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Systems Biology Analysis of Zymomonas mobilis ZM4 Ethanol Stress Responses  [PDF]
Shihui Yang, Chongle Pan, Timothy J. Tschaplinski, Gregory B. Hurst, Nancy L. Engle, Wen Zhou, PhuongAn Dam, Ying Xu, Miguel Rodriguez, Lezlee Dice, Courtney M. Johnson, Brian H. Davison, Steven D. Brown
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0068886
Abstract: Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress.
Research progress of ethanologenic Zymomonas mobilis

LIN Yu-ping,ZHANG Mu-qing {} CHEN Bai-quan,

微生物学报 , 2005,
Abstract: Zymomonas mobilis is one of the natural ethanologenic microbes. With the unique Entner-Doudoroff pathway and some other special pathways of glycolytic and energetic metabolism,Z.mobilis has remarkable characters of higher rate of ethanol production and higher tolerance to ethanol. Glycolytic and energetic metabolism, tolerances (e.g., to ethanol, osmotic stress, etc.) and genetic improvements ofZ.mobilis are reviewed to elucidate the huge potential ofZ.mobilis in fuel ethanol production.
Sheik Abdul Kader Sheik Asraf, K. Narayanan Rajnish, and Paramasamy Gunasekaran
The IIOAB Journal , 2011,
Abstract: Biosynthesis of cellulose has been reported in many species of bacteria. The genes encoding cellulose biosynthetic enzymes of Z. mobilis have not been studied so far. Preliminary sequence analysis of the Z. mobilis ZM4 genome revealed the presence of a cellulose synthase operon comprised of Open Reading Frames (ORFs) ZMO01083 (bcsA), ZMO1084 (bcsB) and ZMO1085 (bcsC). The first gene of the operon bcsA encodes the cellulose synthase catalytic subunit BcsA. The second gene of the operon bcsB encodes the cellulose synthase subunit B (BcsB), which shows the presence of BcsB multi-domain and is inferred to bind c-di-GMP, the regulator of cellulose biosynthesis. The third gene of the operon bcsC encodes the cellulose synthase operon C domain protein (BcsC), which belongs to super family of teratrico peptide repeat (TPR) that are believed to mediate protein – protein interactions for the formation of cellulose. Multiple sequence alignment of the deduced amino acid sequences of BcsA and BcsC with other closely related homologs showed the presence of PVDPYE, HAKAGNLN, DCD motif and TPR motif, the characteristic motifs of bacterial cellulose synthases. Analysis of the nucleotide sequence of the ORF ZMO1085 and neighboring ORFs namely ZMO1083 and ZMO1084 indicated that all the ORFs are translationally linked and form an operon. Transcript analysis using Real-time PCR indicated the expression of the genes involved in cellulose synthase operon in Zymomonas mobilis ZM4. Z. mobilis colonies grown on RM-glucose containing Congo red displayed a characteristic bright red-brown colour. Z. mobilis colonies grown on RM-glucose medium supplemented with Calcoflour exhibited fluorescence. The arrangement of Calcofluor stained microfibrils can be seen in fluorescence microscopy which is an indicative for cellulose biosynthesis. AFM micrograph of the extracellular matrix of Z. mobilis shows a relatively dense matrix with bacterial cell residues. The presence of cellulose was confirmed by the Acetic-Nitric (Updegraff) Cellulose assay. The Bioinformatics and biosynthetic analysis confirm the biosynthesis of cellulose in Z. mobilis.
