Metabolic engineering of Escherichia coli for the production of coenzyme Q10

Coenzyme Q10 is required for respiratory electron transport and protects biological membranes against oxidative damage. As coenzyme Q10 supplements are used to treat or to alleviate symptoms associated with an increasing number of health conditions, there is growing interest in the development of bioprocesses for its production. The biosynthesis of coenzyme Q10 involves the condensation of an isoprenoid, decaprenyl diphosphate, with an aromatic compound, para-hydroxybenzoate, followed by a series of modifications of the aromatic moiety of the molecule via the ubiquinone pathway. Escherichia coli naturally produces coenzyme Q8, but replacement of its octaprenyl diphosphate synthase by a decaprenyl diphosphate synthase is sufficient to eliminate the production of coenzyme Q8 and favor the synthesis of coenzyme Q10. A rational genetic engineering approach was used to create a strain of E. coli capable of producing high levels of coenzyme Q10. First, the endogenous octaprenyl diphosphate synthase gene was deleted and functionally replaced by a decaprenyl diphosphate synthase-encoding gene derived from Sphingomonas baekryungensis. Additionally, this strain was engineered to produce elevated levels of para-hydroxybenzoate by over-expressing genes encoding enzymes of the E. coli shikimate pathway. The production of isoprenoid was increased by introducing a heterologous mevalonate pathway. Decaprenyl diphosphate and para-hydroxybenzoate were further directed toward the ubiquinone pathway by overexpressing a para-hydroxybenzoate prenyltransferase. The resulting recombinant strain was capable of producing elevated levels of coenzyme Q10. In order to further enhance production of this antioxidant, an investigation into the interplay between coenzyme Q10 biosynthesis and primary metabolism was conducted. This investigation revealed a link between sorbitol catabolism and coenzyme Q10 production in the engineered strain. Moreover, abrogating carbon flux to acetate by selected gene knock-outs also enhanced coenzyme Q10 accumulation. However, the engineered strains developed through this research were found to be highly unstable, leading to high variability in coenzyme Q10 production. This instability was hypothesized to result from the burden exerted by the engineered aromatic and foreign mevalonate pathways on primary metabolism. As a result, further optimization of these engineered strains of E. coli will be required in order to develop a suitable platform for coenzyme Q10 production

Analyses directionnelles multivariées de la qualité des précipitations sur la région de Québec

