Physiological effects of overexpressed sigma factors on fermentative stress response of Zymomonas mobilis.

Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z. mobilis ZM4 presents five different sigma factors identified in the genome annotation, but the absence of sigma 38 leads to the questioning of which sigma factors are responsible for its mechanism of fermentative stress response. Thus, in this study, factors sigma 32 and sigma 24, traditionally related to heat shock, were tested for their influence on the response to osmotic and ethanol stress. The overexpression of these sigma factors in Z. mobilis ZM4 strain confirmed that both are associated with heat shock response, as described in other bacteria. Moreover, sigma 32 has also a role in the adaptation to osmotic stress, increasing both growth rate and glucose influx rate. The same strain that overexpresses sigma 32 also showed a decrease in ethanol tolerance, suggesting an antagonism between these two mechanisms. It was not possible to conclude if sigma 24 really affects ethanol tolerance in Z. mobilis, but the overexpression of this sigma factor led to a decrease in ethanol productivity.

Cell Aggregation and Aerobic Respiration Are Important for Zymomonas mobilis ZM4 Survival in an Aerobic Minimal Medium.

Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeast Saccharomyces cerevisiae for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation or about the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation.IMPORTANCE The bacterium Zymomonas mobilis is best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However, Z. mobilis also has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a major Z. mobilis respiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose that Z. mobilis likely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract.

Industrial robustness linked to the gluconolactonase from Zymomonas mobilis.

The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l-1) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l-1) together with sorbitol (65 g l-1) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l-1, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production.

BACKGROUND: The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis. RESULTS: The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain. CONCLUSIONS: Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

Development of corn silk as a biocarrier for Zymomonas mobilis biofilms in ethanol production from rice straw.

Z. mobilis cell immobilization has been proposed as an effective means of improving ethanol production. In this work, polystyrene and corn silk were used as biofilm developmental matrices for Z. mobilis ethanol production with rice straw hydrolysate as a substrate. Rice straw was hydrolyzed by dilute sulfuric acid (H2SO4) and enzymatic hydrolysis. The final hydrolysate contained furfural (271.95 ± 76.30 ppm), 5-hydroxymethyl furfural (0.07 ± 0.00 ppm), vanillin (1.81 ± 0.00 ppm), syringaldehyde (5.07 ± 0.83 ppm), 4-hydroxybenzaldehyde (4-HB) (2.39 ± 1.20 ppm) and acetic acid (0.26 ± 0.08%). Bacterial attachment or biofilm formation of Z. mobilis strain TISTR 551 on polystyrene and delignified corn silk carrier provided significant ethanol yields. Results showed up to 0.40 ± 0.15 g ethanol produced/g glucose consumed when Z. mobilis was immobilized on a polystyrene carrier and 0.51 ± 0.13 g ethanol produced/g glucose consumed when immobilized on delignified corn silk carrier under batch fermentation by Z. mobilis TISTR 551 biofilm. The higher ethanol yield from immobilized, rather than free living, Z. mobilis could possibly be explained by a higher cell density, better control of anaerobic conditions and higher toxic tolerance of Z. mobilis biofilms over free cells.

Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.

Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.

High tolerance and physiological mechanism of Zymomonas mobilis to phenolic inhibitors in ethanol fermentation of corncob residue.

Corncob residue as the lignocellulosic biomass accumulated phenolic compounds generated from xylitol production industry. For utilization of this biomass, Zymomonas mobilis ZM4 was tested as the ethanol fermenting strain and presented a better performance of cell growth (2.8 × 10(8) CFU/mL) and ethanol fermentability (54.42 g/L) in the simultaneous saccharification and fermentation (SSF) than the typical robust strain Saccharomyces cerevisiae DQ1 (cell growth of 2.9 × 10(7) CFU/mL, ethanol titer of 48.6 g/L). The physiological response of Z. mobilis ZM4 to the twelve typical phenolic compounds derived from lignocellulose was assayed and compared with that of S. cerevisiae DQ1. Z. mobilis ZM4 showed nearly the same tolerance to the phenolic aldehydes with S. cerevisiae DQ1, but the stronger tolerance to the phenolic acids existing in corncob residue (2-furoic acid, p-hydroxybenzoic acid, p-coumaric acid, vanillic acid, ferulic acid, and syringic acid). The tolerance mechanism of Z. mobilis was investigated in terms of inhibitor degradation, cell morphology and membrane permeability under the stress of phenolics using GC-MS, scanning and transmission electron microscopies (SEM and TEM), as well as fluorescent probes. The results reveal that Z. mobilis ZM4 has the capability for in situ detoxification of phenolic aldehydes, and the lipopolysaccharide aggregation on the cell outer membrane of Z. mobilis ZM4 provided the permeable barrier to the attack of phenolic acids.

