The inefficient aerobic energetics ofZymomonas mobilis: Identifying the bottleneck

To investigate the mechanisms of Zymomonas mobilis uncoupled aerobic metabolism, growth properties of the wild-type strain Zm6 were compared to those of its respiratory mutants cytB and cydB, and the effects of the ATPase inhibitor DCCD on growth and intracellular ATP concentration were studied. The effects of the ATPase inhibitor DCCD on growth and intracellular ATP concentration strongly indicated that the apparent lack of oxidative phosphorylation in aerobically growing Z. mobilis culture might be caused by the ATP hydrolyzing activity of the H(+) -dependent ATPase in all analyzed strains. Aerobic growth yields of the mutants, and their capacity of oxidative ATP synthesis with ethanol we…

Respiratory behaviour of a Zymomonas mobilis adhB::kan(r) mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions.

AbstractPerturbation of the aerobic steady-state in a chemostat culture of the ethanol-producing bacterium Zymomonas mobilis with a small pulse of ethanol causes a burst of ethanol oxidation, although the reactant ratio of the alcohol dehydrogenase (ADH) reaction ([NADH][acetaldehyde][H+])/([ethanol][NAD+]) remains above the Keq value. Simultaneous catalysis of ethanol synthesis and oxidation by the two ADH isoenzymes, residing in different redox microenvironments, has been proposed previously. In the present study, this hypothesis is verified by construction of an ADH-deficient strain and by demonstration that it lacks the oxidative burst in response to perturbation of its aerobic steady-s…

NADH dehydrogenase deficiency results in low respiration rate and improved aerobic growth of Zymomonas mobilis.

The respiratory chain of the ethanol-producing bacterium Zymomonas mobilis is able to oxidize both species of nicotinamide cofactors, NADH and NADPH. A mutant strain with a chloramphenicol-resistance determinant inserted in ndh (encoding an NADH : CoQ oxidoreductase of type II) lacked the membrane NADH and NADPH oxidase activities, while its respiratory d-lactate oxidase activity was increased. Cells of the mutant strain showed a very low respiration rate with glucose and no respiration with ethanol. The aerobic growth rate of the mutant was elevated; exponential growth persisted longer, resulting in higher biomass densities. For the parent strain a similar effect of aerobic growth stimulat…

Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains

Zymomonas mobilis is the most efficient bacterial ethanol producer and its physiology is potentially applicable to industrial-scale bioethanol production. However, compared to other industrially important microorganisms, the Z. mobilis metabolome and adaptation to various nutritional and genetic perturbations have been poorly characterized. For rational metabolic engineering, it is essential to understand how central metabolism and intracellular redox balance are maintained in Z. mobilis under various conditions. In this study, we applied quantitative mass spectrometry-based metabolomics to explore how glucose-fed non-growing Z. mobilis Zm6 cells metabolically adapt to change of oxygen avai…

Study of Zymomonas mobilis uncoupled energy metabolism

Baktērijām Z. mobilis ir raksturīgs augsts no biosintēzes reakcijām neatkarīgs katabolisma ātrums, jeb atjūgtais enerģētiskais metabolisms. Šīs parādības mehanismi kā arī elpošanas ķēdes loma enerģijas apritē līdz galam nav skaidra. Disertācijas „Zymomonas mobilis atjūgtā enerģētiskā metabolisma pētījumi“ mērķi bija (I) izpētīt Z. mobilis elpošanas ķēdes strukturālās un funkcionālās īpatnības un tās saistību ar atjūgto metabolismu, (II) konstruēt in silico centrāla katabolisma modeli un ar tā palīdzību pētīt katabolisma regulāciju. Darbā secināts: (I) oksidatīvās fosforilēšanas trūkums Z. mobilis ir skaidrojams ar nepietiekamu protondzinējspēka un H+-atkarīgās ATFāzes enerģētisku sajūgšanu,…

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc 1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc 1 complex via cytochrome c 552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc 1 complex (Zm…

Metabolic Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis

Respiratory chain plays a pivotal role in the energy and redox balance of aerobic bacteria. By engineering respiration, it is possible to alter the efficiency of energy generation and intracellular redox state, and thus affect the key bioprocess parameters: cell yield, productivity and stress resistance. Here we summarize the current metabolic engineering and synthetic biology approaches to bacterial respiratory metabolism, with a special focus on the respiratory chain of the ethanologenic bacterium Zymomonas mobilis. Electron transport in Z. mobilis can serve as a model system of bacterial respiration with low oxidative phosphorylation efficiency. Its application for redox balancing and re…

Kinetic modelling of the Zymomonas mobilis Entner-Doudoroff pathway: insights into control and functionality.

