scholarly journals Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida

2005 ◽  
Vol 18 (7) ◽  
pp. 345-357 ◽  
Author(s):  
Petra Siegert ◽  
Michael J. McLeish ◽  
Martin Baumann ◽  
Hans Iding ◽  
Malea M. Kneen ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Substrate Specificities ◽  
Benzoylformate Decarboxylase

scholarly journals Ethanologenesis and respiration in a pyruvate decarboxylase-deficient Zymomonas mobilis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Reinis Rutkis ◽  
Inese Strazdina ◽  
Zane Lasa ◽  
Per Bruheim ◽  
Uldis Kalnenieks
Keyword(s):  
Parent Strain ◽  
Zymomonas Mobilis ◽  
Nadh Oxidase ◽  
Pyruvate Decarboxylase ◽  
Production Ratio ◽  
Pyruvate Node ◽  
Branching Point ◽  
Adh Activity ◽  
Ethanol Synthesis

Abstract Objective Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner–Doudoroff (E–D) glycolysis, pyruvate decarboxylase (Pdc) and two alcohol dehydrogenase (ADH) isoenzymes. Pyruvate is the end-product of the E–D pathway and the substrate for Pdc. Construction and study of Pdc-deficient strains is of key importance for Z. mobilis metabolic engineering, because the pyruvate node represents the central branching point, most novel pathways divert from ethanol synthesis. In the present work, we examined the aerobic metabolism of a strain with partly inactivated Pdc. Results Relative to its parent strain the mutant produced more pyruvate. Yet, it also yielded more acetaldehyde, the product of the Pdc reaction and the substrate for ADH, although the bulk ADH activity was similar in both strains, while the Pdc activity in the mutant was reduced by half. Simulations with the kinetic model of Z. mobilis E-D pathway indicated that, for the observed acetaldehyde to ethanol production ratio in the mutant, the ratio between its respiratory NADH oxidase and ADH activities should be significantly higher, than the measured values. Implications of this finding for the directionality of the ADH isoenzyme operation in vivo and interactions between ADH and Pdc are discussed.

scholarly journals High-Level Acetaldehyde Production in Lactococcus lactis by Metabolic Engineering

2005 ◽  
Vol 71 (2) ◽  
pp. 1109-1113 ◽  
Author(s):  
Roger S. Bongers ◽  
Marcel H. N. Hoefnagel ◽  
Michiel Kleerebezem
Keyword(s):  
Metabolic Engineering ◽  
Lactococcus Lactis ◽  
Zymomonas Mobilis ◽  
Nadh Oxidase ◽  
Pyruvate Decarboxylase ◽  
Resting Cells ◽  
Anaerobic Conditions ◽  
High Level ◽  
Acetaldehyde Production ◽  
Combined Overexpression

ABSTRACT Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.

scholarly journals Genetic and Molecular Organization of the Alkylbenzene Catabolism Operon in the Psychrotrophic Strain Pseudomonas putida 01G3

2001 ◽  
Vol 67 (1) ◽  
pp. 453-458 ◽  
Author(s):  
P. A. Chablain ◽  
A. L. Zgoda ◽  
C.-O. Sarde ◽  
N. Truffaut
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Low Temperatures ◽  
Multiple Alignment ◽  
Molecular Organization ◽  
Substrate Specificities ◽  
Psychrotrophic Strain ◽  
Insight Into

ABSTRACT The 11-kb sequence encompassing the alkylbenzene upper pathway inPseudomonas putida 01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized. Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in P. putida RE204. ebd genes appeared to be preferentially induced by ethylbenzene. Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities. Close to identified genes, remnants of IS5-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster.

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

Alteration of the Substrate Specificity of Benzoylformate Decarboxylase from Pseudomonas putida by Directed Evolution

ChemBioChem ◽  
2003 ◽  
Vol 4 (8) ◽  
pp. 721-726 ◽  
Author(s):  
Bettina Lingen ◽  
Doris Kolter-Jung ◽  
Pascal Dünkelmann ◽  
Ralf Feldmann ◽  
Joachim Grötzinger ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Directed Evolution ◽  
Benzoylformate Decarboxylase
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