The genome-scale metabolic network analysis of Zymomonas mobilis ZM4 explains physiological features and suggests ethanol and succinic acid production strategies
Kyung Lee, Jong Park, Tae Kim, Hongseok Yun, Sang Lee
Microbial Cell Factories , 2010, DOI: 10.1186/1475-2859-9-94
Abstract: The genome-scale metabolic model of Z. mobilis ZM4, ZmoMBEL601, was reconstructed based on its annotated genes, literature, physiological and biochemical databases. The metabolic model comprises 579 metabolites and 601 metabolic reactions (571 biochemical conversion and 30 transport reactions), built upon extensive search of existing knowledge. Physiological features of Z. mobilis were then examined using constraints-based flux analysis in detail as follows. First, the physiological changes of Z. mobilis as it shifts from anaerobic to aerobic environments (i.e. aerobic shift) were investigated. Then the intensities of flux-sum, which is the cluster of either all ingoing or outgoing fluxes through a metabolite, and the maximum in silico yields of ethanol for Z. mobilis and Escherichia coli were compared and analyzed. Furthermore, the substrate utilization range of Z. mobilis was expanded to include pentose sugar metabolism by introducing metabolic pathways to allow Z. mobilis to utilize pentose sugars. Finally, double gene knock-out simulations were performed to design a strategy for efficiently producing succinic acid as another example of application of the genome-scale metabolic model of Z. mobilis.The genome-scale metabolic model reconstructed in this study was able to successfully represent the metabolic characteristics of Z. mobilis under various conditions as validated by experiments and literature information. This reconstructed metabolic model will allow better understanding of Z. mobilis metabolism and consequently designing metabolic engineering strategies for various biotechnological applications.The impact of biotechnology on industry and society is dramatically gaining momentum, particularly in the field of agriculture-food, medicine and chemical production. For the chemical industry, which aims to producing value-added chemicals and fuels in a sustainable way, efforts have been put into strain improvement of microorganisms, utilizing many newly emergin
Transcriptome profiling of Zymomonas mobilis under ethanol stress  [cached]
He Ming-xiong,Wu Bo,Shui Zong-xia,Hu Qi-chun
Biotechnology for Biofuels , 2012, DOI: 10.1186/1754-6834-5-75
Abstract: Background High tolerance to ethanol is a desirable characteristics for ethanologenic strains used in industrial ethanol fermentation. A deeper understanding of the molecular mechanisms underlying ethanologenic strains tolerance of ethanol stress may guide the design of rational strategies to increase process performance in industrial alcoholic production. Many extensive studies have been performed in Saccharomyces cerevisiae and Escherichia coli. However, the physiological basis and genetic mechanisms involved in ethanol tolerance for Zymomonas mobilis are poorly understood on genomic level. To identify the genes required for tolerance to ethanol, microarray technology was used to investigate the transcriptome profiling of the ethanologenic Z. mobilis in response to ethanol stress. Results We successfully identified 127 genes which were differentially expressed in response to ethanol. Ethanol up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. These genes were classified as being involved in a wide range of cellular processes including carbohydrate metabolism, cell wall/membrane biogenesis, respiratory chain, terpenoid biosynthesis, DNA replication, DNA recombination, DNA repair, transport, transcriptional regulation, some universal stress response, etc. Conclusion In this study, genome-wide transcriptional responses to ethanol were investigated for the first time in Z. mobilis using microarray analysis.Our results revealed that ethanol had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to ethanol. Although the molecular mechanism involved in tolerance and adaptation of ethanologenic strains to ethanol is still unclear, this research has provided insights into molecular response to ethanol in Z. mobilis. These data will also be helpful to construct more ethanol resistant strains for cellulosic ethanol production in the future.
Thermotolerant Zymomonas mobilis: Comparison of Ethanol Fermentation Capability with that of an Efficient Type Strain
Zymomonas mobilisZymomonas mobilisSaccharomyces cerevisiaeZ. mobilisadhAadhBpdc
The Open Biotechnology Journal , 2007, DOI: 10.2174/1874070700701010059]
Abstract: Zymomonas mobilis is an alternative microorganism to Saccharomyces cerevisiae for ethanol production. To find a thermotolerant Z. mobilis strain, the growth and ethanol production of four isolates in Thailand were compared with those of the efficient strain ZM4 (NRRL B-14023) at different temperatures. One of the selected strains, TISTR 405, was found to grow and produce ethanol even at 39°C to an extent similar to that at 30°C, and the growth and ethanol productivity at 39°C were better than those of ZM4 at 30°C, suggesting that TISTR 405 is suitable for ethanol fermentation at high temperatures. Analysis of genes directly related to ethanol formation or degradation, adhA, adhB and pdc, encoding alcohol dehydrogenase (Adh) A, AdhB and pyruvate decarboxylase, respectively, revealed that these genes were highly conserved in both strains. Comparison of their gene expression and activity of the products in both TISTR 405 and ZM4 at different temperatures or growth phases indicated that there was not a great difference at the transcriptional level, but the total activity of AdhA and AdhB in TISTR 405 was higher than that in ZM4. Both strains showed a significant increase in AdhB activity in the stationary phase.
