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Improved simultaneous co-fermentation of glucose and xylose by Saccharomyces cerevisiae for efficient lignocellulosic biorefinery.

ABSTRACT: Background:Lignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. Saccharomyces cerevisiae, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery. Results:In this study, we developed a powerful mixed-sugar co-fermenting strain of S. cerevisiae, XUSEA, with improved xylose conversion capacity during simultaneous glucose/xylose co-fermentation. To reinforce xylose catabolism, the overexpression target in the pentose phosphate pathway was selected using a DNA assembler method and overexpressed increasing xylose consumption and ethanol production by twofold. The performance of the newly engineered strain with improved xylose catabolism was further boosted by elevating fermentation temperature and thus significantly reduced the co-fermentation time by half. Through combined efforts of reinforcing the pathway of xylose catabolism and elevating the fermentation temperature, XUSEA achieved simultaneous co-fermentation of lignocellulosic hydrolysates, composed of 39.6 g L-1 glucose and 23.1 g L-1 xylose, within 24 h producing 30.1 g L-1 ethanol with a yield of 0.48 g g-1. Conclusions:Owing to its superior co-fermentation performance and ability for further engineering, XUSEA has potential as a platform in a lignocellulosic biorefinery toward realizing a more economical and sustainable process for large-scale bioethanol production.

SUBMITTER: Hoang Nguyen Tran P

PROVIDER: S-EPMC6975041 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Improved simultaneous co-fermentation of glucose and xylose by <i>Saccharomyces cerevisiae</i> for efficient lignocellulosic biorefinery.

Hoang Nguyen Tran Phuong P Ko Ja Kyong JK Gong Gyeongtaek G Um Youngsoon Y Lee Sun-Mi SM

Biotechnology for biofuels 20200122

<h4>Background</h4>Lignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. <i>Saccharomyces cerevisiae</i>, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery.<h4>Results</h4>In this study, we developed a powerful mixed-sugar co-f ...[more]

PMID: 31993090

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Project description:Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

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.

Dataset Information

Improved simultaneous co-fermentation of glucose and xylose by Saccharomyces cerevisiae for efficient lignocellulosic biorefinery.

Publications

Improved simultaneous co-fermentation of glucose and xylose by <i>Saccharomyces cerevisiae</i> for efficient lignocellulosic biorefinery.

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