Dataset Information

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum.

ABSTRACT: Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.

SUBMITTER: Long TM

PROVIDER: S-EPMC3406140 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum.

Long Tanya M TM Su Yi-Kai YK Headman Jennifer J Higbee Alan A Willis Laura B LB Jeffries Thomas W TW

Applied and environmental microbiology 20120525 16

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spat ...[more]

PMID: 22636012

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Project description:BackgroundCost-effective production of biofuels from lignocellulose requires the fermentation of D-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments.ResultsWe screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis.ConclusionCollectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Project description:BackgroundThe production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion. Xylose fermentation capacity in yeasts is usually linked to xylose reductase (XR) accepting NADH as cofactor. The XR from Scheffersomyces stipitis, which is able to use NADH as cofactor but still prefers NADPH, has been used to generate recombinant xylose-fermenting Saccharomyces cerevisiae. Novel xylose-fermenting yeasts species, as those from the Spathaspora clade, have been described and are potential sources of novel genes to improve xylose fermentation in S. cerevisiae.ResultsXylose fermentation by six strains from different Spathaspora species isolated in Brazil, plus the Sp. passalidarum type strain (CBS 10155(T)), was characterized under two oxygen-limited conditions. The best xylose-fermenting strains belong to the Sp. passalidarum species, and their highest ethanol titers, yields, and productivities were correlated to higher XR activity with NADH than with NADPH. Among the different Spathaspora species, Sp. passalidarum appears to be the sole harboring two XYL1 genes: XYL1.1, similar to the XYL1 found in other Spathaspora and yeast species and XYL1.2, with relatively higher expression level. XYL1.1p and XYL1.2p from Sp. passalidarum were expressed in S. cerevisiae TMB 3044 and XYL1.1p was confirmed to be strictly NADPH-dependent, while XYL1.2p to use both NADPH and NADH, with higher activity with the later. Recombinant S. cerevisiae strains expressing XYL1.1p did not show anaerobic growth in xylose medium. Under anaerobic xylose fermentation, S. cerevisiae TMB 3504, which expresses XYL1.2p from Sp. passalidarum, revealed significant higher ethanol yield and productivity than S. cerevisiae TMB 3422, which harbors XYL1p N272D from Sc. stipitis in the same isogenic background (0.40 vs 0.34 g gCDW (-1) and 0.33 vs 0.18 g gCDW (-1) h(-1), respectively).ConclusionThis work explored a new clade of xylose-fermenting yeasts (Spathaspora species) towards the engineering of S. cerevisiae for improved xylose fermentation. The new S. cerevisiae TMB 3504 displays higher XR activity with NADH than with NADPH, with consequent improved ethanol yield and productivity and low xylitol production. This meaningful advance in anaerobic xylose fermentation by recombinant S. cerevisiae (using the XR/XDH pathway) paves the way for the development of novel industrial pentose-fermenting strains.

Project description:BackgroundThe use of inedible lignocellulosic biomasses for biomanufacturing provides important environmental and economic benefits for society. Efficient co-utilization of lignocellulosic biomass-derived sugars, primarily glucose and xylose, is critical for the viability of lignocellulosic biorefineries. However, the phenomenon of glucose repression prevents co-utilization of both glucose and xylose in cellulosic hydrolysates.ResultsTo circumvent glucose repression, co-utilization of cellobiose and xylose by Bacillus coagulans NL01 was investigated. During co-fermentation of cellobiose and xylose, B. coagulans NL01 simultaneously consumed the sugar mixtures and exhibited an improved lactic acid yield compared with co-fermentation of glucose and xylose. Moreover, the cellobiose metabolism of B. coagulans NL01 was investigated for the first time. Based on comparative genomic analysis, two gene clusters that encode two different operons of the cellobiose-specific phosphoenolpyruvate-dependent phosphotransferase system (assigned as CELO1 and CELO2) were identified. For CELO1, five genes were arranged as celA (encoding EIIAcel), celB (encoding EIIBcel), celC (encoding EIICcel), pbgl (encoding 6-phospho-β-glucosidase), and celR (encoding a transcriptional regulator), and these genes were found to be ubiquitous in different B. coagulans strains. Based on gene knockout results, CELO1 was confirmed to be responsible for the transport and assimilation of cellobiose. For CELO2, the five genes were arranged as celR, celB, celA, celX (encoding DUF871 domain-containing protein), and celC, and these genes were only found in some B. coagulans strains. However, through a comparison of cellobiose fermentation by NL01 and DSM1 that only possess CELO1, it was observed that CELO2 might also play an important role in the utilization of cellobiose in vivo despite the fact that no pbgl gene was found. When CELO1 or CELO2 was expressed in Escherichia coli, the recombinant strain exhibited distinct cellobiose uptake and consumption.ConclusionsThis study demonstrated the cellobiose-assimilating pathway of B. coagulans and provided a new co-utilization strategy of cellobiose and xylose to overcome the obstacles that result from glucose repression in a biorefinery system.

Dataset Information

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum.

Publications

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum.

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