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Improved inhibitor tolerance in xylose-fermenting yeast Spathaspora passalidarum by mutagenesis and protoplast fusion

  • Applied microbial and cell physiology
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Abstract

The xylose-fermenting yeast Spathaspora passalidarum showed excellent fermentation performance utilizing glucose and xylose under anaerobic conditions. But this yeast is highly sensitive to the inhibitors such as furfural present in the pretreated lignocellulosic biomass. In order to improve the inhibitor tolerance of this yeast, a combination of UV mutagenesis and protoplast fusion was used to construct strains with improved performance. Firstly, UV-induced mutants were screened and selected for improved tolerance towards furfural. The most promised mutant, S. passalidarum M7, produced 50% more final ethanol than the wild-type strain in a synthetic xylose medium containing 2 g/l furfural. However, this mutant was unable to grow in a medium containing 75% liquid fraction of pretreated wheat straw (WSLQ), in which furfural and many other inhibitors were present. Hybrid yeast strains, obtained from fusion of the protoplasts of S. passalidarum M7 and a robust yeast, Saccharomyces cerevisiae ATCC 96581, were able to grow in 75% WSLQ and produce around 0.4 g ethanol/g consumed xylose. Among the selected hybrid strains, the hybrid FS22 showed the best fermentation capacity in 75% WSLQ. Phenotypic and partial molecular analysis indicated that S. passalidarum M7 was the dominant parental contributor to the hybrid. In summary, the hybrids are characterized by desired phenotypes derived from both parents, namely the ability to ferment xylose from S. passalidarum and an increased tolerance to inhibitors from S. cerevisiae ATCC 96581.

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Acknowledgments

We thank Inbicon A/S (Dong Energy subsidiary, Denmark) for providing the liquid fraction of pretreated wheat straw. We express our sincere appreciations to Ingelis Larsen and Annette Eva Jensen for their help on HPLC analysis, to Anders Brandt for helpful discussions and suggestions, and to Anders Brandt, Klaus Breddam, and Kim Pilegaard for proofreading the manuscript.

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Authors and Affiliations

  1. Microbial Engineering Group, Biosystems Division, Risoe National Laboratory for Sustainable Energy, Technical University of Denmark, Roskilde, Denmark

    Xiaoru Hou & Shuo Yao

  2. Biosystems Division, Risoe National Laboratory for Sustainable Energy, Technical University of Denmark, Building 330, P.O.B. 49, Frederiksborgvej 399, 4000, Roskilde, Denmark

    Xiaoru Hou

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  1. Xiaoru Hou
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  2. Shuo Yao
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Correspondence to Xiaoru Hou.

Supplementary materials

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Supplementary Figure 1

Partial DNA sequence of 5.8S ribosomal RNA of S. passalidarum. Underlined: primer annealing sites (Spa5.8s_fr and Spa5.8s_re); Italic: HaeIII restriction digestion sites (5’gg^cc3’). Size of the fragments after HaeIII digestion: 400 bp, 99 bp. (PPT 230 kb)

Supplementary Figure 2

Partial DNA sequence of 5.8S ribosomal RNA of S. cerevisiae. Underlined: primer annealing sites (Sac5.8s_fr and Sac5.8s_re); Italic: HaeIII restriction digestion sites (5’gg^cc3’) and EcoRI restriction digestion sites (5’g^aattc3’). Size of the fragments after HaeIII digestion: 311bp, 172 bp, 162 bp and 24 bp. Size of the fragments after EcoRI digestion: 365 bp, 304 bp. (PPT 290 kb)

Supplementary Figure 3

In vitro furfural (A) and hydroxymethylfurfural (B) reduction activities measured from the cell-free extracts of S. passalidarum M7 (M7), S. cerevisiae ATCC 96581 (SC) and hybrid yeast FS22 (FS22) using NADH or NADPH as co-factor; NADH (light column), NADPH (dark column). Yeast strains were anaerobically incubated in YPDX liquid medium for 14 hours before the measurement. One unit of activity was defined as the amount of enzyme catalyzing the oxidation of 1 μmol of NAD(P)H per minute using either furfural or HMF as substrate. Data presented are average of three independent measurements and error bars indicate the standard deviations. (PPT 138 kb)

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Hou, X., Yao, S. Improved inhibitor tolerance in xylose-fermenting yeast Spathaspora passalidarum by mutagenesis and protoplast fusion. Appl Microbiol Biotechnol 93, 2591–2601 (2012). https://doi.org/10.1007/s00253-011-3693-5

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