Transcriptional analysis of adaptation to high glucose concentrations in Zymomonas mobilis.

The ethanologenic bacterium Zymomonas mobilis is usually tolerant to high concentrations of glucose. The addition of sorbitol decreases the lag phase and increases ethanol yield and productivity of the bacteria in high glucose concentrations. The molecular mechanisms of adaptation to high glucose concentrations and the effect of sorbitol are still unclear. In this study, microarray analysis was used to study the global transcriptional adaptation responses of Z. mobilis to high glucose concentrations. A total of 235 genes were differentially expressed when 220 g/L glucose was added with or without 10 mM sorbitol. These genes are involved in diverse aspects of cell metabolism and regulation, including membrane transporters, nitrogen metabolism, and plasmid-encoded genes. However, most differentially expressed genes were downregulated when sorbitol was added. Notably, the transcription of almost all genes involved in the Entner-Doudoroff and ethanol production pathways was not significantly affected. In addition, a prophage and a nitrogen-fixation cluster were significantly induced. These results revealed that Z. mobilis cells responded to high glucose concentrations by regulating the transcriptional levels of genes related to membrane channels and transporters, stress response mechanisms, and metabolic pathways. These data provide insight into the intracellular adaptation responses to high glucose concentrations and reveal strategies to engineer efficient ethanol fermentation in Z. mobilis.

Biofilm production by Zymomonas mobilis enhances ethanol production and tolerance to toxic inhibitors from rice bran hydrolysate.

Microorganisms play a significant role in bioethanol production from lignocellulosic material. A challenging problem in bioconversion of rice bran is the presence of toxic inhibitors in lignocellulosic acid hydrolysate. Various strains of Zymomonas mobilis (ZM4, TISTR 405, 548, 550 and 551) grown under biofilm or planktonic modes were used in this study to examine their potential for bioconversion of rice bran hydrolysate and ethanol production efficiencies. Z. mobilis readily formed bacterial attachment on plastic surfaces, but not on glass surfaces. Additionally, the biofilms formed on plastic surfaces steadily increased over time, while those formed on glass were speculated to cycle through accumulation and detachment phases. Microscopic analysis revealed that Z. mobilis ZM4 rapidly developed homogeneous biofilm structures within 24 hours, while other Z. mobilis strains developed heterogeneous biofilm structures. ZM4 biofilms were thicker and seemed to be more stable than other Z. mobilis strains. The percentage of live cells in biofilms was greater than that for planktonic cells (54.32 ± 7.10% vs. 28.69 ± 3.03%), suggesting that biofilms serve as a protective niche for growth of bacteria in the presence of toxic inhibitors in the rice bran hydrolysate. The metabolic activity of ZM4 grown as a biofilm was also higher than the same strain grown planktonically, as measured by ethanol production from rice bran hydrolysate (13.40 ± 2.43 g/L vs. 0.432 ± 0.29 g/L, with percent theoretical ethanol yields of 72.47 ± 6.13% and 3.71 ± 5.24% respectively). Strain TISTR 551 was also quite metabolically active, with ethanol production by biofilm and planktonically grown cells of 8.956 ± 4.06 g/L and 0.0846 ± 0.064 g/L (percent theoretical yields were 48.37 ± 16.64% and 2.046 ± 1.58%, respectively). This study illustrates the potential for enhancing ethanol production by utilizing bacterial biofilms in the bioconversion of a readily available and normally unusable low value by-product of rice farming.

Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis (pHW20a-fdh).

Toxic compounds, such as formic acid, furfural, and hydroxymethylfurfural (HMF) generated during pretreatment of corn stover (CS) at high temperature and low pH, inhibit growth of Zymomonas mobilis and lower the conversion efficiency of CS to biofuel and other products. The inhibition of toxic compounds is considered as one of the major technical barriers in the lignocellulose bioconversion. In order to detoxify and/or degrade these toxic compounds by the model ethanologenic strain Z. mobilis itself in situ the fermentation medium, we constructed a recombinant Z. mobilis ZM4 (pHW20a-fdh) strain that is capable of degrading toxic inhibitor, formate. This is accomplished by cloning heterologous formate dehydrogenase gene (fdh) from Saccharomyces cerevisiae and by coupling this reaction of NADH regeneration reaction system with furfural and HMF degradation in the recombinant Z. mobilis strain. The NADH regeneration reaction also improved both the energy efficiency and cell physiological activity of the recombinant organism, which were definitely confirmed by the improved cell growth, ethanol yield, and ethanol productivity during fermentation with CS hydrolysate.

Effect of ADH II deficiency on the intracellular redox homeostasis in Zymomonas mobilis.