La question des précipitations acides est devenue une des principales préoccupations environnementales de ce siècle. Les dommages engendrés touchent l'ensemble des composantes atmosphérique, aquatique et terrestre de notre écosystème. Dans cet article, l'intérêt est tourné vers le transport des substances polluantes en faisant intervenir le vecteur des précipitations acides: le vent. Il existe une croyance populaire, largement répandue dans l'est du Canada, consistant à admettre que les vents d'est ou du nord sont associés à des précipitations faiblement chargées en éléments acides, contrairement aux vents du sud-ouest qui transporteraient les charges plus contaminées des industries situées dans le Midwest nord-américain. Une confirmation expérimentale de la réalité du phénomène est présentée ici.Une analyse factorielle des correspondances permet de mettre en évidence des relations entre la direction des vents et la composition chimique des précipitations. Des analyses de variance permettent ensuite de montrer la signification de l'effet de la direction des vents sur les concentrations de sulfates et de nitrates, en plus de mettre en évidence un effet saisonnier significatif pour ces deux variables. Les concentrations de nitrates et de sulfates associées aux vents de l'ouest sont respec- tivement de 0,33 mg/l-¹ et 1,73 mg/l-¹ comparativement à 0,24 mg/l-¹ et 1,48 mg/l-¹ pour les vents provenant de l'est. En ce qui concerne l'effet saisonnier, les concentrations moyennes de nitrates sont plus élevées durant les mois de janvier et de mars alors que les concentrations de sulfates sont plus elevées durant les mois d'été.The problem of acidic precipitation has become an important environmental concern; related damages can affect atmospheric, terrestrial and aquatic components of our ecosystem. This paper focuses on wind-driven atmospheric transport of contaminants, notably niirates and sulfates. Conventional wisdom in eastern Canada holds that winds originating from the east bring precipitation less loaded with acidic components than winds originating from the southwest; the latter carry contarninants generated by industries located in the American Midwest.An experimental confirmation of this phenomenon is presented here. SPERBER (1987) showed that hourly series of piecipitation content and wind direction, measured at a reception site are adequate to represent the lagrangian history of precipitating systems (New York City region). Following this result, we suppose that the northesastern continental atmospheric system is homogeneous enough so that winds measured at our reception site (Québec City region) are representative of the whole system. Thus, our experiment is performed in eulerian coordinates.The data bank used in the statistic alana|yses contains 10 time series: the weekly concentrations of 9 compounds found in the precipitation (H, Ca, Cl, K, Mg, Na, NO3, NH4 and SO4) gathered at the local Montmorency site and a s:ries ofweekly prevailing winds measured at the nearby Québec City airport. The time series contain 312 observations covering a full six year period (December 1981 to December 1987). As the original data bank of corcentrations is episodic, i.e. an obsercvation is available for each day with a significant precipitation event, volumes and loadings are used to derive the average weekly values of concentrations. In contrast, hourly series of direction (projected in 36 directions) and velocity of the prevailing winds are used to build, via a vectorial addition, a weekly series of wind direitions projected on a 12 point wind rose where directions corespond to the nind origin and not its destination.Classic statistical methods are used to deal with this data bank. Principal component analysis studies relationships between series of concentrations in the precipitations, while correspondence analysis highrights the relationships betwlen tile series of precipitation content and the series ofwind direction. The final statistical method, analysis of variance, is used to test the signilïcance of relationships higtrlighted by the correspondence analysis.The principal component analysis shows that all variables were positively correlated with the first component which reflects the fact that a higtrly loaded precipitation event will show high concentrations for each ofthe nine variables. The second component discriminates two groups of variables: one includes NO3, NH4 and SO4, the other Ca, Mg and Cl. The acidity variable, H, is nearer to the acid ion group (NO3, NH4 and SO4) than to the other ion group (Ca, Mg and Cl).The correspondence analysis shows that high concentrations of acidic compounds (NO3, NH4 and SO4) are highly rerated to winds from the W and WSW directions; conversely lower concentrations of the same compounds are associated with winds from the E and ENE directions. The elements Ca, Mg and Cl, reputed to be of oceanic origin, show high functional relationships between high concentrations and E, ENE winds and also between low concentrations and W, WSW winds. These results support the popular belief initially presented that acid precipitation is largely associated with winds from the southwest.The analyses of variance show that precipitation concentrations (NO3 and SO4) are significantly different according to the wind directions. Nitrate and sulfate concentrations associated with winds originating from the west are respectively 0.33-mg.l-l and 1.73 mg.l-l compared, to 0.24 mg.l-l and 1.48 mg.l-l for winds originating from the east. The analyses ofvariance also indicate a significant seasonal effect where mean monthly concentrations in nitrates are hilhest for winter months and early spring whereas sulfate concentrations are highest for the summer months

Metabolomics methods for the synthetic biology of secondary metabolism

Many microbial secondary metabolites are of high biotechnological value for medicine, agriculture, and the food industry. Bacterial genome mining has revealed numerous novel secondary metabolite biosynthetic gene clusters, which encode the potential to synthesize a large diversity of compounds that have never been observed before. The stimulation or “awakening” of this cryptic microbial secondary metabolism has naturally attracted the attention of synthetic microbiologists, who exploit recent advances in DNA sequencing and synthesis to achieve unprecedented control over metabolic pathways. One of the indispensable tools in the synthetic biology toolbox is metabolomics, the global quantification of small biomolecules. This review illustrates the pivotal role of metabolomics for the synthetic microbiology of secondary metabolism, including its crucial role in novel compound discovery in microbes, the examination of side products of engineered metabolic pathways, as well as the identification of major bottlenecks for the overproduction of compounds of interest, especially in combination with metabolic modeling. We conclude by highlighting remaining challenges and recent technological advances that will drive metabolomics towards fulfilling its potential as a cornerstone technology of synthetic microbiology

Opposite Root Growth Phenotypes of hy5 versus hy5 hyh Mutants Correlate with Increased Constitutive Auxin Signaling

