Comparative global metabolite profiling of xylose-fermenting Saccharomyces cerevisiae SR8 and Scheffersomyces stipitis.

Bioconversion of lignocellulosic biomass into ethanol requires efficient xylose fermentation. Previously, we developed an engineered Saccharomyces cerevisiae strain, named SR8, through rational and inverse metabolic engineering strategies, thereby improving its xylose fermentation and ethanol production. However, its fermentation characteristics have not yet been fully evaluated. In this study, we investigated the xylose fermentation and metabolic profiles for ethanol production in the SR8 strain compared with native Scheffersomyces stipitis. The SR8 strain showed a higher maximum ethanol titer and xylose consumption rate when cultured with a high concentration of xylose, mixed sugars, and under anaerobic conditions than Sch. stipitis. However, its ethanol productivity was less on 40 g/L xylose as the sole carbon source, mainly due to the formation of xylitol and glycerol. Global metabolite profiling indicated different intracellular production rates of xylulose and glycerol-3-phosphate in the two strains. In addition, compared with Sch. stipitis, SR8 had increased abundances of metabolites from sugar metabolism and decreased abundances of metabolites from energy metabolism and free fatty acids. These results provide insights into how to control and balance redox cofactors for the production of fuels and chemicals from xylose by the engineered S. cerevisiae.

Deletion of PHO13 improves aerobic L-arabinose fermentation in engineered Saccharomyces cerevisiae.

Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although L-arabinose is a highlighted pentose that has been identified as next-generation biomass, arabinose fermentation has not yet undergone extensive development for industrial utilization. In this study, we integrated a heterologous fungal arabinose pathway with a deletion of PHO13 phosphatase gene. PHO13 deletion increased arabinose consumption rate and specific ethanol productivity under aerobic conditions and consequently depleted sedoheptulose by activation of the TAL1 gene. Global metabolite profiling indicated upregulation of the pentose phosphate pathway and downstream effects such as trehalose accumulation and downregulation of the TCA cycle. Our results suggest that engineering of PHO13 has ample potential for arabinose conversion to ethanol as an industrial source for biofuels.

Simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae.

Pectin-rich biomass has garnered attention as an alternative biomass source. However, some monomers derived from pectin-rich biomass, namely d-galacturonic acid, l-arabinose, and d-xylose, are not fermentable by industrial microorganisms such as Saccharomyces cerevisiae. The purpose of this study is to develop a S. cerevisiae strain capable of fermenting the pectin monomers. Expressions of eight heterologous genes and deletion of two endogenous genes, all of which were successfully completed by Cas9-based in vivo assembly and integration strategy, allowed the consumption of pectin monomers as sole carbon sources. To facilitate the consumption of galacturonic acid, which had the most limitations, the use of a co-substrate was tested using various sugars. As a result, we found that arabinose and xylose allowed simultaneous consumption of galacturonic acid. Based on intracellular metabolite profiling, it was concluded that the five-carbon sugars partially resolve the metabolic bottleneck of galacturonic acid.

Data for simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae expressing heterologous pathways for xylose, arabinose, and galacturonic acid metabolism has been constructed by a Cas9-based genome editing technology [1]. The fermentation performance of the final strain (YE9) was tested under various substrate conditions, and the fermentation parameters were calculated. The dataset can be used for designing bioprocesses for pectin-rich biomass.

Effect of temperature on single- and mixed-strain fermentation of ruminant feeds.

Use of raw feedstuffs for livestock is limited by low digestibility. Recently, fermentation of feedstuffs has been highlighted as a new way to improve nutrient absorption through the production of organic acids using inoculated microorganisms, which can also play a probiotic role. However, standard procedures for feedstuff fermentation have not been clearly defined because the process is influenced by climatic variation, and an analytical standard for fermented feedstuffs is lacking. This study aimed to evaluate the microbiological and biochemical changes of feedstuffs during fermentation at temperatures corresponding to different seasons (10°C, 20°C, 30°C, and 40°C). We also investigated the effects of yeast, lactic acid bacteria (LAB), and Bacillus spp. on fermentation and determined the results of their interactions during fermentation. The viable cells were observed within 8 days in single-strain fermentation. However, when feedstuffs were inoculated with a culture of mixed strains, LAB were predominant at low temperatures (10°C and 20°C), while Bacillus spp. was predominant at high temperatures (30°C and 40°C). A significant drop in pH from 6.5 to 4.3 was observed when LAB was the dominant strain in the culture, which correlated with the concentrations of lactic acid. Slight ethanol production was detected above 20°C regardless of the incubation temperature, suggesting active metabolism of yeast, despite this organism making up a marginal portion of the microbes in the mixed culture. These results suggested that fermentation temperature significantly affects microbiological profiles and biochemical parameters, such as pH and the lactic acid concentration, of fermented feedstuffs. Our data provide valuable information for the determination of industrial standards for fermented feedstuffs.

Metabolic Engineering for Improved Fermentation of L-Arabinose.

L-Arabinose, a five carbon sugar, has not been considered as an important bioresource because most studies have focused on D-xylose, another type of five-carbon sugar that is prevalent as a monomeric structure of hemicellulose. In fact, L-arabinose is also an important monomer of hemicellulose, but its content is much more significant in pectin (3-22%, g/g pectin), which is considered an alternative biomass due to its low lignin content and mass production as juiceprocessing waste. This review presents native and engineered microorganisms that can ferment L-arabinose. Saccharomyces cerevisiae is highlighted as the most preferred engineering host for expressing a heterologous arabinose pathway for producing ethanol. Because metabolic engineering efforts have been limited so far, with this review as momentum, more attention to research is needed on the fermentation of L-arabinose as well as the utilization of pectin-rich biomass.