Glycerol Assimilation Research Articles

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

A novel strain of Yarrowia lipolytica as a platform for value-added product synthesis from glycerol.

BackgroundIncreasing interest of non-conventional yeasts has been observed for many years due to their biochemical characteristics and potential applications. Well-studied, oleaginous yeast Y. lipolytica is an attractive host for converting a low-cost glycerol, into value-added products such as erythritol (sweetener) or citric acid. Glycerol is an important renewable feedstock and is the main co-product of biodiesel production, which is nowadays applied on a large commercial scale. To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain.ResultsIn this light, we enhanced glycerol assimilation by over-expression of the YALI0F00484g gene encoding glycerol kinase (GK) and gene YALI0B02948g encoding glycerol-3-P dehydrogenase (GDH). The modified strains have been tested for glycerol consumption rate and erythritol and citric acid synthesis under various conditions. Here, we show that the overexpression of GK and GDH, increased glycerol consumption resulting in rapid erythritol and citric acid synthesis. Next, we combined the two genes in the tandem gene construct for the simultaneous co-expression of GK and GDH, which further increased the desired product synthesis. The glycerol consumption was explored in a 5-L bioreactor and the engineered strains were able to utilize 150 g/L glycerol within 44–48 hours. The erythritol productivity for GK overexpression and co-expression of GK and DGH was 24 and 35 %, respectively, over the control strain. Moreover, we established conditions for the production of citric acid at pH 3.0, the engineered strains increased citric acid production 14-fold over the control.ConclusionThis work demonstrates the excellent capacity of the engineered strains as a starting platform for further modification for broad-range value-added product biosynthesis from glycerol. This study presents the highest reported titer citric acid at low pH to date. The process parameters such as productivity and yield of erythritol and citric acid were significantly elevated, what is valuable for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0593-z) contains supplementary material, which is available to authorized users.

Effects of culture conditions on the kinetic behavior of 1,3-propanediol fermentation by Clostridium butyricum with a kinetic model

The effects of culture conditions on the kinetic behavior of 1,3-propanediol (PD) fermentation were investigated with a kinetic model. First, with initial glycerol concentration (S0) increasing, μmax and PD inhibition increased. Glycerol assimilation was harder and a little glycerol was consumed on cell maintenance at high S0. Second, with yeast extract concentration increasing, PD inhibition decreased. However, μmax decreased and glycerol assimilation became harder. It seems that the stimulus effect of yeast extract resulted from decreased PD inhibition. Glycerol amount consumed on cell maintenance also decreased. Third, with temperature decreasing, μmax and PD inhibition decreased. Glycerol assimilation was harder and a little more glycerol was consumed on cell maintenance at low temperature. Fourth, with pH increasing, μmax and PD inhibition decreased. Glycerol assimilation was harder and much more glycerol was consumed on cell maintenance at pH 6.5 and 7.5 than 7.0. This work facilitates further fermentation process optimization.

Transketolase activity modulates glycerol-3-phosphate levels in Escherichia coli.

Transketolase activity provides an important link between the metabolic pathways of glycolysis and pentose phosphate shunt and catalyzes inter-conversions between pentose phosphates and glycolytic intermediates. It is widely conserved in life forms. A genetic screen for suppression of the growth defect of Escherichia coli tktA tktB mutant in LB medium revealed two mutations, one that rendered the glpK expression constitutive and another that inactivated deoB. Characterizing these mutations aided in uncovering the role of ribose-5-P (a transketolase substrate) as an inhibitor of glycerol assimilation and de novo glycerol-3-P synthesis. Using lacZ fusions, we show that ribose-5-P enhances GlpR-mediated repression of the glpFKX operon and inhibits glycerol assimilation. Electrophoretic Mobility Shift Assay (EMSA) showed ribose-5-P made the DNA-GlpR complex less sensitive to the inducer glycerol-3-P. In addition to inhibition of glycerol assimilation, obstruction of ribose-5-P metabolism retards growth from glycerol-3-P limitation. Glucose helps to overcome this limitation through a mechanism involving catabolite repression. To our knowledge, this report is the first to show ribose-5-P can modulate glycerol-3-P concentration in the cell by regulation of glycerol assimilation as well as its de novo synthesis. This regulation could be prevalent in other organisms.

Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production.

We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l(-1) and 28.4-44.6% cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l(-1) and 28.3-38.4% CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

Metabolic engineering of Mortierella alpina for arachidonic acid production with glycerol as carbon source.

