Exploring xylose metabolism in Spathaspora species: XYL1.2 from Spathaspora passalidarum as the key for efficient anaerobic xylose fermentation in metabolic engineered Saccharomyces cerevisiae.

Carla Ferreira,Carlos A. Rosa,Alejandro M. de las Heras,Francisco Gírio,Anders G. Sandström,Marie-Françoise Gorwa-Grauslund,César Fonseca,Raquel M. Cadete

doi:10.1186/s13068-016-0570-6

Abstract

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 Scheffersomycesstipitis, 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 10155T), 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0570-6) contains supplementary material, which is available to authorized users.

Highlights

The production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion
Among the modification induced or selected, the N272D mutation in XYL1 from Sc. stipitis resulted in an increased preference for NADH in this yeast [14], and a recombinant S. cerevisiae expressing the same gene with N272D was able to grow anaerobically in xylose [15]
Xylose fermentation by Spathaspora species under two different oxygen‐limited conditions Six yeasts strains isolated from Brazilian habitats, Sp. arborariae UFMG-CM-Y352T, Sp. brasiliensis UFMGCM-Y353T, Sp. passalidarum UFMG-CM-Y469, Sp. roraimanensis UFMG-CM-Y477T, Sp. suhii UFMGCM-Y475T, and Sp. xylofermentans UFMG-CM-Y478T, plus the reference strain Sp. passalidarum Centraalbureau voor Schimmelcultures (CBS) 10155T, were studied under two different oxygen-limited conditions corresponding to an oxygen transfer rate (OTR) of approximately 1–2 mmol L−1 min−1 and 10–15 mmol L−1 min−1

Summary

Introduction

The production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion. Lignocellulosic substrates are the largest source of fermentable sugars for the production of fuels and chemicals and the economic viability of second generation (2G) or lignocellulosic ethanol technology is dependent on the complete and efficient conversion of the carbohydrates, including those from the cellulosic and hemicellulosic fractions, primarily glucose and xylose [3, 4]. One of the key challenges for cost-effective lignocellulosic ethanol is the availability of robust microorganisms able to efficiently ferment all sugars present in lignocellulosic hydrolysates [1] To address this bottleneck, Saccharomyces cerevisiae, the most commonly microorganism used in industrial alcoholic fermentations, has been engineered towards efficient xylose fermentation capacity [1], since other microorganisms, including native xylosefermenting yeasts, can hardly convert xylose into ethanol at high titers, yields or productivities under industrially relevant ethanol production conditions, such as strict anaerobic environment, high osmotic stress, or high concentration of ethanol and inhibitors present in lignocellulosic hydrolysates [5]. Among the modification induced or selected, the N272D mutation in XYL1 from Sc. stipitis resulted in an increased preference for NADH in this yeast [14], and a recombinant S. cerevisiae expressing the same gene with N272D was able to grow anaerobically in xylose [15]

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