Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene ( pdc ) and Comparison to Bacterial Homologues

Abstract

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 K m 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.Pyruvate decarboxylase (PDC) is the key enzyme for all homo-fermentative ethanol pathways. This enzyme catalyzes the nonoxidative decarboxylation of pyruvate to acetaldehyde using Mg 2ϩ and thiamine pyrophosphate (TPP) as cofactors. Acetaldehyde is reduced to ethanol by alcohol dehydrogenase (ADH) during NADH oxidation. ADH enzymes are widely distributed throughout nature (41). In contrast, PDC is common to only plants, yeasts, and fungi; it is absent in animals and rare in prokaryotes (25). PDC has been established by cloning and purification for only three bacteria, Zymomonas mobilis (6,7,11,19,33,34), Sarcina ventriculi (28,45), and Acetobacter pasteurianus (40). Though cloned from plants and fungi (25,38), only bacterial PDC enzymes are produced at high levels as active recombinant products (6,10,11,34,40,45). Recombinant Z. mobilis PDC has been used for the metabolic engineering of ethanol pathways in many organisms (4,8,14,16,22,44).An ethanologenic bacterium, Zymobacter palmae, has been reported which appears to contain a PDC enzyme (37). Z. palmae was originally isolated from palm sap (37) and produces ethanol as a primary fermentation product from a variety of hexose sugars and saccharides (20,36). This bacterium is distinct from other ethanol-fermenting bacteria including those belonging to the genera Zymomonas. Zymomonas is well known for production of ethanol from plant sap in tropical areas; however, its fermentable carbohydrates are limited to glucose, fructose, and saccharose (22). In addition to substrate utilization, Zymobacter (55.8 Ϯ 0.4 mol% GϩC) differs dramatically from Zymomonas (48.5 Ϯ 0.5 mol% GϩC) in genome composition. This is consistent with the other phenotypic differences that have been observed, including the type of ubiquinone synthesized and peritrichous versus polar flagellation (37).In this paper, we have establis...

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

“…The enzyme also displayed a ±20-fold decrease in the K M for pyruvate with a decrease in pH from 7 to 5, without an equivalent change in the catalytic rate (k cat showed an approximate 2 fold decrease). This is in line with previous observations in related enzymes (Raj et al, 2002; Meyer e t a l . , 2010) and supports the interpretation that PDCs requires a protonated residue for efficient binding of the substrate.…”

“…A similar prediction that Gram negative PDCs should express well in an E. coli background and poorly in a Gram positive host is supported by the wealth of expression data of these genes in various E. coli hosts Chandra Raj et al, 2001;Talarico et al, 2001;Raj et al, 2002;Talarico et al, 2005).…”

“…PDCs are common in the plant and fungal kingdoms and at least in the latter, together with alcohol dehydrogenase (ADH, EC 1.1.1.1) form part of an ethanol fermentation pathway (Konig, 1998). Several plant and yeast PDC's have been isolated and characterized, but as yet only four of bacterial origin have been described; from Zymomonas mobilis, Zymobacter palmae, Acetobacter pasteurianus and Sarcina ventriculi (Raj et al, 2002). As in yeast, these bacterial PDCs participate in ethanol production, but are incorporated into the Entner Doudoroff pathway as opposed to glycolysis for pyruvate production.…”

“…Incompatibilities in codon usage and their effect on expression of PDCs have been reported (Lowe et al, 1992;Talarico et al, 2001;Talarico et al, 2005). Two examples of the effect of codon usage on PDC production include the 5-10 fold increase in soluble SvePDC when expressed in an E. coli strain with or without accessory tRNAs (specifically those which are rarely used in E. coli), as well as the superior production of this PDC relative to those from A. pasteurianus and Z. mobilis in B. megatarium (Talarico et al, 2001;Raj et al, 2002;Talarico et al, 2005).…”

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

“…Recently, however, the gene sequences encoding functional PDCs from three additional bacteria have become available, including that of the Gram-positive Sarcina ventriculi (Sv), and Gram-negative Acetobacter pasteurianus and Zymobacter palmae (Talarico et al, 2001;Raj et al, 2001Raj et al, , 2002. Expression of these genes in recombinant Escherichia coli results in different levels of PDC protein; in particular, the levels of S. ventriculi PDC are reduced in comparison to the other PDCs (Talarico et al, 2001;Raj et al, 2001Raj et al, , 2002. This deficiency in SvPDC protein is remedied by the inclusion of accessory tRNA genes (Talarico et al, 2001;Raj et al, 2002).…”