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Physiology and Growth |
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: John R. Guest. Tel: +44 114 2224406. Fax: +44 114 2222787. e-mail: j.r.guest{at}sheffield.ac.uk
The metabolic importance of pyruvate oxidase (PoxB), which converts pyruvate directly to acetate and CO2, was assessed using an isogenic set of genetically engineered strains of Escherichia coli. In a strain lacking the pyruvate dehydrogenase complex (PDHC), PoxB supported acetate-independent aerobic growth when the poxB gene was expressed constitutively or from the IPTG-inducible tac promoter. Using aerobic glucose-limited chemostat cultures of PDH-null strains, it was found that steady-states could be maintained at a low dilution rate (0·05 h-1) when PoxB is expressed from its natural promoter, but not at higher dilution rates (up to at least 0·25 h-1) unless expressed constitutively or from the tac promoter. The poor complementation of PDH-deficient strains by poxB plasmids was attributed to several factors including the stationary-phase-dependent regulation of the natural poxB promoter and deleterious effects of the multicopy plasmids. As a consequence of replacing the PDH complex by PoxB, the growth rate (µmax), growth yield (Ymax) and the carbon conversion efficiency (flux to biomass) were lowered by 33%, 9–25% and 29–39% (respectively), indicating that more carbon has to be oxidized to CO2 for energy generation. Extra energy is needed to convert PoxB-derived acetate to acetyl-CoA for further metabolism and enzyme analysis indicated that acetyl-CoA synthetase is induced for this purpose. In similar experiments with a PoxB-null strain it was shown that PoxB normally makes a significant contribution to the aerobic growth efficiency of E. coli. In glucose minimal medium, the respective growth rates (µmax), growth yields (Ymax) and carbon conversion efficiencies were 16%, 14% and 24% lower than the parental values, and correspondingly more carbon was fluxed to CO2 for energy generation. It was concluded that PoxB is used preferentially at low growth rates and that E. coli benefits from being able to convert pyruvate to acetyl-CoA by a seemingly wasteful route via acetate.
Keywords: pyruvate metabolism, pyruvate dehydrogenase complex, growth rate, biomass yield and energetics, acetyl-CoA synthetase, continuous culture
Abbreviations: ACK, acetokinase; ACS, acetyl-CoA synthetase; PDH, pyruvate dehydrogenase; PDH-E1p, E1 component of the pyruvate dehydrogenase complex; PDHC, pyruvate dehydrogenase complex; PFL, pyruvate formate-lyase; PoxB, pyruvate oxidase; PTA, phosphotransacetylase; ThDP, thiamin diphosphate
a Present address: Botany Department, Faculty of Sciences, Minia University, Minia, Egypt.
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