The genome-scale metabolic network analysis of Zymomonas mobilis ZM4 explains physiological features and suggests ethanol and succinic acid production strategies

BackgroundZymomonas mobilis ZM4 is a Gram-negative bacterium that can efficiently produce ethanol from various carbon substrates, including glucose, fructose, and sucrose, via the Entner-Doudoroff pathway. However, systems metabolic engineering is required to further enhance its metabolic performance for industrial application. As an important step towards this goal, the genome-scale metabolic model of Z. mobilis is required to systematically analyze in silico the metabolic characteristics of this bacterium under a wide range of genotypic and environmental conditions.ResultsThe genome-scale metabolic model of Z. mobilis ZM4, ZmoMBEL601, was reconstructed based on its annotated genes, literature, physiological and biochemical databases. The metabolic model comprises 579 metabolites and 601 metabolic reactions (571 biochemical conversion and 30 transport reactions), built upon extensive search of existing knowledge. Physiological features of Z. mobilis were then examined using constraints-based flux analysis in detail as follows. First, the physiological changes of Z. mobilis as it shifts from anaerobic to aerobic environments (i.e. aerobic shift) were investigated. Then the intensities of flux-sum, which is the cluster of either all ingoing or outgoing fluxes through a metabolite, and the maximum in silico yields of ethanol for Z. mobilis and Escherichia coli were compared and analyzed. Furthermore, the substrate utilization range of Z. mobilis was expanded to include pentose sugar metabolism by introducing metabolic pathways to allow Z. mobilis to utilize pentose sugars. Finally, double gene knock-out simulations were performed to design a strategy for efficiently producing succinic acid as another example of application of the genome-scale metabolic model of Z. mobilis.ConclusionThe genome-scale metabolic model reconstructed in this study was able to successfully represent the metabolic characteristics of Z. mobilis under various conditions as validated by experiments and literature information. This reconstructed metabolic model will allow better understanding of Z. mobilis metabolism and consequently designing metabolic engineering strategies for various biotechnological applications.

[1]  S. Rhee,et al.  A novel aerobic respiratory chain-linked NADH oxidase system in Zymomonas mobilis , 1995, Journal of bacteriology.

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

[3]  L. Ingram,et al.  Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli , 1989, Applied and environmental microbiology.

[4]  Sunwon Park,et al.  MetaFluxNet, a program package for metabolic pathway construction and analysis, and its use in large-scale metabolic flux analysis of Escherichia coli. , 2003, Genome informatics. International Conference on Genome Informatics.

[5]  S. Lee,et al.  Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation , 2007, Proceedings of the National Academy of Sciences.

[6]  Erin Beck,et al.  The comprehensive microbial resource , 2000, Nucleic Acids Res..

[7]  R. K. Finn,et al.  Effect of oxygen on the metabolism of Zymomonas mobilis , 1984, Archives of Microbiology.

[8]  Ian T. Paulsen,et al.  TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels , 2006, Nucleic Acids Res..

[9]  S. Lee,et al.  Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers , 2010, Biotechnology and bioengineering.

[10]  김정현,et al.  Method for mass production of primary metabolites, strain for mass production of primary metabolites, and method for preparation thereof , 2007 .

[11]  R. Moreau,et al.  News & Notes: The Effect of Ethanol and Oxygen on the Growth of Zymomonas mobilis and the Levels of Hopanoids and Other Membrane Lipids , 1997, Current Microbiology.

[12]  田中 俊典.  National Center for Biotechnology Information (NCBI) , 2012 .

[13]  Á. Bastida,et al.  Synthesis of the aminocyclopentitol moieties of the hopanoids of Zymomonas mobilis and 'Anacystis montana'. , 2003, Chemical communications.

[14]  H. Sahm,et al.  Content and composition of hopanoids in Zymomonas mobilis under various growth conditions , 1991, Journal of bacteriology.

[15]  Sang Yup Lee,et al.  Prediction of metabolic fluxes by incorporating genomic context and flux-converging pattern analyses , 2010, Proceedings of the National Academy of Sciences.

[16]  J. Bu’lock,et al.  High productivity ethanol fermentation on a mineral medium using a flocculent strain ofZymomonas mobilis , 1986, Biotechnology Letters.

[17]  J Swings,et al.  The biology of Zymomonas , 1977, Bacteriological reviews.

[18]  B. Palsson,et al.  Toward Metabolic Phenomics: Analysis of Genomic Data Using Flux Balances , 1999, Biotechnology progress.

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

[20]  Milton H. Saier,et al.  TCDB: the Transporter Classification Database for membrane transport protein analyses and information , 2005, Nucleic Acids Res..

[21]  J. Nielsen,et al.  Analysis of Aspergillus nidulans metabolism at the genome-scale , 2008, BMC Genomics.

[22]  S. Lee,et al.  Towards systems metabolic engineering of microorganisms for amino acid production. , 2008, Current opinion in biotechnology.

[23]  Dong-Yup Lee,et al.  Flux-sum analysis: a metabolite-centric approach for understanding the metabolic network , 2009, BMC Systems Biology.

[24]  L. Ingram,et al.  Genetic engineering of ethanol production in Escherichia coli , 1987, Applied and environmental microbiology.

[25]  Sang Yup Lee,et al.  Systems-level analysis of genome-scalein silico metabolic models using MetaFluxNet , 2005 .