Magnesium Ions Improve Growth and Ethanol Production of Zymomonas mobilis under Heat or Ethanol Stress  [PDF]
Pornthap Thanonkeo,Pattana Laopaiboon,Kaewta Sootsuwan,Mamoru Yamada
Biotechnology , 2007,
Abstract: Like other ethanologenic organisms, Zymomonas mobilis showed a significant decrease in cell viability and fermentation capability when the initial ethanol concentration or temperature was elevated. The cell mass and ethanol production were largely decreased at more than 35°C or in the presence of higher than 7% (by vol) ethanol at an initial concentration. Neither growth nor ethanol production was observed at more than 40°C or at higher than 14% (by vol) ethanol. The supplementation of magnesium (10-20 mM) dramatically improved the negative effects by these stresses. Magnesium also increased in fermentation ability and repressed the synthesis of stress proteins under such conditions. These results clearly demonstrated the usefulness of magnesium for ethanol fermentation in Z. mobilis under heat or ethanol stress.
The influence of centrifugation on Zymomonas mobilis aggregation
Perez Fernandez Palha,María de los Angeles; Edison Lopes,Carlos; Gomes de Andrade Lima,Maria Alice; Pereira Junior,Nei;
Electronic Journal of Biotechnology , 2002,
Abstract: flocculent zymomonas mobilis breaks down in smaller flocs and individual cells when centrifuged. the main consequence of it is an increase in the dispersion of the sample, suggesting that the influence of the centrifugal force on the aggregation of cells is worth to study. the experiments showed that the degree of dispersion varied between 30% and 100% when the centrifugal acceleration changed from 25 g to 2500 g. observation under the electronic microscope showed that a slimy material covered the cells recovered by gentle gravitational settling and, that the centrifuged cells presented a bare cell wall.
Optimization of levan production by Zymomonas mobilis
Ananthalakshmy, V. K;Gunasekaran, P;
Brazilian Archives of Biology and Technology , 1999, DOI: 10.1590/S1516-89131999000300004
Abstract: effect of different fermentation conditions on levan production by zymomonas mobilis b-4286 was studied. levan production increased from 5.7-g/l to 12.6-g/l with an increase in initial sucrose concentration (50-150 g/l). above 15% (20 and 25%) sucrose concentration, there was no increase in the biomass. the sucrose hydrolysis and levan production occurred even in the absence of significant growth of cells. maximum amount of levan was produced (14.5 g/l) at ph 5 and 15 g /l at 250c temperature. at temperature between 350c and 400c, levan production was not detected. presence of glucose in the medium considerably reduced levan production (2.8 g/l) than fructose 6.7g/l.
Zymomonas mobilis levan production in the presence of antimetabolic angents  [cached]
Cristina Sturzoiu,Andrei Tanase,Anca Dinischiotu,Gheorghe Stoian
Analele ?tiin?ifice Ale Universit??ii Alexandru Ioan Cuza din Ia?i,Sectiunea II A : Genetica si Biologie Moleculara , 2010,
Abstract: Our studies have focused on the screening of Zymomonas mobilis bacterial strains capable to produce levan under the action of three antimetabolic agents (sulfafurozol, sulfametaxazol, trimetoprim). The experiments were carried out using spontaneous mutant strains derived from Z.mobilis NCIB 11163 and 11163/70, obtained by supplemented media with methotrexate (600 μg/mL) and trimethoprim (1000 μg/mL). Exponential growth profiles of bacterial cells and the production of levan were assessed in the absence and presence of antimetabolites of different concentrations. The studies have shown that Z.mobilis 11163/70 strains manifest a progressive growth in the presence of trimethoprim (50 μg/mL), an inhibition growth in the presence of sulfametoxazol (100 μg/mL). and also good resistance in the presence of sulfafurazol (100 μg/mL). Sulphonamides can inhibit the production of levan (Z.mobilis CP4PRRif, Z.mobilis NCIB 11163/70). On the other hand, a stimulation of levan production has been observed in the presence of trimetoprim (50 μg/mL) (Z.mobilis CP4Rif and 10988).
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