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

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.

Anaerobic poly-3-d-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase

“…This results confirmed what previous works showed about the importance of a more balanced co-factor ratio in the xylose metabolisation steps (see e.g. [31–33]). In particular, strains expressing XR mut have higher affinity for NADH than XR wt and therefore demand less usage of NADPH for xylose reduction thereby increasing the availability of NADPH for biomass formation and a cofactor balanced xylose conversion to xylulose [34].…”

Efficient production of second generation ethanol and xylitol by yeasts from Amazonian beetles (Coleoptera) and their galleries

“…Regarding the xylitol producers, isolate P8-2.12 -S. roraimanensis was not efficient when compared to that reported by Cadete et al (2016) for the same species. Our isolate produced 12.25 g.L -1 of xylitol.…”

“…It suggests that S. passalidarum may use different xylose transport systems under anaerobic and aerobic conditions. Cadete et al (2016), under severe oxygen limitation conditions, obtained ethanol production above 20 g.L -1 for S. passalidarum. Compared with this study, the isolates P16-1.1 (18.04 g.L ).…”

“…Previous research (Cadete et al, 2009;Hou, 2012) has shown that all species of the Spathaspora clade isolated from decomposing wood trunks or insects associated with this substrate have converted xylose into ethanol more efficiently than the species of reference Pichia stipitis. Cadete et al (2016) classified some species of Spathaspora as ethanol producers and xylitol producers, according to the main product of xylose metabolism. In their research, ethanol was the main product for S. passalidarum.…”

“…In fact, the gut of beetles and other insects is considered a hotspot of yeast diversity (Suh and Blackwell, 2004;Boekhout, 2005;Rivera et al, 2009;Cadete et al, 2009Cadete et al, , 2012Urbina et al, 2013;Gouliamova et al, 2015;Cadete et al, 2015Cadete et al, , 2016.…”