title: 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: BACKGROUND
  The 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.
  
  
  RESULTS
  Xylose 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).
  
  
  CONCLUSIONS
  This 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.
authors: Departamento de Microbiologia, ICB, C.P. 486, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901 Brazil ; Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisbon, Portugal ; Department of Applied Microbiology, Lund University, PO Box 124, 22100 Lund, Sweden.
authors: Raquel M
authors: RM
authors: Cadete
authors: Department of Applied Microbiology, Lund University, PO Box 124, 22100 Lund, Sweden.
authors: Alejandro M
authors: AM
authors: de Las Heras
authors: Department of Applied Microbiology, Lund University, PO Box 124, 22100 Lund, Sweden ; Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark.
authors: Anders G
authors: AG
authors: Sandström
authors: Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisbon, Portugal.
authors: Carla
authors: C
authors: Ferreira
authors: Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisbon, Portugal.
authors: Francisco
authors: F
authors: Gírio
authors: Department of Applied Microbiology, Lund University, PO Box 124, 22100 Lund, Sweden.
authors: Marie-Françoise
authors: MF
authors: Gorwa-Grauslund
authors: Departamento de Microbiologia, ICB, C.P. 486, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901 Brazil.
authors: Carlos A
authors: CA
authors: Rosa
authors: Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisbon, Portugal ; Section for Sustainable Biotechnology, Aalborg University Copenhagen, A. C. Meyers Vænge 15, 2450 Copenhagen SV, Denmark.
authors: César
authors: 0000-0003-2448-7063
authors: C
authors: Fonseca
keywords: Bioethanol; NADH-preferring xylose reductase; Saccharomyces cerevisiae; Spathaspora passalidarum; Spathaspora species; XYL1.2; Xylose fermentation