Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

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.

Enhancement of bioethanol production from Gracilaria verrucosa by Saccharomyces cerevisiae through the overexpression of SNR84 and PGM2.

A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.

Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains.

Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A) exhibited a 14% higher ethanol yield and 46% lower byproduct yield than the IIK1 strain from anaerobic fermentation of the Miscanthus hydrolysate. Our results demonstrate that industrial yeast strains can be engineered via haploid isolation. The isolated haploid strain (4124-S60) can be used for metabolic engineering to produce fuels and chemicals.

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc.

PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

UNLABELLED: Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ß-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. IMPORTANCE: In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ß-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

Deletion of PHO13, encoding haloacid dehalogenase type IIA phosphatase, results in upregulation of the pentose phosphate pathway in Saccharomyces cerevisiae.

The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.

Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR.

Genomic integration of expression cassettes containing heterologous genes into yeast with traditional methods inevitably deposits undesirable genetic elements into yeast chromosomes, such as plasmid-borne multiple cloning sites, antibiotic resistance genes, Escherichia coli origins, and yeast auxotrophic markers. Specifically, drug resistance genes for selecting transformants could hamper further industrial usage of the resulting strains because of public health concerns. While we constructed an efficient and rapid xylose-fermenting Saccharomyces cerevisiae, the engineered strain (SR8) might not be readily used for a large-scale fermentation because the SR8 strain contained multiple copies of drug resistance genes. We utilized the Cas9/CRISPR-based technique to refactor an efficient xylose-fermenting yeast strain without depositing any undesirable genetic elements in resulting strains. In order to integrate genes (XYL1, XYL2, and XYL3) coding for xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis, and delete both PHO13 and ALD6, a double-strand break formation by Cas9 and its repair by homologous recombination were exploited. Specifically, plasmids containing guide RNAs targeting PHO13 and ALD6 were sequentially co-transformed with linearized DNA fragments containing XYL1, XYL2, and XYL3 into S. cerevisiae expressing Cas9. As a result, two copies of XYL1, XYL2, and XYL3 were integrated into the loci of PHO13 and ALD6 for achieving overexpression of heterologous genes and knockout of endogenous genes simultaneously. With further prototrophic complementation, we were able to construct an engineered strain exhibiting comparable xylose fermentation capabilities with SR8 within 3 weeks. We report a detailed procedure for refactoring xylose-fermenting yeast using any host strains. The refactored strains using our procedure could be readily used for large-scale fermentations since they have no antibiotic resistant markers.

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.

Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular ß-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production.

Evaluating the Nutritional Composition of Unripe Citrus and Its Effect on Inhibiting Adipogenesis and Adipocyte Differentiation.

Citrus fruits offer a range of health benefits due to their rich nutritional profile, including vitamin C, flavonoids, carotenoids, and fiber. It is known that unripe citrus has higher levels of vitamin C, dietary fiber, polyphenols, and flavonoids compared to mature fruits. In this study, we assessed the nutritional components of unripe citrus peel and pressed juices, as well as their anti-obesity potential through the modulation of adipocyte differentiation and the expression of adipogenesis-related genes, specifically PPARγ and C/EBPα, in 3T3-L1 preadipocytes. Our analysis revealed that unripe citrus peel exhibited elevated levels of fiber and protein compared to pressed juice, with markedly low levels of free sugar, particularly sucrose. The content of hesperidin, a representative flavonoid in citrus fruits, was 3,157.6 mg/kg in unripe citrus peel and 455.5 mg/kg in pressed juice, indicating that it was approximately seven times higher in unripe citrus peel compared to pressed juice. Moreover, we observed that the peel had a dose-dependently inhibitory effect on adipocyte differentiation, which was linked to a significant downregulation of adipogenesis-related gene expression. Thus, our findings suggest that unripe citrus possesses anti-obesity effects by impeding adipogenesis and adipocyte differentiation, with the peel demonstrating a more pronounced effect compared to pressed juice.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Deletion of FPS1, encoding aquaglyceroporin Fps1p, improves xylose fermentation by engineered Saccharomyces cerevisiae.

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD(+)/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

Feasibility of xylose fermentation by engineered Saccharomyces cerevisiae overexpressing endogenous aldose reductase (GRE3), xylitol dehydrogenase (XYL2), and xylulokinase (XYL3) from Scheffersomyces stipitis.

