Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae

The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na+) tolerance and not acetate (Ac-) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na+ and Ac- ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.

[1]  Fabrice Armougom,et al.  Expresso: automatic incorporation of structural information in multiple sequence alignments using 3D-Coffee , 2006, Nucleic Acids Res..

[2]  Min Zhang,et al.  Metabolic Engineering of a Pentose Metabolism Pathway in Ethanologenic Zymomonas mobilis , 1995, Science.

[3]  Zhang-liang Chen,et al.  Over-expression of the bacterial nhaA gene in rice enhances salt and drought tolerance , 2005 .

[4]  E. Padan,et al.  NhaA crystal structure: functional–structural insights , 2009, Journal of Experimental Biology.

[5]  Gwo-Liang Chen,et al.  Improved genome annotation for Zymomonas mobilis , 2009, Nature Biotechnology.

[6]  Peter L. Rogers,et al.  A mutant of Zymomonas mobilis ZM4 capable of ethanol production from glucose in the presence of high acetate concentrations , 1998, Biotechnology Letters.

[7]  W. Verstraete,et al.  Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400 , 2006, Applied and Environmental Microbiology.

[8]  M. Zhang,et al.  Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering , 1996, Applied and environmental microbiology.

[9]  Hyun Seok Park,et al.  The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4 , 2005, Nature Biotechnology.

[10]  J. Souciet,et al.  The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux. , 1998, Microbiology.

[11]  E. Blumwald,et al.  Identification and Characterization of Vnx1p, a Novel Type of Vacuolar Monovalent Cation/H+ Antiporter of Saccharomyces cerevisiae* , 2007, Journal of Biological Chemistry.

[12]  Cédric Notredame,et al.  3DCoffee: combining protein sequences and structures within multiple sequence alignments. , 2004, Journal of molecular biology.

[13]  Brent A. Gregersen,et al.  Mechanism of Na+/H+ Antiporting , 2007, Science.

[14]  Hartmut Michel,et al.  Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH , 2005, Nature.

[15]  The effect of acetic acid on fuel ethanol production byZymomonas , 1992 .

[16]  Julian Parkhill,et al.  Microbiology in the post-genomic era , 2008, Nature Reviews Microbiology.

[17]  Michael E. Himmel,et al.  Enzymatic conversion of biomass for fuels production. , 1994 .

[18]  P. Rogers,et al.  Nuclear magnetic resonance studies of acetic acid inhibition of rec Zymomonas mobilis ZM4(pZB5). , 2000, Applied biochemistry and biotechnology.

[19]  G. Stephanopoulos,et al.  Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? , 2009, Nature Reviews Microbiology.

[20]  C. Nusbaum,et al.  Quality scores and SNP detection in sequencing-by-synthesis systems. , 2008, Genome research.

[21]  Parmjit S. Panesar,et al.  Zymomonas mobilis : an alternative ethanol producer , 2006 .

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

[23]  J. Souciet,et al.  Characterization of the NHA1 gene encoding a Na+/H+‐antiporter of the yeast Saccharomyces cerevisiae , 1996, FEBS letters.

[24]  Anne-Kristin Kaster,et al.  Methanogenic archaea: ecologically relevant differences in energy conservation , 2008, Nature Reviews Microbiology.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  D. Botstein,et al.  Comparing whole genomes using DNA microarrays , 2008, Nature Reviews Genetics.

[27]  David K. Johnson,et al.  Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production , 2007, Science.

[28]  Peter L. Rogers,et al.  Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis , 2002, Biotechnology Letters.

[29]  Yi-Song Wang,et al.  A general system for studying protein-protein interactions in Gram-negative bacteria. , 2008, Journal of proteome research.

[30]  Ryan T Gill,et al.  Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli , 2009, Biotechnology for biofuels.

[31]  H G Lawford,et al.  Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate , 2001, Applied biochemistry and biotechnology.

[32]  S. Lee,et al.  Application of systems biology for bioprocess development. , 2008, Trends in biotechnology.

[33]  B. Palsson,et al.  An evaluation of Comparative Genome Sequencing (CGS) by comparing two previously-sequenced bacterial genomes , 2007, BMC Genomics.

[34]  S. Hemmingsen,et al.  Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast. , 1992, The EMBO journal.

[35]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[36]  T. W. Jeffries,et al.  Bacteria engineered for fuel ethanol production: current status , 2003, Applied Microbiology and Biotechnology.

[37]  P. Rogers,et al.  Zymomonas mobilis for fuel ethanol and higher value products. , 2007, Advances in biochemical engineering/biotechnology.

[38]  R. Helm,et al.  Identification of inhibitory components toxic toward zymomonas mobilis CP4(pZB5) xylose fermentation , 1997 .

[39]  Kay Hofmann,et al.  Tmbase-A database of membrane spanning protein segments , 1993 .

[40]  H. Lawford,et al.  Improving fermentation performance of recombinant zymomonas in acetic acid-containing media , 1998, Applied biochemistry and biotechnology.

[41]  J. D. McMillan,et al.  Conversion of hemicellulose hydrolyzates to ethanol , 1994 .

[42]  Manuel C. Peitsch,et al.  SWISS-MODEL: an automated protein homology-modeling server , 2003, Nucleic Acids Res..

[43]  Brian H Davison,et al.  Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations , 2009, BMC Genomics.

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

[45]  J. Bailey,et al.  Toward a science of metabolic engineering , 1991, Science.

[46]  P. Young,et al.  Functional expression of the Schizosaccharomyces pombe Na+/H+ antiporter gene, sod2, in Saccharomyces cerevisiae. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[47]  G. Stephanopoulos.  Challenges in Engineering Microbes for Biofuels Production , 2007, Science.