Lahiru N. Jayakody

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

ABSTRACT Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO2 production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.

Improvement of tolerance of Saccharomyces cerevisiae to hot-compressed water-treated cellulose by expression of ADH1

Hot-compressed water treatment of cellulose and hemicellulose for subsequent bioethanol production is a novel, economically feasible, and nonhazardous method for recovering sugars. However, the hot-compressed water-treated cellulose and hemicellulose inhibit subsequent ethanol fermentation by the yeast Saccharomyces cerevisiae. To overcome this problem, we engineered a yeast strain with improved tolerance to hot-compressed water-treated cellulose. We first determined that glycolaldehyde has a greater inhibitory effect than 5-HMF and furfural and a combinational effect with them. On the basis of the hypothesis that the reduction of glycolaldehyde to ethylene glycol should detoxify glycolaldehyde, we developed a strain overexpressing the alcohol dehydrogenase gene ADH1. The ADH1-overexpressing strain exhibits an improved fermentation profile in a glycolaldehyde-containing medium. The conversion ratio of glycolaldehyde to ethylene glycol is 30 ± 1.9% when the control strain is used; this ratio increases to 77 ± 3.6% in the case of the ADH1-overexpressing strain. A glycolaldehyde treatment and the overexpression of ADH1 cause changes in the fermentation products so as to balance the metabolic carbon flux and the redox status. Finally, the ADH1-overexpressing strain shows a statistically significantly improved fermentation profile in a hot-compressed water-treated cellulose-containing medium. The conversion ratio of glycolaldehyde to ethylene glycol is 33 ± 0.85% when the control strain is used but increases to 72 ± 1.7% in the case of the ADH1-overexpressing strain. These results show that the reduction of glycolaldehyde to ethylene glycol is a promising strategy to decrease the toxicity of hot-compressed water-treated cellulose. This is the first report on the improvement of yeast tolerance to hot-compressed water-treated cellulose and glycolaldehyde.

SUMO expression shortens the lag phase of Saccharomyces cerevisiae yeast growth caused by complex interactive effects of major mixed fermentation inhibitors found in hot-compressed water-treated lignocellulosic hydrolysate

The complex inhibitory effects of inhibitors present in lignocellulose hydrolysate suppress the ethanol fermentation of Saccharomyces cerevisiae. Although the interactive inhibitory effects play important roles in the actual hydrolysate, few studies have investigated glycolaldehyde, the key inhibitor of hot-compressed water-treated lignocellulose hydrolysate. Given this challenge, we investigated the interactive effects of mixed fermentation inhibitors, including glycolaldehyde. First, we confirmed that glycolaldehyde was the most potent inhibitor in the hydrolysate and exerted interactive inhibitory effects in combination with major inhibitors. Next, through genome-wide analysis and megavariate data modeling, we identified SUMOylation as a novel potential mechanism to overcome the combinational inhibitory effects of fermentation inhibitors. Indeed, overall SUMOylation was increased and Pgk1, which produces an ATP molecule in glycolysis by substrate-level phosphorylation, was SUMOylated and degraded in response to glycolaldehyde. Augmenting the SUMO-dependent ubiquitin system in the ADH1-expressing strain significantly shortened the lag phase of growth, released cells from G2/M arrest, and improved energy status and glucose uptake in the inhibitor-containing medium. In summary, our study was the first to establish SUMOylation as a novel platform for regulating the lag phase caused by complex fermentation inhibitors.

Structural Determination of Glucosylceramides in the Distillation Remnants of Shochu, the Japanese Traditional Liquor, and Its Production by Aspergillus kawachii

Shochu is traditional Japanese liquor produced from various crops and fungi Aspergillus kawachi or A. awamorii . The amount of unutilized shochu distillation remnants is increasing because of the recent prohibition of ocean dumping of these remnants. In this Article, we first describe the structures of glucosylceramides contained in shochu distillation remnants by fragment ion analysis using ESI-tandem mass spectrometry. Shochu distillation remnant produced from barley contained glucosylceramides d18:2/C16:0h, d18:2/C20:0h, d19:2/C18:1h, and d18:2/C18:0h. Koji (barley fermented with A. kawachii) contained the same glucosylceramides. Shochu distillation remnants produced from rice contained glucosylceramides d18:2/C18:0h and d19:2/C18:1h. The culture broth of A. kawachii contained glucosylceramides d19:2/C18:1h and d19:2/C18:0h. These results indicate that the glucosylceramides contained in crops and those produced by A. kawachii transfer through the processes of fermentation with yeast and distillation to the shochu distillation remnant. This information will enable utilization of shochu distillation remnants and koji as novel sources of sphingolipids.