[26]  G. Stephanopoulos,et al.  Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. , 2005, Metabolic engineering.

[27]  W. Wiechert,et al.  Metabolic state of Zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13C- and 31P-NMR spectroscopy , 1999, Archives of Microbiology.

[28]  K. Poralla,et al.  Cloning of conserved genes from Zymomonas mobilis and Bradyrhizobium japonicum that function in the biosynthesis of hopanoid lipids. , 1998, Biochimica et biophysica acta.

[29]  Markus J. Herrgård,et al.  Reconstruction and validation of Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale metabolic model. , 2004, Genome research.

[30]  S. Lee,et al.  Genome-scale metabolic network analysis and drug targeting of multi-drug resistant pathogen Acinetobacter baumannii AYE. , 2010, Molecular bioSystems.

[31]  Sang Yup Lee,et al.  Prediction of Maximum Yields of Metabolites and Optimal Pathways for Their Production by Metabolic Flux Analysis , 2003 .

[32]  Bernhard O. Palsson,et al.  Connecting Extracellular Metabolomic Measurements to Intracellular Flux States in Yeast , 2022 .

[33]  Ron D. Appel,et al.  ExPASy: the proteomics server for in-depth protein knowledge and analysis , 2003, Nucleic Acids Res..

[34]  H. Sahm,et al.  Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli , 1986, Archives of Microbiology.

[35]  Sang Yup Lee,et al.  BioSilico: an integrated metabolic database system , 2004, Bioinform..

[36]  Markus J. Herrgård,et al.  Network-based prediction of human tissue-specific metabolism , 2008, Nature Biotechnology.

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

[38]  A. Varsaki,et al.  Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture , 2000, Journal of applied microbiology.

[39]  L. Quek,et al.  AraGEM, a Genome-Scale Reconstruction of the Primary Metabolic Network in Arabidopsis1[W] , 2009, Plant Physiology.

[40]  Miguel Rocha,et al.  Data Integration Issues in the Reconstruction of the Genome-Scale Metabolic Model of Zymomonas Mobillis , 2008, IWPACBB.

[41]  V. Vinci,et al.  Improvement of microbial strains and fermentation processes , 2000, Applied Microbiology and Biotechnology.

[42]  H. Doelle,et al.  Purification and kinetic characteristics of pyruvate decarboxylase and ethanol dehydrogenase from Zymomonas mobilis in relation to ethanol production , 2004, European journal of applied microbiology and biotechnology.

[43]  Adam M. Feist,et al.  A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information , 2007, Molecular systems biology.

[44]  J. Förster,et al.  In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production. , 2006, Metabolic engineering.

[45]  Hyohak Song,et al.  Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production , 2006, Applied and Environmental Microbiology.

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

[47]  Peter L. Rogers,et al.  Ethanol production by Zymomonas mobilis , 1982 .

[48]  Sang Yup Lee,et al.  Systems biotechnology for strain improvement. , 2005, Trends in biotechnology.

[49]  Dong-Yup Lee,et al.  Genome-scale modeling and in silico analysis of mouse cell metabolic network. , 2009, Molecular bioSystems.

[50]  Sang Yup Lee,et al.  MFAML: a standard data structure for representing and exchanging metabolic flux models , 2005, Bioinform..

[51]  S. Lee,et al.  Production of succinic acid by bacterial fermentation , 2006 .

[52]  G. Church,et al.  Genome-Scale Metabolic Model of Helicobacter pylori 26695 , 2002, Journal of bacteriology.

[53]  L. Ingram,et al.  Lipid composition of Zymomonas mobilis: effects of ethanol and glucose , 1983, Journal of bacteriology.

[54]  S. Lee,et al.  Systems metabolic engineering of Escherichia coli for L-threonine production , 2007, Molecular systems biology.

[55]  Maria I Klapa,et al.  Quantifying the metabolic capabilities of engineered Zymomonas mobilis using linear programming analysis , 2007, Microbial cell factories.

[56]  Jens Nielsen,et al.  Reconstruction of the central carbon metabolism of Aspergillus niger. , 2003, European journal of biochemistry.

[57]  C. W. Jones,et al.  The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus , 1984, Archives of Microbiology.

[58]  Monica L. Mo,et al.  Global reconstruction of the human metabolic network based on genomic and bibliomic data , 2007, Proceedings of the National Academy of Sciences.

[59]  Peter L. Rogers,et al.  Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilisZM4(pZB5) , 2000, Applied and Environmental Microbiology.

[60]  Sang Yup Lee,et al.  The genome sequence of the capnophilic rumen bacterium Mannheimia succiniciproducens , 2004, Nature Biotechnology.

[61]  Paramasamy Gunasekaran,et al.  Ethanol fermentation technology - Zymomonas mobilis. , 1999 .

[62]  J. Bu’lock,et al.  Zymomonas mobilis: a bacterium for ethanol production. , 1986, Biotechnology advances.

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

[64]  Sang Yup Lee,et al.  Metabolite essentiality elucidates robustness of Escherichia coli metabolism , 2007, Proceedings of the National Academy of Sciences.

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

[66]  Kiyoko F. Aoki-Kinoshita,et al.  From genomics to chemical genomics: new developments in KEGG , 2005, Nucleic Acids Res..

[67]  K. Shanmugam,et al.  Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase , 2010, Applied and Environmental Microbiology.