Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli

ABSTRACT Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low Km values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low Km for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

[1]  K. Shanmugam,et al.  Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli , 2009, Biotechnology Letters.

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

[3]  H. Eklund,et al.  Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme. , 2004, Journal of molecular biology.

[4]  F. A. Shamsi,et al.  Mutagenicity of furfural in plasmid DNA. , 1995, Cancer letters.

[5]  Xueli Zhang,et al.  Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C , 2008, Biotechnology and bioengineering.

[6]  Wei Shen,et al.  Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design , 2006 .

[7]  L. Ingram,et al.  Purification and characterization of a furfural reductase (FFR) from Escherichia coli strain LYO1--an enzyme important in the detoxification of furfural during ethanol production. , 2006, Journal of biotechnology.

[8]  Mark Holtzapple,et al.  Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover. , 2005, Bioresource technology.

[9]  Jeffrey H. Miller,et al.  A short course in bacterial genetics , 1992 .

[10]  B. Dien,et al.  Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae , 2006, Applied Microbiology and Biotechnology.

[11]  J. Barciszewski,et al.  A mechanism for the in vivo formation of N6‐furfuryladenine, kinetin, as a secondary oxidative damage product of DNA , 1997, FEBS letters.

[12]  D. Schomburg,et al.  BRENDA: a resource for enzyme data and metabolic information. , 2002, Trends in biochemical sciences.

[13]  Bärbel Hahn-Hägerdal,et al.  Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification , 2000 .

[14]  B. Um,et al.  Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover. , 2003, Applied biochemistry and biotechnology.

[15]  A Martinez,et al.  Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli , 2000, Biotechnology and bioengineering.

[16]  Mark Holtzapple,et al.  Coordinated development of leading biomass pretreatment technologies. , 2005, Bioresource technology.

[17]  Peter D. Karp,et al.  Multidimensional annotation of the Escherichia coli K-12 genome , 2007, Nucleic acids research.

[18]  M. Galbe,et al.  Bio-ethanol--the fuel of tomorrow from the residues of today. , 2006, Trends in biotechnology.

[19]  Chankyu Park,et al.  Conversion of Methylglyoxal to Acetol by Escherichia coli Aldo-Keto Reductases , 2005, Journal of bacteriology.

[20]  C. Nakamura,et al.  Metabolic engineering for the microbial production of 1,3-propanediol. , 2003, Current opinion in biotechnology.

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

[22]  A Martinez,et al.  Detoxification of Dilute Acid Hydrolysates of Lignocellulose with Lime , 2001, Biotechnology progress.

[23]  Mohammad J Taherzadeh,et al.  In situ detoxification and continuous cultivation of dilute-acid hydrolyzate to ethanol by encapsulated S. cerevisiae. , 2006, Journal of biotechnology.

[24]  L. Ingram,et al.  Use of UV Absorbance To Monitor Furans in Dilute Acid Hydrolysates of Biomass , 2000, Biotechnology progress.

[25]  A. Angelov,et al.  Effect of furfural on carbon metabolism key enzymes of lactose-assimilating yeasts , 2006 .

[26]  L. Ingram,et al.  Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. , 1999, Biotechnology and bioengineering.

[27]  Sonia Rodriguez,et al.  Purification and Identification of an Escherichiacoli β‐Keto Ester Reductase as 2,5‐Diketo‐d‐gluconate Reductase YqhE , 2002, Biotechnology progress.

[28]  Akihiko Kondo,et al.  Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain , 2006, Applied Microbiology and Biotechnology.

[29]  K. Shanmugam,et al.  Low salt medium for lactate and ethanol production by recombinant Escherichia coli B , 2007, Biotechnology Letters.

[30]  D. White.  The Physiology and Biochemistry of Prokaryotes , 1999 .

[31]  K. Shanmugam,et al.  Re-engineering Escherichia coli for ethanol production , 2008, Biotechnology Letters.

[32]  Yutaka Tokiwa,et al.  Biological production of functional chemicals from renewable resources , 2008 .

[33]  P. Slininger,et al.  Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae , 2008, Applied Microbiology and Biotechnology.

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

[35]  N. Singh,et al.  Acetaldehyde: genotoxicity and cytotoxicity in human lymphocytes. , 1995, Mutation research.

[36]  M. Taherzadeh,et al.  Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. , 2002, The Biochemical journal.

[37]  I. S. Horváth,et al.  Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae. , 2001, Biotechnology and bioengineering.

[38]  A Martinez,et al.  Effects of Ca(OH)(2) treatments ("overliming") on the composition and toxicity of bagasse hemicellulose hydrolysates. , 2000, Biotechnology and bioengineering.

[39]  D. Yum,et al.  Identification of the yqhE and yafB genes encoding two 2, 5-diketo-D-gluconate reductases in Escherichia coli. , 1999, Applied and environmental microbiology.

[40]  L. Jarboe,et al.  Development of ethanologenic bacteria. , 2007, Advances in biochemical engineering/biotechnology.

[41]  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.

[42]  J. Liu,et al.  Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes. , 2001, Nucleic acids research.

[43]  J. M. Sandoval,et al.  Escherichia coli YqhD Exhibits Aldehyde Reductase Activity and Protects from the Harmful Effect of Lipid Peroxidation-derived Aldehydes* , 2008, Journal of Biological Chemistry.

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

[45]  G. Lidén,et al.  NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae , 2008, Applied Microbiology and Biotechnology.

[46]  K. Shanmugam,et al.  Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II , 1991, Applied and environmental microbiology.

[47]  J. Claverie,et al.  Crystal structure of Escherichia coli DkgA, a broad‐specificity aldo‐keto reductase , 2005, Proteins.

[48]  Ramaraj Boopathy,et al.  Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria , 1993, Journal of Industrial Microbiology.

[49]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.