Mutant strain of the facultatively anaerobic, ethanol-producing bacterium Zymomonas 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.

Transcriptome profiling of Zymomonas mobilis under furfural stress.

Furfural from lignocellulosic hydrolysates is the prevalent inhibitor to microorganisms during cellulosic ethanol production, but the molecular mechanisms of tolerance to this inhibitor in Zymomonas mobilis are still unclear. In this study, genome-wide transcriptional responses to furfural were investigated in Z. mobilis using microarray analysis. We found that 433 genes were differentially expressed in response to furfural. Furfural up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. However, furfural has a subtle negative effect on Entner-Doudoroff pathway mRNAs. Our results revealed that furfural had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to furfural. This research has provided insights into the molecular response to furfural in Z. mobilis, and it will be helpful to construct more furfural-resistant strains for cellulosic ethanol production.

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. mobilis lectotype strain ATCC 10988.

Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.

The genome-scale metabolic network analysis of Zymomonas mobilis ZM4 explains physiological features and suggests ethanol and succinic acid production strategies.

BACKGROUND: Zymomonas mobilis ZM4 is a Gram-negative bacterium that can efficiently produce ethanol from various carbon substrates, including glucose, fructose, and sucrose, via the Entner-Doudoroff pathway. However, systems metabolic engineering is required to further enhance its metabolic performance for industrial application. As an important step towards this goal, the genome-scale metabolic model of Z. mobilis is required to systematically analyze in silico the metabolic characteristics of this bacterium under a wide range of genotypic and environmental conditions. RESULTS: 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. CONCLUSION: 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.

Efecto de adición de iones hierro y zinc sobre la producción de etanol de dos cepas recombinantes de Saccharomyces cerevisiae / The effect of adding iron and zinc ions to ethanol production from two recombinant Saccharomyces cerevisiae strains

La producción de etanol por fermentación es influenciada por la presencia de iones metálicos como hierro y zinc dado que son cofactores de la enzima alcohol deshidrogenasa. El estudio de este efecto permitiría identificar el comportamiento de los microorganismos fermentadores en sustratos industriales que contienen altas concentraciones de este tipo de iones. Este trabajo evaluó la producción de biomasa, los azúcares residuales y la producción de etanol por fermentación de tres cepas de S. cerevisiae, CBS8066, recombinantes GG570-CIBI y GG570-CIBII, bajo el efecto de la adición de hierro a 0, 50 y 150 M, y zinc a 0 y 50 M. Las cepas presentaron inhibición en la producción de biomasa y etanol bajo efecto de iones de hierro y zinc, siendo dicha inhibición mayor al estar en presencia de zinc o alta concentración de hierro. GG570-CIBI mostró disminución en producción de biomasa de 4 g/L y una caída en producción de etanol de 40% en el tratamiento 150 M hierro-50 M zinc (con respecto al tratamiento basal). GG570-CIBII fue la menos afectada con inhibición en la producción de etanol inferior a 11% a las 20 h de fermentación. Adicionalmente, presentó la mayor producción de etanol cuando hubo adición de 150 M Fe con o sin adición de zinc, siendo dicha producción entre un 9 y 14% superior a la de las cepas CBS8066 y GG570-CIBI respectivamente, bajo las mismas condiciones. Posteriormente, GG570-CIBII será evaluada en sustratos industriales debido a su menor inhibición en la producción de etanol, permitiendo así obtener mejores rendimientos.

The ethanol production by fermentation is influenced by the presence of metallic ions like iron and zinc because these are alcohol dehydrogenase enzyme cofactors. The study of this effect would allow for identifying the behavior of microorganisms in industrial substrates that contain high concentrations of this kind of ions. This work evaluated biomass production, residual sugars and ethanol production by fermentation of three S. cerevisiae strains, CBS8066, recombinants GG570-CIBI and GG570-CIBII, under the effect of the addition of ferrous ion at 0, 50 and 150 M and zinc ion at 0 and 50 M. The strains showed inhibition on biomass and ethanol production under the effect of zinc and ferrous ions, however, this inhibition was greater in the presence of zinc or iron at high concentration. GG570-CIBI showed reduction in biomass production of 4 g/L and an ethanol production drop of 40 % in the treatment 150 M iron–50 M zinc (with respect to the basal treatment). GG570-CIBII was the less affected with an inhibition on ethanol production below 11 % at 20 h of fermentation. Additionally, GG570-CIBII presented the greatest ethanol production when 150 M iron was added to the culture medium with or without zinc addition. In this case, the production was 9 and 14 % greater than ethanol production of CBS8066 and GG570-CIBI respectively, at the same conditions. Later, GG570-CIBII will be evaluated in industrial substrates due to its lower ethanol production inhibition, allowing for obtaining better yields.

