Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Bringer-Meyer, Stephanie; Schimz, Karl-Ludwig; Sahm, Hermann

doi:10.1007/bf00402334

Cited by 97 publications

(56 citation statements)

References 28 publications

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“…Still another possibility for acetate formation from pyruvate is via pyruvate decarboxylase (EC 4.1.1.1) and acetaldehyde dehydrogenase (EC 1.2.1.4). It has been known for a long time that acetic acid bacteria possess pyruvate decarboxylase (5,9). While pyruvate decarboxylase is widely distributed in plants (44), in bacteria it has been detected in only a few species (37), such as Zymomonas mobilis (8), Sarcina ventriculi (2,12), and the acetic acid bacteria (9).…”

Section: Resultsmentioning

confidence: 99%

Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

Krajewski

Šimić²,

Mouncey³

et al. 2010

Appl Environ Microbiol

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Gluconobacter oxydans N44-1, an obligatory aerobic acetic acid bacterium, oxidizes glucose primarily in the periplasm to the end products 2-ketogluconate and 2,5-diketogluconate, with intermediate formation of gluconate. Only a minor part of the glucose (less than 10%) is metabolized in the cytoplasm after conversion to gluconate or after phosphorylation to glucose-6-phosphate via the only functional catabolic routes, the pentose phosphate pathway and the Entner-Doudoroff pathway. This unusual method of glucose metabolism results in a low growth yield. In order to improve it, we constructed mutants of strain N44-1 in which the gene encoding the membrane-bound glucose dehydrogenase was inactivated either alone or together with the gene encoding the cytoplasmic glucose dehydrogenase. The growth and product formation from glucose of the resulting strains, N44-1 mgdH::kan and N44-1 ⌬mgdH sgdH::kan, were analyzed. Both mutant strains completely consumed the glucose but produced neither gluconate nor the secondary products 2-ketogluconate and 2,5-diketogluconate. Instead, carbon dioxide formation of the mutants increased by a factor of 4 (N44-1 mgdH::kan) or 5.5 (N44-1 ⌬mgdH sgdH::kan), and significant amounts of acetate were produced, presumably by the activities of pyruvate decarboxylase and acetaldehyde dehydrogenase. Most importantly, the growth yields of the two mutants increased by 110% (N44-1 mgdH::kan) and 271% (N44-1 ⌬mgdH sgdH::kan). In addition, the growth rates improved by 39% (N44-1 mgdH::kan) and 78% (N44-1 ⌬mgdH sgdH::kan), respectively, compared to the parental strain. These results show that the conversion of glucose to gluconate and ketogluconates has a strong negative impact on the growth of G. oxydans.As the Gram-negative acetic acid bacterium Gluconobacter oxydans is able to oxidize sugars and sugar alcohols regioselectively, it is a valuable and versatile biocatalyst and has been used in industry for a long time, e.g., for the production of vitamin C via Reichstein synthesis (32) and of 1-deoxynojirimycin, a precursor of the antidiabetic drug miglitol (35). Both processes are combined biotechnological-chemical syntheses carried out on a large scale (10,35). The key biotechnological reactions in these two examples are the regioselective oxidation of D-sorbitol to L-sorbose and N-formyl-1-amino-1-deoxy-D-sorbitol to N-formyl-6-amino-6-deoxy-L-sorbose, respectively. The latter conversion is performed in whole-cell biotransformations with resting cells of G. oxydans as a catalyst.A characteristic trait of G. oxydans is the presence of parallel but spatially separated pathways for the oxidation of nonphosphorylated substrates and intermediates in both the periplasmic and cytoplasmic compartments (Fig.

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Section: Resultsmentioning

confidence: 99%

Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

Krajewski

Šimić²,

Mouncey³

et al. 2010

Appl Environ Microbiol

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“…Expression of the Z. mobilis adhA, adhB, and pdc genes under control of the tac promoter was first examined in recombinants of E. coli S17-1(Apir) ( Fig. 2A; 85 to 181 IU of PDC per mg (6,21,31). On the basis of these values, the maximal amounts of ADHI, ADHII, and PDC proteins were estimated to be 14.5, 5, and 17% to 36% of total cellular protein, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The glycolytic and ethanologenic enzymes in Z. mobilis represent the sole route for energy generation, and together they constitute approximately 50% of soluble cellular protein (2,3,34). The biochemistry and kinetics of the enzymes involved have been characterized (6,21,30,31,34,38,41), and some have recently been proposed to form complexes in vivo (1). In contrast to Embden-Meyerhof glycolysis, which exhibits considerable allosteric control, the ED pathway lacks two key allosteric enzymes, namely, phosphofructokinase and an allosteric hexokinase (2, 29, 41).…”

mentioning

confidence: 99%

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis

1992

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The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase H (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobUis by using a broad-host-range vector containing the tac promoter and the lacP repressor gene. Maximal IPIG (isopropyl-1%-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase H activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression ofadfA, adhB, orpdc at high specific activities (above 8 lU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50%o decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.Zymomonas mobilis represents an excellent model system for metabolic flux control analysis (8,33). This organism is an obligately fermentative gram-negative bacterium that utilizes the Entner-Douderoff (ED) pathway for glycolysis. More than 95% of the glucose metabolized is converted into ethanol and carbon dioxide with a low ATP yield (29). The glycolytic and ethanologenic enzymes in Z. mobilis represent the sole route for energy generation, and together they constitute approximately 50% of soluble cellular protein (2,3,34). The biochemistry and kinetics of the enzymes involved have been characterized (6,21,30,31,34,38,41), and some have recently been proposed to form complexes in vivo (1). In contrast to Embden-Meyerhof glycolysis, which exhibits considerable allosteric control, the ED pathway lacks two key allosteric enzymes, namely, phosphofructokinase and an allosteric hexokinase (2, 29, 41). On the basis of biochemical characterizations and the small metabolite pools in Z. mobilis, it has been proposed that little allosteric regulation operates within the Z. mobilis ED glycolytic pathway (2, 5). Consequently, carbon flux may be limited by the specific activities of pathway enzymes.Most of the Z. mobilis genes encoding ED enzymes and ethanologenic enzymes have been cloned and sequenced (4,(8)(9)(10)(11)(12)24). To facilitate the study of flux control by using these genes, a regulated expression vector is needed for Z. mobilis and none have been previously reported. In this article, we describe the modification and use of a broad-hostrange vector (18) that allows partial control of plasmid-borne genes. This vector was used to investigate the effects of the ethanologenic genes (adhA, adhB, and pdc) on metabolic activity and g...

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“…cerevisiae PDC1 (ScPDC1) was analysed based on its close relationship to SvPDC ( Talarico ) and its use in corn-to-ethanol production (Dien et al, 2002). The A. pasteurianus pdc (Appdc) (Raj et al, 2001) and Z. mobilis pdc (Zmpdc) were also included (Hoppner & Doelle, 1983;Braü & Sahm, 1986;Bringer-Meyer et al, 1986;Conway et al, 1987;Neale et al, 1987). The latter two genes are from Gram-negative bacteria and have high levels of expression and activity in Gram-negative hosts (Raj et al, 2001(Raj et al, , 2002.…”

Section: Construction Of Gram-positive Pdc Expression Plasmidsmentioning

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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Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Cited by 97 publications

References 28 publications

Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis

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

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