Electron transport chain in aerobically cultivated Zymomonas mobilis

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ENERGY COUPLING SITES IN THE ELECTRON-TRANSPORT CHAIN OF ZYMOMONAS-MOBILIS

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Modeling of Zymomonas mobilis central metabolism for novel metabolic engineering strategies

Mathematical modeling of metabolism is essential for rational metabolic engineering. The present work focuses on several types of modeling approach to quantitative understanding of central metabolic network and energetics in the bioethanol-producing bacterium Zymomonas mobilis. Combined use of Flux Balance, Elementary Flux Mode, and thermodynamic analysis of its central metabolism, together with dynamic modeling of the core catabolic pathways, can help to design novel substrate and product pathways by systematically analyzing the solution space for metabolic engineering, and yields insights into the function of metabolic network, hardly achievable without applying modeling tools

Application of FT-IR Spectroscopy for Fingerprinting of Zymomonas mobilis Respiratory Mutants

Abstract. Z. mobilis ATCC 29191 and its respiratory knockout mutants, kat-, ndh-, cytB-, and cydB-, were grown under anaerobic and aerobic conditions. FT-IR spectroscopy was used to study the variations of the cell macromolecular composition. Quantitative analysis showed that the concentration ratios-nucleic acids to lipids, for Z. mobilis parent strain, kat-, ndh-, cytB-, and cydB-strains, clearly distinguished Z. mobilis parent strain from its mutant derivatives and corresponded fairly well to the expected degree of biochemical similarity between the strains. Two different FT-IR-spectra hierarchical cluster analysis (HCA) methods were created to differentiate Z. mobilis parent strain and respiratory knockout mutant strains. HCA based on discriminative spectra ranges of carbohydrates, nucleic acids, and lipids allowed to evaluate the influence of growth environment (aeration, growth phase) on the macromolecular composition of cells and differentiate the strains. HCA based on IR spectra of inoculums, in a diagnostic region including the characteristic nucleic acid vibration modes, clearly discriminated the strains under study. Thus it was shown that FT-IR spectroscopy can distinguish various alterations of Z. mobilis respiratory metabolism by HCA of biomass spectra

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 catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers

Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations

<p>Abstract</p> <p>Background</p> <p><it>Zymomonas mobilis </it>ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. <it>Z. mobilis </it>performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly.</p> <p>Results</p> <p>In this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.</p> <p>Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (<it>glk, zwf, pgl, pgk, and eno</it>) and gene <it>pdc</it>, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.</p> <p>Conclusion</p> <p>High oxygen concentrations present during <it>Z. mobilis </it>fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.</p

Phenotype MicroArray Profiling of Zymomonas mobilis ZM4

In this study, we developed a Phenotype MicroArray™ (PM) protocol to profile cellular phenotypes in Zymomonas mobilis, which included a standard set of nearly 2,000 assays for carbon, nitrogen, phosphorus and sulfur source utilization, nutrient stimulation, pH and osmotic stresses, and chemical sensitivities with 240 inhibitory chemicals. We observed two positive assays for C-source utilization (fructose and glucose) using the PM screen, which uses redox chemistry and cell respiration as a universal reporter to profile growth phenotypes in a high-throughput 96-well plate-based format. For nitrogen metabolism, the bacterium showed a positive test results for ammonia, aspartate, asparagine, glutamate, glutamine, and peptides. Z. mobilis appeared to use a diverse array of P-sources with two exceptions being pyrophosphate and tripolyphosphate. The assays suggested that Z. mobilis uses both inorganic and organic compounds as S-sources. No stimulation by nutrients was detected; however, there was evidence of partial inhibition by purines and pyrimidines, NAD, and deferoxamine. Z. mobilis was relatively resistant to acid pH, tolerating a pH down to about 4.0. It also tolerated phosphate, sulfate, and nitrate, but was rather sensitive to chloride and nitrite. Z. mobilis showed resistance to a large number of diverse chemicals that inhibit most bacteria. The information from PM analysis provides an overview of Z. mobilis physiology and a foundation for future comparisons of other wild-type and mutant Z. mobilis strains

Zymomonas mobilis metabolism: Novel tools and targets for its rational engineering

Zymomonas mobilis is an α-proteobacterium that interests the biofuel industry due to its perfect ethanol fermentation yields. From its first description as a bacterial isolate in fermented alcoholic beverages to date, Z. mobilis has been rigorously studied in directions basic and applied. The Z. mobilis powerful Entner-Doudoroff glycolytic pathway has been the center of rigorous biochemical studies and, aside from ethanol, it has attracted interest in terms of high-added-value chemical manufacturing. Energetic balances and the effects of respiration have been explored in fundamental directions as also in applications pursuing strain enhancement and the utilization of alternative carbon sources. Metabolic modeling has addressed the optimization of the biochemical circuitry at various conditions of growth and/or substrate utilization; it has been also critical in predicting desirable end-product yields via flux redirection. Lastly, stress tolerance has received particular attention, since it directly determines biocatalytical performance at challenging bioreactor conditions. At a genetic level, advances in the genetic engineering of the organism have brought forth beneficial manipulations in the Z. mobilis gene pool, e.g., knock-outs, knock-ins and gene stacking, aiming to broaden the metabolic repertoire and increase robustness. Recent omic and expressional studies shed light on the genomic content of the most applied strains and reveal landscapes of activity manifested at ambient or reactor-based conditions. Studies such as those reviewed in this work, contribute to the understanding of the biology of Z. mobilis, enable insightful strain development, and pave the way for the transformation of Z. mobilis into a consummate organism for biomass conversion. © 2020 Elsevier Lt