Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA 1 Author for correspondence: Julie A. Maupin-Furlow. Tel: +1 352 392 4095. Fax: +1 352 392 5922. e-mail: jmaupin{at}ufl.edu Sarcina ventriculi grows in a remarkable range of mesophilic environments from pH 2 to pH 10. During growth in acidic environments, where acetate is toxic, expression of pyruvate decarboxylase (PDC) serves to direct the flow of pyruvate into ethanol during fermentation. PDC is rare in bacteria and absent in animals, although it is widely distributed in the plant kingdom. The pdc gene from S. ventriculi is the first to be cloned and characterized from a Gram-positive bacterium. In Escherichia coli , the recombinant pdc gene from S. ventriculi was poorly expressed due to differences in codon usage that are typical of low-G+C organisms. Expression was improved by the addition of supplemental codon genes and this facilitated the 136-fold purification of the recombinant enzyme as a homo-tetramer of 58 kDa subunits. Unlike Zymomonas mobilis PDC, which exhibits Michaelis–Menten kinetics, S. ventriculi PDC is activated by pyruvate and exhibits sigmoidal kinetics similar to fungal and higher plant PDCs. Amino acid residues involved in the allosteric site for pyruvate in fungal PDCs were conserved in S. ventriculi PDC, consistent with a conservation of mechanism. Cluster analysis of deduced amino acid sequences confirmed that S. ventriculi PDC is quite distant from Z. mobilis PDC and plant PDCs. S. ventriculi PDC appears to have diverged very early from a common ancestor which included most fungal PDCs and eubacterial indole-3-pyruvate decarboxylases. These results suggest that the S. ventriculi pdc gene is quite ancient in origin, in contrast to the Z. mobilis pdc , which may have originated by horizontal transfer from higher plants. Keywords: pyruvate metabolism, thiamin pyrophosphate-dependent enzymes, alcohol dehydrogenase, ethanol fermentation, allosteric regulation Abbreviations: ADH, alcohol dehydrogenase; E1, decarboxylase component of pyruvate dehydrogenase; IPD, indole-3-pyruvate decarboxylase; PDC, pyruvate decarboxylase; TPP, thiamin pyrophosphate; TK, transketolase. The GenBank accession number for the sequence reported in this paper is AF354297 .