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

Sarcina ventriculi grows in a remarkable range of mesophilic environments f rom pH 2 to pH 10. During growth in acidic environments, where acetate is t oxic, expression of pyruvate decarboxylase (PDC) serves to direct the flow of pyruvate into ethanol during fermentation. PDC is rare in bacteria and a bsent in animals, although it is widely distributed in the plant kingdom. T he pdc gene from S. ventriculi is the first to be cloned and characterized from a Grampositive bacterium. In Escherichia coli, the recombinant pdc gen e 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 addi tion of supplemental codon genes and this facilitated the 136-fold purifica tion of the recombinant enzyme as a homo-tetramer of 58 kDa subunits. Unlik e Zymomonas mobilis PDC, which exhibits Michael is-Menten kinetics, S. vent riculi PDC is activated by pyruvate and exhibits sigmoidal kinetics similar to fungal and higher plant PDCs. Amino acid residues involved in the allos teric site for pyruvate in fungal PDCs were conserved in S. ventriculi PDC, consistent with a conservation of mechanism. Cluster analysis of deduced a mino acid sequences confirmed that S. ventriculi PDC is quite distant from Z. mobilis PDC and plant PDCs. S. ventriculi PDC appears to have diverged v ery early from a common ancestor which included most fungal PDCs and eubact erial indole-3-pyruvate decarboxylases. These results suggest that the S. v entriculi pdc gene is quite ancient in origin, in contrast to the Z mobilis pdc, which may have originated by horizontal transfer from higher plants.