The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors.

BACKGROUND: Zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions. RESULTS: We generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions. CONCLUSIONS: We have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

In vivo assessment of possible probiotic properties of Zymomonas mobilis in a Wistar rat model

In recent years the incorporation of probiotic bacteria into foods has received increasing scientific interest for health promotion and disease prevention. The safety and probiotic properties of Zymomonas mobilis CP4 (UFPEDA-202) was studied in a Wistar rat model fed the 10(9) colony forming units (cfu)/mL-1 of the assayed strain for 30 days. No abnormal clinical signs were noted in the group receiving viable cells of Z. mobilis and water (control) during the period of the experiment. There were no significant difference (p > 0.05) in feed intake and weight gain among mice fed the Z. mobilis in comparison to the control group. No bacteria were found in blood, liver and spleen of any animals. Mice receiving Z. mobilis showed significantly differences (p < 0.05) in total and differential leucocytes count, excepting for neutrophils, after the experimental period. Otherwise, it was not found in control group. Histological examination showed that feeding mice with Z. mobilis caused no signs of adverse effects on gut, liver and spleen. From these results, Z. mobilis CP4 (UFEPEDA-202) is likely to be nonpathogenic and safe for consumption, and could have a slight modulating effect on immunological performance in mice.

Engineering of bio-hybrid materials by electrospinning polymer-microbe fibers.

Although microbes have been used in industrial and niche applications for several decades, successful immobilization of microbes while maintaining their usefulness for any desired application has been elusive. Such a functionally bioactive system has distinct advantages over conventional batch and continuous-flow microbial reactor systems that are used in various biotechnological processes. This article describes the use of polyethylene oxide(99)-polypropylene oxide(67)-polyethylene oxide(99) triblock polymer fibers, created via electrospinning, to encapsulate microbes of 3 industrially relevant genera, namely, Pseudomonas, Zymomonas, and Escherichia. The presence of bacteria inside the fibers was confirmed by fluorescence microscopy and SEM. Although the electrospinning process typically uses harsh organic solvents and extreme conditions that generally are harmful to bacteria, we describe techniques that overcome these limitations. The encapsulated microbes were viable for several months, and their metabolic activity was not affected by immobilization; thus they could be used in various applications. Furthermore, we have engineered a microbe-encapsulated cross-linked fibrous polymeric material that is insoluble. Also, the microbe-encapsulated active matrix permits efficient exchange of nutrients and metabolic products between the microorganism and the environment. The present results demonstrate the potential of the electrospinning technique for the encapsulation and immobilization of bacteria in the form of a synthetic biofilm, while retaining their metabolic activity. This study has wide-ranging implications in the engineering and use of novel bio-hybrid materials or biological thin-film catalysts.

Zymomonas mobilis for fuel ethanol and higher value products.

High oil prices, increasing focus on renewable carbohydrate-based feedstocks for fuels and chemicals, and the recent publication of its genome sequence, have provided continuing stimulus for studies on Zymomonas mobilis. However, despite its apparent advantages of higher yields and faster specific rates when compared to yeasts, no commercial scale fermentations currently exist which use Z. mobilis for the manufacture of fuel ethanol. This may change with the recent announcement of a Dupont/Broin partnership to develop a process for conversion of lignocellulosic residues, such as corn stover, to fuel ethanol using recombinant strains of Z. mobilis. The research leading to the construction of these strains, and their fermentation characteristics, are described in the present review. The review also addresses opportunities offered by Z. mobilis for higher value products through its metabolic engineering and use of specific high activity enzymes.

Cloning and transcriptional analysis of groES and groEL in ethanol-producing bacterium Zymomonas mobilis TISTR 548.

Heat and ethanol had an affect not only on growth and cell viability of an obligatorily fermentative Gram-negative bacterium Zymomonas mobilis, but also on protein synthesis. Analysis by SDS-polyacrylamide gel electrophoresis revealed pronounced increasing of two dominant proteins designated as groES and groEL. Molecular cloning of the gene encoding groES and groEL was performed by PCR technique using specific primers synthesized based on the Z. mobilis groESL gene. Sequencing analysis of 2179 bp led to the detection of two open reading frames encoded for 95 and 549 amino acids, respectively. The deduced amino acid sequence of the Z. mobilis groES and groEL shows a high degree of identity with other. The strongly conserved carboxyl-terminus Gly-Gly-Met motif and two small segments, which appear more conserved between ethanol-producing organisms, were found, suggesting that their may be related to stability of protein under heat or ethanol stress. Induction of groES and groEL occurs in response to heat and ethanol, but not to salt stress.