BackgroundAlthough some microorganisms can convert glycerol into valuable products such as polyunsaturated fatty acids, the yields are relative low due primarily to an inefficient assimilation of glycerol. Mortierella alpina is an oleaginous fungus which preferentially uses glucose over glycerol as the carbon source for fatty acid synthesis.ResultsIn the present study, we metabolically engineered M. alpina to increase the utilization of glycerol. Glycerol kinase and glycerol-3-phosphate dehydrogenase control the first two steps of glycerol decomposition. GK overexpression increased the total fatty acid content by 35 %, whereas G3PD1, G3PD2 and G3PD3 had no significant effect. Overexpression of malic enzyme (ME1) but not glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase significantly increased fatty acid content when glycerol was used as carbon source. Simultaneous overexpression of GK and ME1 enabled M. alpina to accumulate fatty acids efficiently, with a 44 % increase in fatty acid content (% of dry weight), a 57 % increase in glycerol to fatty acid yield (g/g glycerol) and an 81 % increase in fatty acid production (g/L culture). A repeated batch process was applied to relieve the inhibitory effect of raw glycerol on arachidonic acid synthesis, and under these conditions, the yield reached 52.2 ± 1.9 mg/g.ConclusionsThis study suggested that GK is a rate-limiting step in glycerol assimilation in M. alpina. Another restricting factor for fatty acid accumulation was the supply of cytosolic NADPH. We reported a bioengineering strategy by improving the upstream assimilation and NADPH supply, for oleaginous fungi to efficiently accumulate fatty acid with glycerol as carbon source.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0392-4) contains supplementary material, which is available to authorized users.

Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates.

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK Ec or glpK Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.

Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment

Yarrowia lipolytica, an ascomycete with biotechnological potential, is able to form either yeast cells or hyphae and pseudohyphae in response to environmental conditions. This study shows that the morphology of Y. lipolytica, cultivated in batch cultures on hydrophilic (glucose and glycerol) and hydrophobic (olive oil) media, was not affected by the nature of the carbon source, nor by the nature or the concentration of the nitrogen source. By contrast, dissolved oxygen concentration (DOC) should be considered as the major factor affecting yeast morphology. Specifically, when growth occurred at low or zero DOC the mycelial and/or pseudomycelial forms predominated over the yeast form independently of the carbon and nitrogen sources used. Experimental data obtained from a continuous culture of Y. lipolytica on glycerol, being used as carbon and energy source, demonstrated that the mycelium-to-yeast form transition occurs when DOC increases from 0.1 to 1.5 mg l(-1). DOC also affected the yeast physiology, as the activity of enzymes implicated in lipid biosynthesis (i.e. ATP-citrate lyase, malic enzyme) was upregulated at high DOC whereas the activity of enzymes implicated in glycerol assimilation (such as glycerol dehydrogenase and kinase) remained fundamentally unaffected in the cell-free extract.

The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation

The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L⁻¹ depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L⁻¹ of CA with the mass yield coefficient of 0.64 g g⁻¹ and isocitric acid (ICA) amounted to 4.6 g L⁻¹; in the medium with raw glycerol, CA production was 112 g L⁻¹ with the mass yield coefficient of 0.90 g g⁻¹ and ICA amounted to 5.3 g L⁻¹. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.

Acceleration effect of amino acid supplementation on glycerol assimilation by Escherichia coli in minimal medium

Growth of Escherichia coli BL21 in a glycerol minimal medium was accelerated following supplementation with trace amounts of amino acid (0.35mM). Of 12 amino acids tested, Arg and Ser gave the highest response, increasing cell growth by 63 and 53%, respectively, compared to control cells. The ability of amino acids to accelerate cell growth was "switch-like" and was achieved by promoting glycerol utilization, which may be applied to shorten the long lag-phase when glycerol is used as carbon source. Acceleration of cell growth following amino acid supplementation was also observed using lactose minimal medium.

Expression and functional studies of genes involved in transport and metabolism of glycerol in Pachysolen tannophilus

BackgroundPachysolen tannophilus is a non-conventional yeast, which can metabolize many of the carbon sources found in low cost feedstocks including glycerol and xylose. The xylose utilisation pathways have been extensively studied in this organism. However, the mechanism behind glycerol metabolism is poorly understood. Using the recently published genome sequence of P. tannophilus CBS4044, we searched for genes with functions in glycerol transport and metabolism by performing a BLAST search using the sequences of the relevant genes from Saccharomyces cerevisiae as queries.ResultsQuantitative real-time PCR was performed to unveil the expression patterns of these genes during growth of P. tannophilus on glycerol and glucose as sole carbon sources. The genes predicted to be involved in glycerol transport in P. tannophilus were expressed in S. cerevisiae to validate their function. The S. cerevisiae strains transformed with heterologous genes showed improved growth and glycerol consumption rates with glycerol as the sole carbon source.ConclusionsP. tannophilus has characteristics relevant for a microbial cell factory to be applied in a biorefinery setting, i.e. its ability to utilise the carbon sources such as xylose and glycerol. However, the strain is not currently amenable to genetic modification and transformation. Heterologous expression of the glycerol transporters from P. tannophilus, which has a relatively high growth rate on glycerol, could be used as an approach for improving the efficiency of glycerol assimilation in other well characterized and applied cell factories such as S. cerevisiae.

