Construction and expression of an ethanol production operon in Gram-positive bacteria.

Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15-fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.

Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase gene (pdc) and comparison to bacterial homologues.

Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein(-1)) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

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

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.