Identification and detoxification of glycolaldehyde, an unattended bioethanol fermentation inhibitor

Abstract Although there have been approximately 60 chemical compounds identified as potent fermentation inhibitors in lignocellulose hydrolysate, our research group recently discovered glycolaldehyde as a key fermentation inhibitor during second generation biofuel production. Accordingly, we have developed a yeast S. cerevisiae strain exhibiting tolerance to glycolaldehyde. During this glycolaldehyde study, we established novel approaches for rational engineering of inhibitor-tolerant S. cerevisiae strains, including engineering redox cofactors and engineering the SUMOylation pathway. These new technical dimensions provide a novel platform for engineering S. cerevisiae strains to overcome one of the key barriers for industrialization of lignocellulosic ethanol production. As such, this review discusses novel biochemical insight of glycolaldehyde in the context of the biofuel industry.

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

ABSTRACT In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.

Metabolic Engineering of Probiotic Saccharomyces boulardii

ABSTRACT Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.

The Role of GroE Chaperonins in Developing Biocatalysts for Biofuel andChemical Production

As the need and interest for producing renewable biofuels and biochemical has grown, new avenues to improve product yields and productivity have been explored. Specifically, improving the tolerance of host microbes towards stressors, such as heat shock or the presence of harmful solvents, has been an especially important route to improve industrial-scale chemical production. In this review, we discuss recent advances in microbial engineering for renewable chemical production through the introduction and expression of chaperonins, especially the bacterial GroE complex. The GroE complex provides a closed-off environment and allows vital proteins to enter and engage in post-translational folding or refolding in a more-ideal environment, allowing the microbe to possess increased survival rates in low/high temperatures or in high concentrations of otherwise harmful end-products. Overall, we highlighted how chaperonin systems such as the GroE complex could have many industrially-relevant uses in the coming years.

Mitigating health risks associated with alcoholic beverages through metabolic engineering

Epidemiological studies have established a positive relationship between the occurrence of cancer and consumption of alcoholic beverages. Metabolic engineering of brewing yeast to reduce potential carcinogenic compounds in alcoholic beverage is technically feasible as well as economically promising. This review presents the mechanisms of formation of potentially carcinogenic components in alcoholic beverages, such as formaldehyde, acetaldehyde, ethyl carbamate, acrylamide, and heavy metals, and introduces effective genetic perturbations to minimize the concentrations of these harmful components. As precise and effective genome editing tools for polyploid yeast are now available, we envision that yeast metabolic engineering might open up new research directions for improving brewing yeast in order to ensure product safety as well as to increase overall quality of alcoholic beverages.

Expression of Gre2p improves tolerance of engineered xylose-fermenting Saccharomyces cerevisiae to glycolaldehyde under xylose metabolism

Engineered S. cerevisiae employing the xylose reductase pathway enables efficient xylose valorization to fuels and chemicals. However, toxicity of thermochemically pretreated biomass hydrolysate on S. cerevisiae is one of the key technical challenges to upgrade biomass-derived sugars including xylose and glucose into high-value products. We investigated the effect of glycolaldehyde, one of the biomass-derived highly toxic aldehyde compounds, and its combinatorial inhibitory effect with other major fermentation inhibitors commonly found in plant hydrolysate such as methylglyoxal, 5-HMF, furfural, vanillin, and acetic acid on engineered xylose-fermenting S. cerevisiae in xylose and/or glucose media. We elucidated that glycolaldehyde and methylglyoxal are the key inhibitory short-aliphatic aldehydes on engineered xylose-fermenting S. cerevisiae in xylose-containing medium. Indeed, the degree of toxicity of these tested fermentation inhibitors varies with the sole carbon source of the medium. We demonstrate that genome integration of an extra copy of autologous GRE2 with its native promotor substantially improved the toxic tolerance of engineered xylose-fermenting S. cerevisiae to major inhibitory compounds including glycolaldehyde in the xylose-containing medium, and xylose-rich, lignocellulosic hydrolysate derived from Miscanthus giganteus, and concurrently improved the ethanol fermentation profile. Outcomes of this study will aid the development of next-generation robust S. cerevisiae strains for efficient fermentation of hexose and pentose sugars found in biomass hydrolysate.