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h(-1) where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. (13)C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO2 production and oxygen consumption rates which were in good agreement.

Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water-gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B12 (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B12 was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.

Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media

Aim of the present study was to evaluate the effect of exogenous additions of 1,3-propanediol (1,3-PDO) on microbial growth and metabolites production of Clostridium butyricum VPI 1718 strain, during crude glycerol fermentation. Preliminary batch cultures in anaerobic Duran bottles revealed that early addition of 1,3-PDO caused growth cessation in rather low quantities (15g/L), while 1,3-PDO additions during the middle exponential growth phase up to 70g/L resulted in an almost linear decrease of the specific growth rate (μ), accompanied by reduced glycerol assimilation, with substrate consumption being used mainly for energy of maintenance requirements. During batch trials in a 3-L bioreactor, the strain proved able to withstand more than 70g/L of both biologically produced and externally added 1,3-PDO, whereas glycerol assimilation and metabolite production were carried on at a lower rate. Adaptation of the strain in high 1,3-PDO concentration environments was validated during its continuous cultivation with pulses of 1,3-PDO in concentrations of 31 and 46g/L, where no washout phenomena were noticed. As far as C. butyricum cellular lipids were concerned, during batch bioreactor cultivations, 1,3-PDO addition was found to favor the biosynthesis of unsaturated fatty acids. Also, fatty acid composition was studied during continuous cultures, in which additions of 1,3-PDO were performed at steady states. Lipids were globally more saturated compared to batch cultures, while by monitoring of the transitory phases, it was noticed that the gradual diol washout had an evident impact in the alteration of the fatty acid composition, by rendering them more unsaturated.

Tartronate Semialdehyde Reductase Defines a Novel Rate-Limiting Step in Assimilation and Bioconversion of Glycerol in Ustilago maydis

BackgroundGlycerol is a by-product of biodiesel production. Currently, it has limited applications with low bioconversion efficiency to most metabolites reported. This is partly attributed to the poor knowledge on the glycerol metabolic pathway in bacteria and fungi.Methodology/Principal FindingsWe have established a fast screening method for identification of genes that improve glycerol utilization in Ustilago maydis. This was done by comparing the growth rates of T-DNA tagged mutant colonies on solid medium using glycerol as the sole carbon source. We present a detailed characterization of one of the mutants, GUM1, which contains a T-DNA element inserted into the promoter region of UM02592 locus (MIPS Ustilago maydis database, MUMDB), leading to enhanced and constitutive expression of its mRNA. We have demonstrated that um02592 encodes a functional tartronate semialdehyde reductase (Tsr1), which showed dual specificity to cofactors NAD+ and NADP+ and strong substrate specificity and enantioselectivity for D-glycerate. Improved glycerol assimilation in GUM1 was associated with elevated expression of tsr1 mRNA and this could be phenocopied by over-expression of the gene. Glycolipid accumulation was reduced by 45.2% in the knockout mutant whereas introduction of an extra copy of tsr1 driven by the glyceraldehyde phosphate dehydrogenase promoter increased it by 40.4%.Conclusions/SignificanceOur results demonstrate that tartronate semialdehyde reductase (TSR) plays an important role in glycerol assimilation in U. maydis and defines a novel target in genetic engineering for improved conversion of glycerol to higher value products. Our results add significant depth to the understanding of the glycerol metabolic pathway in fungi. We have demonstrated, for the first time, a biological role of a eukaryotic TSR.

Diagnosis and Quantification of Glycerol Assimilating Denitrifying Bacteria in an Integrated Fixed-Film Activated Sludge Reactor via13C DNA Stable-Isotope Probing

