Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi

Lowe, Susan E.; Zeikus, J. G.

doi:10.1099/00221287-138-4-803

Cited by 35 publications

(30 citation statements)

References 21 publications

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“…Previous studies have demonstrated that the PDC purified from S. ventriculi also retains the N-terminal methionine which is amino to a lysine residue (28,45). In contrast, the N-terminal sequence of PDC purified from A. pasteurianus (TYTVGMYLAERL) lacked an N-terminal methionine, suggesting cleavage by a native methionine aminopeptidase.…”

Section: Resultsmentioning

confidence: 83%

“…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).…”

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confidence: 99%

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

Krishnan

Talarico

Ingram

et al. 2002

Appl Environ Microbiol

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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...

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Section: Resultsmentioning

confidence: 83%

mentioning

confidence: 99%

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

Krishnan

Talarico

Ingram

et al. 2002

Appl Environ Microbiol

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“…Alternatively, a pdc gene with optimized codon usage could be synthesized for high-level production of alternative PDCs. SvPDC has qualities that make it unique among bacterial PDCs, including its substrate activation and elevated pH optimum (Lowe & Zeikus, 1992;Talarico et al, 2001). However, in comparison to other PDCs, the K M of SvPDC for pyruvate is over tenfold higher (Raj et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, only low levels of the ZmPDC, ApPDC and ScPDC1 proteins were detected. To determine if the PDC proteins were produced in an active form, the cell lysate of the recombinant B. megaterium strains was assayed for PDC activity ( (Lowe & Zeikus, 1992;Talarico et al, 2001). The results demonstrate that SvPDC is not only produced in very high quantity, but is produced in an active form within the B. megaterium host cell.…”

Section: Expression Of Pdc In Recombinant B Megateriummentioning

confidence: 99%

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

et al. 2005

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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.

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“…It has been demonstrated that the position and usage frequency of codons, together, play a role in correct protein folding and that "codon harmonization" could be used to overcome poor expression, at least in E. coli (Angov et al, 2008;Rosano et al, 2009). 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).…”

Section: Introductionmentioning

confidence: 99%

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

Zyl

Taylor

Eley

et al. 2013

Appl Microbiol Biotechnol

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This study reports the expression, purification and kinetic characterization of a PDC from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120µM at pH 5), high catalytic efficiency (4.75 x 10 5 M -1 s -1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a b a c t e r i a l P D C ) . D u e t o g o o d in vitro thermostablity (approximately 40% enzyme activity retained after 30 minutes at 65°C) this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius. Initial studies using a variety of methods failed to detect activity at any growth temperature. However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection and ethanol productivity). Here we describe the successful expression of PDC in a true thermophile. Yields as high as 0.35 g/g ±0.04 ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights that the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription / translation coupling.

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Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi

Cited by 35 publications

References 21 publications

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

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

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

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

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