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ABSTRACT Engineered Saccharomyces cerevisiae can reduce xylose to xylitol. However, in S.cerevisiae, there are several endogenous enzymes including xylitol dehydrogenase encoded by XYL2, sorbitol dehydrogenases encoded by SOR1/SOR2 and xylulokinase encoded by XKS1 may lead to the assimilation of xylitol. In this study, to increase xylitol accumulation, these genes were separately deleted through CRISPR/Cas9 system. Their effects on xylitol yield of an industrial S. cerevisiae CK17 overexpressing Candida tropicalis XYL1 (encoding xylose reductase) were investigated. Deletion of SOR1/SOR2 or XKS1 increased the xylitol yield in both batch and fed-batch fermentation with different concentrations of glucose and xylose. The analysis of the transcription level of key genes in the mutants during fed-batch fermentation suggests that SOR1/SOR2 are more crucially responsible for xylitol oxidation than XYL2 under the genetic background of S.cerevisiae CK17. The deletion of XKS1 gene could also weaken SOR1/SOR2 expression, thereby increasing the xylitol accumulation. The XKS1-deleted strain CK17ΔXKS1 produced 46.17 g/L of xylitol and reached a xylitol yield of 0.92 g/g during simultaneous saccharification and fermentation (SSF) of pretreated corn stover slurry. Therefore, the deletion of XKS1 gene provides a promising strategy to meet the industrial demands for xylitol production from lignocellulosic biomass. This study highlights the role of sorbitol dehydrogenase genes for xylitol-oxidation in Saccharomyces cerevisiae and provides a strategy through deleting xylulokinase gene for improved xylitol production.

Rhodosporidium toruloides is an oleaginous yeast capable of producing a variety of biofuels and bioproducts from diverse carbon sources. Despite numerous studies showing its promise as a platform microorganism, little is known about its metabolism and physiology. In this work, we investigated the central carbon metabolism in R. toruloides IFO0880 using transcriptomics and metabolomics during growth on glucose, xylose, acetate, or soybean oil. These substrates were chosen because they can be derived from plants. Significant changes in gene expression and metabolite concentrations were observed during growth on these four substrates. We mapped these changes onto the governing metabolic pathways to better understand how R. toruloides reprograms its metabolism to enable growth on these substrates. One notable finding concerns xylose metabolism, where poor expression of xylulokinase induces a bypass leading to arabitol production. Collectively, these results further our understanding of central carbon metabolism in R. toruloides during growth on different substrates. They may also help guide the metabolic engineering and development of better models of metabolism for R. toruloides.Key points• Gene expression and metabolite concentrations were significantly changed.• Reduced expression of xylulokinase induces a bypass leading to arabitol production.• R. toruloides reprograms its metabolism to allow growth on different substrates.

Background Xylitol, a white or transparent polyol or sugar alcohol, is digestible by colonic microorganisms and promotes the proliferation of beneficial bacteria and the production of short-chain fatty acids (SCFAs), but the mechanism underlying these effects remains unknown. We studied mice fed with 0%, 2% (2.17 g/kg/day), or 5% (5.42 g/kg/day) (weight/weight) xylitol in their chow for 3 months. In addition to the in vivo digestion experiments in mice, 3% (weight/volume) (0.27 g/kg/day for a human being) xylitol was added to a colon simulation system (CDMN) for 7 days. We performed 16S rRNA sequencing, beneficial metabolism biomarker quantification, metabolome, and metatranscriptome analyses to investigate the prebiotic mechanism of xylitol. The representative bacteria related to xylitol digestion were selected for single cultivation and co-culture of two and three bacteria to explore the microbial digestion and utilization of xylitol in media with glucose, xylitol, mixed carbon sources, or no-carbon sources. Besides, the mechanisms underlying the shift in the microbial composition and SCFAs were explored in molecular contexts. Results In both in vivo and in vitro experiments, we found that xylitol did not significantly influence the structure of the gut microbiome. However, it increased all SCFAs, especially propionate in the lumen and butyrate in the mucosa, with a shift in its corresponding bacteria in vitro. Cross-feeding, a relationship in which one organism consumes metabolites excreted by the other, was observed among Lactobacillus reuteri , Bacteroides fragilis , and Escherichia coli in the utilization of xylitol. At the molecular level, we revealed that xylitol dehydrogenase (EC 1.1.1.14), xylulokinase (EC 2.7.1.17), and xylulose phosphate isomerase (EC 5.1.3.1) were key enzymes in xylitol metabolism and were present in Bacteroides and Lachnospiraceae . Therefore, they are considered keystone bacteria in xylitol digestion. Also, xylitol affected the metabolic pathway of propionate, significantly promoting the transcription of phosphate acetyltransferase (EC 2.3.1.8) in Bifidobacterium and increasing the production of propionate. Conclusions Our results revealed that those key enzymes for xylitol digestion from different bacteria can together support the growth of micro-ecology, but they also enhanced the concentration of propionate, which lowered pH to restrict relative amounts of Escherichia and Staphylococcus. Based on the cross-feeding and competition among those bacteria, xylitol can dynamically balance proportions of the gut microbiome to promote enzymes related to xylitol metabolism and SCFAs. CGRfu2_UbvGGd6F7x5LTJk Video Abstract

Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min−1 mg−1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides.Key points• Overexpressing xylA or xks1 increased the xylose consumption and the lipid content.• The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI.• Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S . cerevisiae , which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S . cerevisiae . Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S . cerevisiae are also addressed.

Previously, we presented a novel approach for increasing the consumption of xylose and the lipid yield by overexpressing the genes coding for xylose isomerase (XI) and xylulokinase (XK) in Mucor circinelloides. In the present study, an in-depth analysis of lipid accumulation by xylose metabolism engineered M. circinelloides strains (namely Mc-XI and Mc-XK) using corn straw hydrolysate was to be explored. The results showed that the fatty acid contents of the engineered M. circinelloides strains were, respectively, increased by 19.8% (in Mc-XI) and 22.3% (in Mc-XK) when compared with the control strain, even though a slightly decreased biomass in these engineered strains was detected. Moreover, the xylose uptake rates of engineered strains in the corn straw hydrolysate were improved significantly by 71.5% (in Mc-XI) and 68.8% (in Mc-XK), respectively, when compared with the control strain. Maybe the increased utilization of xylose led to an increase in lipid synthesis. When the recombinant M. circinelloides strains were cultured in corn straw hydrolysate medium with the carbon-to-nitrogen ratio (C/N ratio) of 50 and initial pH of 6.0, at 30 °C and 500 rpm for 144 h, a total biomass of 12.6–12.9 g/L with a lipid content of 17.2–17.7% (corresponding to a lipid yield of 2.17–2.28 g/L) was achieved. Our study provides a foundation for the further application of the engineered M. circinelloides strains to produce lipid from lignocelluloses.

Abstract The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides. This review examines the unique xylose metabolism of Rhodotorula toruloides and explores strategies to improve its efficiency for sustainable biomanufacturing.

Only trace amount of isobutanol is produced by the native Saccharomyces cerevisiae via degradation of amino acids. Despite several attempts using engineered yeast strains expressing exogenous genes, catabolite repression of glucose must be maintained together with high activity of downstream enzymes, involving iron–sulfur assimilation and isobutanol production. Here, we examined novel roles of nonfermentable carbon transcription factor Znf1 in isobutanol production during xylose utilization. RNA-seq analysis showed that Znf1 activates genes in valine biosynthesis, Ehrlich pathway and iron–sulfur assimilation while coupled deletion or downregulated expression of BUD21 further increased isobutanol biosynthesis from xylose. Overexpression of ZNF1 and xylose-reductase/dehydrogenase (XR-XDH) variants, a xylose-specific sugar transporter, xylulokinase, and enzymes of isobutanol pathway in the engineered S. cerevisiae pho13gre3Δ strain resulted in the superb ZNXISO strain, capable of producing high levels of isobutanol from xylose. The isobutanol titer of 14.809 ± 0.400 g/L was achieved, following addition of 0.05 g/L FeSO4.7H2O in 5 L bioreactor. It corresponded to 155.88 mg/g xylose consumed and + 264.75% improvement in isobutanol yield. This work highlights a new regulatory control of alternative carbon sources by Znf1 on various metabolic pathways. Importantly, we provide a foundational step toward more sustainable production of advanced biofuels from the second most abundant carbon source xylose. The engineered yeast strain could be used as a cell factory to produce high levels of isobutanol from the second most abundant sugar xylose due to activation of Znf1 target genes in the the valine biosynthesis, Ehrlich pathway and iron–sulfur assimilation.

Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d -xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d -xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1 , encoding a Golgi Ca 2+ /Mn 2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d -xylose. In pmr1 strains, intracellular Mn 2+ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn 2+ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast.