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.

Upstream processes of citrus fruit waste biorefinery for complete valorization.

Citrus fruit waste (CW) is a useful biomass and its valorization into fuels and biochemicals has received much attention. For economic feasibility, increased efficiency of the preceding extraction and enzyme saccharification processes is necessary. However, at present, there is a lack of systematic reviews addressing these two integral upstream processes in concert for CW biorefinery. Here, the state-of-the-art advancements in enzyme extraction and saccharification processes-using which relevant essential oils, flavonoids, and sugars can be obtained-are reviewed. Specifically, the extraction options for two commercially available CW-derived products, essential oils and pectin, are discussed. With respect to enzyme saccharification, the use of an undefined commercial mixture routinely results in suboptimal sugar production. In this respect, applicable strategies for enzyme mixture customization are suggested for maximizing the hydrolytic efficiency of CW. The enzyme degradation system for CW-derived carbohydrates and its extensive application for sugar production are also discussed.

l-Lactic Acid Production Using Engineered Saccharomyces cerevisiae with Improved Organic Acid Tolerance.

Lactic acid is mainly used to produce bio-based, bio-degradable polylactic acid. For industrial production of lactic acid, engineered Saccharomyces cerevisiae can be used. To avoid cellular toxicity caused by lactic acid accumulation, pH-neutralizing agents are used, leading to increased production costs. In this study, lactic acid-producing S. cerevisiae BK01 was developed with improved lactic acid tolerance through adaptive laboratory evolution (ALE) on 8% lactic acid. The genetic basis of BK01 could not be determined, suggesting complex mechanisms associated with lactic acid tolerance. However, BK01 had distinctive metabolomic traits clearly separated from the parental strain, and lactic acid production was improved by 17% (from 102 g/L to 119 g/L). To the best of our knowledge, this is the highest lactic acid titer produced by engineered S. cerevisiae without the use of pH neutralizers. Moreover, cellulosic lactic acid production by BK01 was demonstrated using acetate-rich buckwheat husk hydrolysates. Particularly, BK01 revealed improved tolerance against acetic acid of the hydrolysates, a major fermentation inhibitor of lignocellulosic biomass. In short, ALE with a high concentration of lactic acid improved lactic acid production as well as acetic acid tolerance of BK01, suggesting a potential for economically viable cellulosic lactic acid production.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Recent advances in the biological valorization of citrus peel waste into fuels and chemicals.

In the quest to reduce global food loss and waste, fruit processing wastes, particularly citrus peel waste (CPW), have emerged as a promising and sustainable option for biorefinery without competing with human foods and animal feeds. CPW is largely produced and, as recent studies suggest, has the industrial potential of biological valorization into fuels and chemicals. In this review, the promising aspects of CPW as an alternative biomass were highlighted, focusing on its low lignin content. In addition, specific technical difficulties in fermenting CPW are described, highlighting that citrus peel is high in pectin that consist of non-fermentable sugars, mainly galacturonic acid. Last, recent advances in the metabolic engineering of yeast and other microbial strains that ferment CPW-derived sugars to produce value-added products, such as ethanol and mucic acid, are summarized. For industrially viable CPW-based biorefinery, more studies are needed to improve fermentation efficiency and to diversify product profiles.

Inhibitory Effect of Steamed Soybean Wastewater Against DSS-Induced Intestinal Inflammation in Mice.

This study was performed to examine the beneficial potential of steamed soybean wastewater (SSW), which is generated during the manufacture of fermented soybean products and usually discarded as a by-product. The SSW was found to contain considerable amounts of isoflavones and had concentration-dependent radical scavenging capabilities. Moreover, oral administration of SSW effectively prevented colonic damage induced by dextran sulfate sodium (DSS), based on improvement of morphological and histological features, reduction of oxidative stress indicators, suppression of proinflammatory cytokine production, downregulation of inflammatory marker expression in the colonic tissue, and inhibition of the inflammatory activation of macrophages. It suggests that SSW could prevent intestinal inflammation in humans, although its efficacy should be verified through careful study design in humans. These findings have implications for enhancement of the value-added of SSW and for reduction of wastewater treatment costs incurred by the food industry.

Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae.

Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.

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.

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.

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.

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.

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.

Glucose repression can be alleviated by reducing glucose phosphorylation rate in Saccharomyces cerevisiae.

Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.

Development and Metabolite Profiling of Elephant Garlic Vinegar.

Elephant garlic (Allium ampeloprasum var. ampeloprasum), which belongs to the Alliaceae family along with onion and garlic, has a flavor and shape similar to those of normal garlic but is not true garlic. Additionally, its properties are largely unknown, and its processing and product development have not been reported. In this study, we focused on using elephant garlic to produce a new type of vinegar, for which the market is rapidly growing because of its health benefits. First, we evaluated the effects of elephant garlic addition on acetic acid fermentation of rice wine by Acetobacter pasteurianus. In contrast to normal garlic, for which 2% (w/v) addition completely halted fermentation, addition of elephant garlic enabled slow but successful fermentation of ethanol to acetic acid. Metabolite analysis suggested that sulfur-containing volatile compounds were less abundant in elephant garlic than in normal garlic; these volatile compounds may be responsible for inhibiting acetic acid fermentation. After acetic acid fermentation, vinegar with elephant garlic did not have any sulfur-containing volatile compounds, which could positively contribute to the vinegar flavor. Moreover, the amino acid profile of the vinegar suggested that nutritional and sensory properties were more enhanced following addition of elephant garlic. Thus, elephant garlic may have applications in the development of a new vinegar product with improved flavor and quality and potential health benefits.

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.