Zymomonas mobilis, an ethanol-producing bacterium, possesses the Entner-Doudoroff (E-D) pathway, pyruvate decarboxylase and two alcohol dehydrogenase isoenzymes for the fermentative production of ethanol and carbon dioxide from glucose. Using available kinetic parameters, we have developed a kinetic model that incorporates the enzymic reactions of the E-D pathway, both alcohol dehydrogenases, transport reactions and reactions related to ATP metabolism. After optimizing the reaction parameters within likely physiological limits, the resulting kinetic model was capable of simulating glycolysis in vivo and in cell-free extracts with good agreement with the fluxes and steady-state intermediate …

Zymomonas mobilis atjūgtā enerģētiskā metabolisma pētījumi

Baktērijām Z. mobilis ir raksturīgs augsts no biosintēzes reakcijām neatkarīgs katabolisma ātrums, jeb atjūgtais enerģētiskais metabolisms. Šīs parādības mehanismi kā arī elpošanas ķēdes loma enerģijas apritē līdz galam nav skaidra. Disertācijas „Zymomonas mobilis atjūgtā enerģētiskā metabolisma pētījumi“ mērķi bija (I) izpētīt Z. mobilis elpošanas ķēdes strukturālās un funkcionālās īpatnības un tās saistību ar atjūgto metabolismu, (II) konstruēt in silico centrāla katabolisma modeli un ar tā palīdzību pētīt katabolisma regulāciju. Darbā secināts: (I) oksidatīvās fosforilēšanas trūkums Z. mobilis ir skaidrojams ar nepietiekamu protondzinējspēka un H+-atkarīgās ATFāzes enerģētisku sajūgšanu,…

Translocation of Zymomonas mobilis pyruvate decarboxylase to periplasmic compartment for production of acetaldehyde outside the cytosol

Abstract Acetaldehyde, a valuable commodity chemical, is a volatile inhibitory byproduct of aerobic fermentation in Zymomonas mobilis and in several other microorganisms. Attempting to improve acetaldehyde production by minimizing its contact with the cell interior and facilitating its removal from the culture, we engineered a Z. mobilis strain with acetaldehyde synthesis reaction localized in periplasm. For that, the pyruvate decarboxylase (PDC) was transferred from the cell interior to the periplasmic compartment. This was achieved by the construction of a Z. mobilis Zm6 PDC‐deficient mutant, fusion of PDC with the periplasmic signal sequence of Z. mobilis gluconolactonase, and the follow…

Does Probiotic Yeast Act as Antigenotoxin?

The effect of probiotic yeast Saccharomyces boulardii on genotoxicity induced by the well-known mutagen 4-nitroquinoline-N-oxide (4-NQO), as well as antibacterial (furazolidone) and antibiotic (nalidixic acid) drugs, has been studied using the short-term bacterial assay, SOS chromotest, with Escherichia coli PQ 37 as the test organism. It has been shown that S. boulardii possesses antigenotoxic activity, revealed by SOS chromotest, when coincubated with these genotoxins. A weaker antigenotoxic activity against the same compounds was observed with S. carlsbergensis, too.

Effect of ADH II Deficiency on the Intracellular Redox Homeostasis in Zymomonas mobilis

Mutant strain of the facultatively anaerobic, ethanol-producing bacteriumZymomonas mobilis, deficient in the Fe-containing alcohol dehydrogenase isoenzyme (ADH II), showed impaired homeostasis of the intracellular NAD(P)H during transition from anaerobic to aerobic conditions, and also in steady-state continuous cultures at various oxygen supplies. At the same time, ADH II deficiency in aerobically grown cells was accompanied by a threefold increase of catalase activity and by about 50% increase of hydrogen peroxide excretion. It is concluded that ADH II under aerobic conditions functions to maintain intracellular redox homeostasis and to protect the cells from endogenous hydrogen peroxide.

The Low Energy-Coupling Respiration in Zymomonas mobilis Accelerates Flux in the Entner-Doudoroff Pathway.

Performing oxidative phosphorylation is the primary role of respiratory chain both in bacteria and eukaryotes. Yet, the branched respiratory chains of prokaryotes contain alternative, low energy-coupling electron pathways, which serve for functions other than oxidative ATP generation (like those of respiratory protection, adaptation to low-oxygen media, redox balancing, etc.), some of which are still poorly understood. We here demonstrate that withdrawal of reducing equivalents by the energetically uncoupled respiratory chain of the bacterium Zymomonas mobilis accelerates its fermentative catabolism, increasing the glucose consumption rate. This is in contrast to what has been observed in o…

High aerobic growth with low respiratory rate: The ndh-deficient Zymomonas mobilis

Improvement of acetaldehyde production in Zymomonas mobilis by engineering of Its aerobic metabolism

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catab…

Aerobic catabolism and respiratory lactate bypass in Ndh-negative Zymomonas mobilis

Ability to ferment in the presence of oxygen increases the robustness of bioprocesses and opens opportunity for novel industrial setups. The ethanologenic bacterium Zymomonas mobilis performs rapid and efficient anaerobic ethanol fermentation, yet its respiratory NADH dehydrogenase (Ndh)-deficient strain (ndh-) is known to produce ethanol with high yield also under oxic conditions. Compared to the wild type, it has a lower rate of oxygen consumption, and an increased expression of the respiratory lactate dehydrogenase (Ldh). Here we present a quantitative study of the product spectrum and carbon balance for aerobically growing ndh-. Ldh-deficient and Ldh-overexpressing ndh- strains were con…