Evolutionary engineering strategies to enhance tolerance of xylose utilizing recombinant yeast to inhibitors derived from spruce biomass

BackgroundOne of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation.ResultsA long-term adaptation in repetitive batch cultures in shake flasks using a cocktail of 12 different inhibitors and a long-term chemostat adaptation using spruce hydrolysate were used as evolutionary engineering strategies to improve the inhibitor tolerance in the metabolically engineered xylose utilizing Saccharomyces cerevisiae strain, TMB3400. The yeast was evolved for a period of 429 and 97 generations in repetitive batch cultures and chemostat cultivation, respectively. During the evolutionary engineering in repetitive batch cultures the maximum specific growth rate increased from 0.18 h-1 to 0.33 h-1 and the time of lag phase was decreased from 48 h to 24 h. In the chemostat adaptation, after 97 generations, the specific conversion rates of HMF and furfural were found to be 3.5 and 4 folds higher respectively, compared to rates after three generations. Two evolved strains (RK60-5, RKU90-3) and one evolved strain (KE1-17) were isolated from evolutionary engineering in repetitive batches and chemostat cultivation, respectively. The strains displayed significantly improved growth performance over TMB3400 when cultivated in spruce hydrolysate under anaerobic conditions, the evolved strains exhibited 25 to 38% increase in specific consumption rate of sugars and 32 to 50% increased specific ethanol productivity compared to TMB3400. The evolved strains RK60-5 and RKU90-3 were unable to consume xylose under anaerobic conditions, whereas, KE1-17 was found to consume xylose at similar rates as TMB3400.ConclusionUsing evolutionary engineering strategies in batch and chemostat cultivations we have generated three evolved strains that show significantly better tolerance to inhibitors in spruce hydrolysate and displayed a shorter time for overall fermentation of sugars compared to the parental strain.

[1]  U. Sauer,et al.  Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain , 2008, Microbial biotechnology.

[2]  Leif J. Jönsson,et al.  Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce , 1999 .

[3]  Z Lewis Liu,et al.  Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. , 2005, Applied biochemistry and biotechnology.

[4]  Andreas Wagner,et al.  Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae. , 2011, FEMS yeast research.

[5]  A. Wheals,et al.  Fuel ethanol after 25 years. , 1999, Trends in biotechnology.

[6]  L. Olsson,et al.  Fermentation of lignocellulosic hydrolysates for ethanol production. , 1996 .

[7]  U. Sauer.  Evolutionary engineering of industrially important microbial phenotypes. , 2001, Advances in biochemical engineering/biotechnology.

[8]  Mohammad J. Taherzadeh,et al.  Conversion of dilute-acid hydrolyzates of spruce and birch to ethanol by fed-batch fermentation , 1999 .

[9]  J. Bautista,et al.  Effects of furfural and 5-hydroxymethylfurfural on the fermentation of Saccharomyces cerevisiae and biomass production from Candida guilliermondii , 1988 .

[10]  Mats Galbe,et al.  Comparison of SO2 and H2SO4 impregnation of softwood prior to steam pretreatment on ethanol production , 1998 .

[11]  X. Parés,et al.  Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction. , 2002, The Biochemical journal.

[12]  L. Jönsson,et al.  Comparison of the resistance of industrial and laboratory strains of Saccharomyces and Zygosaccharomyces to lignocellulose-derived fermentation inhibitors , 2003 .

[13]  Anneli Petersson,et al.  Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae , 2007 .

[14]  Leif J. Jönsson,et al.  Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae , 2000, Applied biochemistry and biotechnology.

[15]  B. Hahn-Hägerdal,et al.  Cofactor Dependence in Furan Reduction by Saccharomyces cerevisiae in Fermentation of Acid-Hydrolyzed Lignocellulose , 2005, Applied and Environmental Microbiology.

[16]  G. Zacchi,et al.  The generation of fermentation inhibitors during dilute acid hydrolysis of softwood , 1999 .

[17]  B. Hahn-Hägerdal,et al.  Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, andXKS1 in Mineral Medium Chemostat Cultures , 2000, Applied and Environmental Microbiology.

[18]  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.

[19]  Ye Sun,et al.  Hydrolysis of lignocellulosic materials for ethanol production: a review. , 2002, Bioresource technology.

[20]  N. Meinander,et al.  Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. , 1999, Biotechnology and bioengineering.

[21]  B. Hahn-Hägerdal,et al.  A 5‐hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance , 2006, Yeast.

[22]  L. Gomez,et al.  Sustainable liquid biofuels from biomass: the writing's on the walls. , 2008, The New phytologist.

[23]  L. Gorton,et al.  Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors. , 2002, Biotechnology and bioengineering.

[24]  G. Zacchi,et al.  Comparison of S 02 and H 2 S 0 4 Impregnation of Softwood Prior to Steam Pretreatment on Ethanol Production , 2022 .

[25]  L. Gustafsson,et al.  Characterization and fermentation of dilute-acid hydrolyzates from wood , 1997 .

[26]  Leif J Jönsson,et al.  Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors. , 2007, Bioresource technology.

[27]  Lo Gorton,et al.  Effect of different forms of alkali treatment on specific fermentation inhibitors and on the fermentability of lignocellulose hydrolysates for production of fuel ethanol. , 2002, Journal of agricultural and food chemistry.

[28]  O. Berg.  Periodic selection and hitchhiking in a bacterial population. , 1995, Journal of theoretical biology.

[29]  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.

[30]  B. Ahring,et al.  Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass , 2004, Applied Microbiology and Biotechnology.

[31]  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.

[32]  A. Stams,et al.  Utilisation of biomass for the supply of energy carriers , 1999, Applied Microbiology and Biotechnology.

[33]  G. Lidén,et al.  Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate , 2008, Biotechnology and bioengineering.

[34]  L. Gustafsson,et al.  Conversion of furfural in aerobic and anaerobic batch fermentation of glucose by Saccharomyces cerevisiae. , 1999, Journal of bioscience and bioengineering.

[35]  L. Gustafsson,et al.  Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae , 2000, Applied Microbiology and Biotechnology.

[36]  Guido Zacchi,et al.  Cost Analysis of Ethanol Production from Willow Using Recombinant Escherichia coli , 1994, Biotechnology progress.

[37]  B. Hahn-Hägerdal,et al.  Towards industrial pentose-fermenting yeast strains , 2007, Applied Microbiology and Biotechnology.

[38]  Bärbel Hahn-Hägerdal,et al.  Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. , 2000 .