Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

ABSTRACT Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The Vmax of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] × h versus 0.007 g/g [DW] × h), and the anaerobic growth rate was increased from ∼0 h−1 to 0.08 h−1. The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

[1]  B. Hahn-Hägerdal,et al.  The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. , 2002, FEMS yeast research.

[2]  A. Willems,et al.  Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure , 1995, Yeast.

[3]  K. Bohren,et al.  Expression of Human Aldose and Aldehyde Reductases , 1991 .

[4]  Gunnar Lidén,et al.  Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae. , 2003, Biotechnology and bioengineering.

[5]  M. Galbe,et al.  Pretreatment of lignocellulosic materials for efficient bioethanol production. , 2007, Advances in biochemical engineering/biotechnology.

[6]  H. Schneider,et al.  Growth of yeasts on D-xylulose , 1980 .

[7]  Jung-Kul Lee,et al.  Cloning and Characterization of the xyl1 Gene, Encoding an NADH-Preferring Xylose Reductase from Candida parapsilosis, and Its Functional Expression in Candida tropicalis , 2003, Applied and Environmental Microbiology.

[8]  Jack T Pronk,et al.  High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae? , 2003, FEMS yeast research.

[9]  A. Cornish-Bowden.  Fundamentals of Enzyme Kinetics , 1979 .

[10]  P. Kötter,et al.  Xylose fermentation by Saccharomyces cerevisiae , 1993, Applied Microbiology and Biotechnology.

[11]  B. Hahn-Hägerdal,et al.  Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae , 2009, Microbial cell factories.

[12]  Peter J. Butterworth,et al.  Fundamentals of enzyme kinetics (3rd edn) A. Cornish-Bowden. Portland Press Ltd, London, 422 + xvi pp., ISBN 1 85578 158 1 (2000) , 2005 .

[13]  Wayne M Patrick,et al.  Strategies and computational tools for improving randomized protein libraries. , 2005, Biomolecular engineering.

[14]  Barbara M. Bakker,et al.  Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry. , 2000, European journal of biochemistry.

[15]  J. Pronk,et al.  Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component. , 2007, Advances in biochemical engineering/biotechnology.

[16]  T. Flynn,et al.  Aldose reductase from human psoas muscle. Purification, substrate specificity, immunological characterization, and effect of drugs and inhibitors. , 1989, The Journal of biological chemistry.

[17]  K. Bohren,et al.  Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262. , 1991, The Journal of biological chemistry.

[18]  Hung Lee,et al.  Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase. , 1998, FEMS microbiology letters.

[19]  W. A. Scheffers,et al.  Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation , 1992, Yeast.

[20]  P Argos,et al.  NADP‐Dependent enzymes. I: Conserved stereochemistry of cofactor binding , 1997, Proteins.

[21]  H. Nojima,et al.  High efficiency transformation of Escherichia coli with plasmids. , 1990, Gene.

[22]  Merja Penttilä,et al.  Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases , 2007, Applied Microbiology and Biotechnology.

[23]  B. Hahn-Hägerdal,et al.  Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae. , 2003, FEMS yeast research.

[24]  Oskar Bengtsson,et al.  The expression of a Pichia stipitis xylose reductase mutant with higher KM for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae , 2006, Biotechnology and bioengineering.

[25]  B. Hahn-Hägerdal,et al.  Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering , 2005, Yeast.

[26]  R. Schiestl,et al.  Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method , 2007, Nature Protocols.

[27]  T. Jeffries,et al.  Engineering yeasts for xylose metabolism. , 2006, Current opinion in biotechnology.

[28]  B. Hahn-Hägerdal,et al.  Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae , 2009, Biotechnology for biofuels.

[29]  Uwe Sauer,et al.  Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose , 2003, Applied and Environmental Microbiology.

[30]  Bärbel Hahn-Hägerdal,et al.  Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. , 2007, Advances in biochemical engineering/biotechnology.

[31]  A A ANDREASEN,et al.  Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. , 1953, Journal of cellular and comparative physiology.

[32]  W. V. van Zyl,et al.  Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources , 1999, Applied Microbiology and Biotechnology.

[33]  K. Kavanagh,et al.  Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases. , 2003, The Biochemical journal.

[34]  M. Wallner,et al.  Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization , 2010, Microbial cell factories.

[35]  H. Schneider,et al.  Growth of yeasts on D-xylulose 1. , 1980, Canadian journal of microbiology.

[36]  W. V. van Zyl,et al.  Role of cultivation media in the development of yeast strains for large scale industrial use , 2005, Microbial cell factories.

[37]  B. Hahn-Hägerdal,et al.  Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose , 2002, Applied and Environmental Microbiology.

[38]  W. V. van Zyl,et al.  Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054. , 2003, FEMS yeast research.

[39]  Kentaro Miyazaki,et al.  Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. , 2002, BioTechniques.

[40]  K. Kavanagh,et al.  The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography. , 2005, The Biochemical journal.

[41]  Seung Pil Pack,et al.  Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis. , 2007, Microbiology.

[42]  W. J. Dower,et al.  High efficiency transformation of E. coli by high voltage electroporation , 1988, Nucleic Acids Res..

[43]  T. Flynn,et al.  Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine. , 1989, The Journal of biological chemistry.

[44]  Jack T Pronk,et al.  Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. , 2005, FEMS yeast research.

[45]  J. Pronk,et al.  Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose, Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains , 2008, Applied and Environmental Microbiology.

[46]  G. Piétu,et al.  Effect of plasmid size on transformation efficiency by electroporation of Escherichia coli DH5 alpha. , 1994, BioTechniques.

[47]  Andrew E. Firth,et al.  Statistics of protein library construction , 2005, Bioinform..

[48]  Bernd Nidetzky,et al.  Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae , 2008, Microbial cell factories.

[49]  F. Hynne,et al.  Full-scale model of glycolysis in Saccharomyces cerevisiae. , 2001, Biophysical chemistry.