass during the chemical pretreatments used to improve the accessibility of cellulose and hemicellulose to hydrolysis [9]. After enzymatic treatment and the application of the ammonia fiber expansion (AFEX) method used to deconstruct corn stover [10], phenolic amides, phenolic acids, furans, and other small inhibitory molecules are generated [11]; these molecules are collectively termed “hydrolysate toxins (HTs).” Proposed mechanisms for their toxicity include the inhibition of key enzymatic steps, such as glutamine PRPP amidotransferase (PurF), which is important for de novo purine biosynthesis but inhibited by feruloyl amide in Escherichia coli [12]; decreased energy availability due to costly efflux pumps [13]; and redox imbalances caused by the detoxification of acids and aldehydes [9] After enzymatic treatment and the application of the ammonia fiber expansion (AFEX) method used to deconstruct corn stover [10], phenolic amides, phenolic acids, furans, and other small inhibitory molecules are generated [11]; these molecules are collectively termed “hydrolysate toxins (HTs).” Proposed mechanisms for their toxicity include the inhibition of key enzymatic steps, such as glutamine PRPP amidotransferase (PurF), which is important for de novo purine biosynthesis but inhibited by feruloyl amide in Escherichia coli [12]; decreased energy availability due to costly efflux pumps [13]; and redox imbalances caused by the detoxification of acids and aldehydes [9]
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