Saccharomyces cerevisiae has been engineered for producing ethanol from xylose, the second most abundant sugar in cellulosic biomass hydrolyzates. Heterologous expressions of xylose reductase (XYL1) and xylitol dehydrogenase (XYL2), or of xylose isomerase (xylA), either case of which being accompanied by overexpression of xylulokinase (XKS1 or XYL3), are known as the prevalent strategies for metabolic engineering of S. cerevisiae to ferment xylose. In this study, we propose an alternative strategy that employs overexpression of GRE3 coding for endogenous aldose reductase instead of XYL1 to construct efficient xylose-fermenting S. cerevisiae. Replacement of XYL1 with GRE3 has been regarded as an undesirable approach because NADPH-specific aldose reductase (GRE3) would aggravate redox balance with xylitol dehydrogenase (XYL2) using NAD(+) exclusively. Here, we demonstrate that engineered S. cerevisiae overexpressing GRE3, XYL2, and XYL3 can ferment xylose as well as a mixture of glucose and xylose with higher ethanol yields (0.29-0.41 g g(-1) sugars) and productivities (0.13-0.85 g L(-1) h(-1)) than those (0.23-0.39 g g(-1) sugars, 0.10-0.74 g L(-1) h(-1)) of an isogenic strain overexpressing XYL1, XYL2, and XYL3 under oxygen-limited conditions. We found that xylose fermentation efficiency of a strain overexpressing GRE3 was dramatically increased by high expression levels of XYL2. Our results suggest that optimized expression levels of GRE3, XYL2, and XYL3 could overcome redox imbalance during xylose fermentation by engineered S. cerevisiae under oxygen-limited conditions.

Characterization and structural analysis of the endo-1,4-ß-xylanase GH11 from the hemicellulose-degrading Thermoanaerobacterium saccharolyticum useful for lignocellulose saccharification.

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-ß-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg-1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL-1 and 34,015.3 s-1, respectively, resulting in kcat/Km value of 2658.7 mL mg-1 s-1, higher by 102-103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a ß-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation.

Physicochemical and Functional Properties of Yeast-Fermented Cabbage.

Microbial fermentation is often used to improve the functionality of plant-based food materials. Herein, we investigated changes in the physicochemical and functional properties of cabbage during yeast fermentation to develop new products using fermented cabbage. Among the 8 types of food-grade yeast, both Saccharomyces cerevisiae and Saccharomyces boulardii fermented 10% cabbage powder solution (w/w) the most effectively, leaving no soluble sugars after 12 h of fermentation. In addition, the yeast fermentation of cabbage resulted in functionally positive outcomes in terms of sulforaphane content, antioxidant properties, and anti-inflammatory activity. Specifically, the yeast-fermented cabbages contained about 500% more sulforaphane. The soluble fraction (5 µg/ml) of yeast-fermented cabbage had no cytotoxicity in murine RAW 264.7 cells, and the radical-scavenging capacity was equivalent to 1 µg/ml of ascorbic acid. Moreover, cabbage fermented with S. boulardii significantly suppressed both lipopolysaccharides (LPS)-induced nitric oxide production and LPS-induced reactive oxygen species production in RAW 264.7 cells, suggesting a potential anti-inflammatory effect. These results support the idea that yeast fermentation is promising for developing functionally improved cabbage products.

Dietary protocatechuic acid redistributes tight junction proteins by targeting Rho-associated protein kinase to improve intestinal barrier function.

Inflammatory bowel disease (IBD) is continuously increasing globally and caused by intestinal barrier dysfunction. Although protocatechuic acid (PCA) has a protective effect on colitis, the molecular mechanisms underlying its contribution to intestinal barrier function remain unknown. Transepithelial electrical resistance (TEER) and FITC-dextran permeability measurements reveled that PCA suppresses lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α-induced increase in intestinal permeability; zonula occludens (ZO)-1 and claudin-2 redistribution was also suppressed in the epithelial cell membranes of differentiated Caco-2 cells. PCA was found to directly bind Rho-associated coiled-coil containing protein kinase (ROCK), subsequently suppressing myosin light chain (MLC) phosphorylation. Notably, PCA binds ROCK to a similar degree as Y27632, a selective ROCK inhibitor. Orally administering PCA (5 or 25 mg per kg per day) to C57BL/6 mice alleviated the 3% dextran sulfate sodium (DSS)-induced colitis symptoms including reduced colon length, disrupted intestinal barrier structure, and increased proinflammatory cytokines expressions, such as interleukin (IL)-1ß, TNF-α, and IL-6. Furthermore, orally administering PCA suppressed DSS-induced ZO-1 and claudin-2/4 redistribution in mice colon membrane fractions. Therefore, PCA may serve as a promising nutraceutical to improve gut health and alleviate IBD by maintaining intestinal barrier function in vitro and in vivo.