Abstract
A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.
The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.
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References
Axelsen NH, Kroll J, Weeke B (1973) A manual of quantitative immunoelectrophoresis. Methods and applications. Universitetsforlaget. Oslo Bergen Tromsø
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL, Boyer HW (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Bräu B, Piepersberg W (1983) Cointegrational transduction and mobilization of gentamicin resistance plasmid pWP14a is mediated by IS140. Mol Gen Genet 189:298–303
Brill WJ (1980) Biochemical genetics of nitrogen fixation. Microbiol Rev 44:449–467
Bringer S, Sahm H, Swyzen W (1984a) Ethanol production by Zymomonas mobilis and its application on an industrial scale. Biotechnol Bioeng Symp 14:311–319
Bringer S, Finn RK, Sahm H (1984b) Effect of oxygen on the metabolism of Zymomonas mobilis. Arch Microbiol 139:376–381
Carey VC, Walia SK, Ingram LO (1983) Expression of a lactose transposon (Tn951) in Zymomonas mobilis. J Bacteriol 46: 1163–1168
Clarke L, Carbon J (1976) A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome. Cell 9:91–99
Dawes EA, Ribbons DW, Large PJ (1966) The route of ethanol formation in Zymomonas mobilis. Biochem J 98:795–803
Everaerts FM, Beckers JL, Verheggen TPEM (1976) Isotachophoresis. Theory, instrumentation and applications. J Chromatogr Library, vol 6. Elsevier Scientific Publishing Company, Amsterdam Oxford New York
Feiss M, Fisher RA, Crayton MA, Egner C (1977) Packaging of the bacteriophage lambda chromosome: effect of chromosome length. Virology 77:281–293
Finn RK, Bringer S, Sahm H (1984) Fermentation of arabinose to ethanol by Sarcina ventriculi. Appl Microbiol Biotechnol 19:161–166
Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298
Hoppner TC, Doelle HW (1983) Purification and kinetic characteristics of pyruvate decarboxylase and ethanol dehydrogenase from Zymomonas mobilis in relation to ethanol production. Eur J Appl Microbiol Biotechnol 17:152–157
Hudson L, Hay FC (1976) Practical immunology. Blackwell Scientific Publications. Oxford
Ingram LO, Buttke TM (1984) Effects of alcohols on micro-organisms. Adv Microb Physiol 25:253–300
Knappe J, Blaschkowski HP, Gröbner P, Schmitt T (1974) Pyruvate formate-lyase of Escherichia coli: the acetyl-enzyme intermediate. Eur J Biochem 50:253–263
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning A laboratory manual. Cold Spring Harbor Laboratory, New York
Murray NE, Brammar WJ, Murray K (1977) Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet 150:53–61
Pecher A, Blaschkowski HP, Knappe J, Böck A (1982) Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene. Arch Microbiol 132:365–371
Rogers PL, Skotnicki ML, Lee KJ, Lee JH (1984) Recent developments in the Zymomonas process for ethanol production. Crit Rev Biotechnol 1:273–288
Rossi JJ, Soberon X, Marumoto Y, McMahon J, Itakura K (1983) Biological expression of an Escherichia coli consensus sequence promoter and some mutant derivatives. Proc Natl Acad Sci USA 80:3203–3207
Saito H, Miura K-I (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Schmitt HD, Ciriacy M, Zimmermann FK (1983) The synthesis of yeast pyruvate decarboxylase is regulated by large variations in the messenger RNA level. Mol Gen Genet 192:247–252
Schmitt HD, Zimmermann FK (1982) Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis. J Bacteriol 151:1146–1152
Scrutton MC (1971) Assay of enzymes of CO2 metabolism. In: Morris JR, Ribbons DW (eds) Methods in enzymology, vol 6A. Academic Press, New York San Francisco London, pp 479–541
Skotnicki ML, Tribe DE, Rogers PL (1980) R-plasmid transfer in Zymomonas mobilis. Appl Environ Microbiol 40:7–12
Stokes HW, Picataggio SK, Eveleigh DE (1983) Recombinant genetic approaches for efficient ethanol production. Adv Solar Energy 1:113–132
Swings J, De Ley J (1977) The biology of Zymomonas. Bacteriol Rev 41:1–46
Tarmy EM, Kaplan NO (1968) Chemical characterization of d-lactate dehydrogenase from Escherichia coli B. J Biol Chem 243:2579–2586
Varenne S, Casse F, Chippaux M, Pascal MC (1975) A mutant of Escherichia coli deficient in pyruvate formate-lyase. Mol Gen Genet 141:181–184
Wong P-K, Barrett EL (1983) Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli. FEMS Microbiol Lett 22:143–148
Yu EKC, Saddler JN (1982) Power solvent production by Klebsiella pneumoniae grown on sugars present in wood hemicellulose. Biotechnol Lett 4:121–126
Zehender H, Ullrich J (1985) Amino acid composition of α- and β-chains of yeast and wheat germ pyruvate decarboxylase. FEBS Lett 180:51–54
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Bräu, B., Sahm, H. Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli . Arch. Microbiol. 144, 296–301 (1986). https://doi.org/10.1007/BF00410966
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DOI: https://doi.org/10.1007/BF00410966