Glycerol, a byproduct of biodiesel and oleo-chemical manufacturing operations, represents an attractive alternate to methanol as a carbon and electron donor for enhanced denitrification. However, unlike methanol, little is known about the diversity and activity of glycerol assimilating bacteria in activated sludge. In this study, the microbial ecology of glycerol assimilating denitrifying bacteria in a sequencing batch integrated fixed film activated sludge (SB-IFAS) reactor was investigated using (13)C-DNA stable isotope probing (SIP). During steady state SB-IFAS reactor operation, near complete nitrate removal (92.7 ± 5.8%) was achieved. Based on (13)C DNA clone libraries obtained after 360 days of SB-IFAS reactor operation, bacteria related to Comamonas spp. and Diaphorobacter spp. dominated in the suspended phase communities. (13)C assimilating members in the biofilm community were phylogenetically more diverse and were related to Comamonas spp., Bradyrhizobium spp., and Tessaracoccus spp. Possibly owing to greater substrate availability in the suspended phase, the glycerol-assimilating denitrifying populations (quantified by real-time PCR) were more abundant therein than in the biofilm phase. The biomass in the suspended phase also had a higher specific denitrification rate than the biofilm phase (p = 4.33e-4), and contributed to 69.7 ± 4.5% of the overall N-removal on a mass basis. The kinetics of glycerol based denitrification by suspended phase biomass were approximately 3 times higher than with methanol. Previously identified methanol assimilating denitrifying bacteria were not associated with glycerol assimilation, thereby suggesting limited cross-utilization of these two substrates for denitrification in the system tested.

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 (+)), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1 Delta cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 (+) is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 (+) and dak2 (+) encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1 Delta strain showed a more severe reduction of growth on glycerol and DHA than the dak2 Delta strain because the expression of dak1 (+) mRNA was higher than that of dak2 (+). In wild-type S. pombe, expression of the gld1 (+), dak1 (+), and dak2 (+) genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 (+) was regulated by glucose repression and that it was derepressed in scr1 Delta and tup12 Delta strains.

Genome-wide transcriptional response of Yersinia pestis to stressful conditions simulating phagolysosomal environments

Yersinia pestis is a Gram-negative coccobacillus causing the dangerous disease, plague. Survival of Y. pestis within host macrophages is important in the initial stages of infection. In our present work, DNA microarray was used to determine the expression profiles of Y. pestis strain 201 in response to in vitro simulating conditions of Mg 2+ limitation, polymyxin treatment and oxidative stress that could be found in phagolysosomal environment. It was demonstrated that Y. pestis made appropriate adaptive/protective responses to survive the stressful environments. There are the induced expression of antiphagocytic factors and Mg 2+ transporters under Mg 2+ limitation condition, the stimulation of drug/analogue sensitivity and glycerol assimilation after polymyxin treatment, and the differential expression in genes encoding stress-responsive proteins, components of cell envelope, iron assimilation and regulatory functions in response to both Mg 2+ limitation and polymyxin treatment. Under oxidative stress, Y. pestis uses several mechanisms, especially including the induced expression of detoxification enzymes and DNA repair proteins, to protect from or repair the oxidative cell damages. This microarray analysis would provide the candidates for identifying genes or pathways required for growth and proliferation of Y. pestis in macrophages.

Integrating the Microkinetics of the L-Carnitine and Central Metabolism in Escherichia coli with the Macrokinetics of a High-Cell Density Reactor

A combined E. coli cell metabolism-bioreactor system is described using an S-system model developed within the Biochemical System Theory framework. Such a model is able to represent the activities of the enzymes involved in the biotransformation process, the biotransformation compound concentrations and their transporter. In addition, the transporter is described as an ATP-dependent antiport of betaines and by considering the difference between the concentration of substrate (crotronobetaine) and product (L-carnitine) inside/outside (reactor bulk) the cell. The central metabolism, including glycerol assimilation through the glycolytic and the tricarboxylic acid pathway (TCA) and the synthesis of lactate, formate and acetate has also been modelled. Simulation results matched experiments run with the high-cell density reactor system.

Assimilation of glycerol by a strain of Lactobacillus collinoides isolated from cider

A collection of glycerol-degrading lactic acid bacteria from spoiled cider was isolated. The most widespread species was Lactobacillus collinoides, a heterofermentative species which mainly produced the D -lactic acid isomer from glucose. Conditions of glycerol assimilation by such bacteria were investigated for strain Lb. collinoides IOEB 9527. It could not be cultivated with glycerol alone as carbon source. Sugars were necessary for growth, and fructose was preferred to glucose. Glycerol was degraded, mainly to 1,3-propanediol. Small amounts of acrolein, produced from 3-hydroxypropion-aldehyde (3-HPA) during distillation of samples, were detected. When Lb. collinoides was cultivated with glucose or fructose alone, it produced the usual compounds of the heterofermentative pathway, notably D -lactic acid and small amounts of L -lactic acid, acetic acid and ethanol. When glycerol was present, the amounts of lactic acid were surprisingly much lower and acetic acid + ethanol much higher than expected. Oxidation of D -lactic acid, and to a lesser extent of L -lactic acid, was coupled to degradation of glycerol to 1,3-propanediol. This co-metabolism leading to acetic acid provided energy. Consequently, in fermented beverages spoiled by such strains, glycerol is degraded and 3-HPA plus acetic acid are produced. Both components are undesirable for health and sensorial quality.