The Arabidopsis transcription factor HY5 controls light-induced gene expression downstream of photoreceptors and plays an important role in the switch of seedling shoots from dark-adapted to light-adapted development. In addition, HY5 has been implicated in plant hormone signaling, accounting for the accelerated root system growth phenotype of hy5 mutants. Mutants in the close HY5 homolog HYH resemble wild-type, despite the largely similar expression patterns and levels of HY5 and HYH, and the functional equivalence of the respective proteins. Moreover, the relative contribution of HYH to the overall activity of the gene pair is increased by an alternative HYH transcript, which encodes a stabilized protein. Consistent with the enhanced root system growth observed in hy5 loss-of-function mutants, constitutively overexpressed alternative HYH inhibits root system growth. Paradoxically, however, in double mutants carrying hy5 and hyh null alleles, the hy5 root growth phenotype is suppressed rather than enhanced. Even more surprisingly, compared to wild-type, root system growth is diminished in hy5 hyh double mutants. In addition, the double mutants display novel shoot phenotypes that are absent from either single mutant. These include cotyledon fusions and defective vasculature, which are typical for mutants in genes involved in the transcriptional response to the plant hormone auxin. Indeed, many auxin-responsive and auxin signaling genes are misexpressed in hy5 mutants, and at a higher number and magnitude in hy5 hyh mutants. Therefore, auxin-induced transcription is constitutively activated at different levels in the two mutant backgrounds. Our data support the hypothesis that the opposite root system phenotypes of hy5 single and hy5 hyh double mutants represent the morphological response to a quantitative gradient in the same molecular process, that is gradually increased constitutive auxin signaling. The data also suggest that HY5 and HYH are important negative regulators of auxin signaling amplitude in embryogenesis and seedling development

Multi-time-scale hydroclimate dynamics of a regional watershed and links to large-scale atmospheric circulation:Application to the Seine river catchment, France

(IF 3.73; Q1)International audienceIn the present context of global changes, considerable efforts have been deployed by the hydrological scientific community to improve our understanding of the impacts of climate fluctuations on water resources. Both observational and modeling studies have been extensively employed to characterize hydrological changes and trends, assess the impact of climate variability or provide future scenarios of water resources. In the aim of a better understanding of hydrological changes, it is of crucial importance to determine how and to what extent trends and long-term oscillations detectable in hydrological variables are linked to global climate oscillations.In this work, we develop an approach associating correlation between large and local scales, empirical statistical downscaling and wavelet multiresolution decomposition of monthly precipitation and streamflow over the Seine river watershed, and the North Atlantic sea level pressure (SLP) in order to gain additional insights on the atmospheric patterns associated with the regional hydrology. We hypothesized that: (i) atmospheric patterns may change according to the different temporal wavelengths defining the variability of the signals; and (ii) definition of those hydrological/circulation relationships for each temporal wavelength may improve the determination of large-scale predictors of local variations.The results showed that the links between large and local scales were not necessarily constant according to time-scale (i.e. for the different frequencies characterizing the signals), resulting in changing spatial patterns across scales. This was then taken into account by developing an empirical statistical downscaling (ESD) modeling approach, which integrated discrete wavelet multiresolution analysis for reconstructing monthly regional hydrometeorological processes (predictand: precipitation and streamflow on the Seine river catchment) based on a large-scale predictor (SLP over the Euro-Atlantic sector). This approach basically consisted in three steps: 1 – decomposing large-scale climate and hydrological signals (SLP field, precipitation or streamflow) using discrete wavelet multiresolution analysis, 2 – generating a statistical downscaling model per time-scale, 3 – summing up all scale-dependent models in order to obtain a final reconstruction of the predictand. The results obtained revealed a significant improvement of the reconstructions for both precipitation and streamflow when using the multiresolution ESD model instead of basic ESD. In particular, the multiresolution ESD model handled very well the significant changes in variance through time observed in either precipitation or streamflow. For instance, the post-1980 period, which had been characterized by particularly high amplitudes in interannual-to-interdecadal variability associated with alternating flood and extremely low-flow/drought periods (e.g., winter/spring 2001, summer 2003), could not be reconstructed without integrating wavelet multiresolution analysis into the model. In accordance with previous studies, the wavelet components detected in SLP, precipitation and streamflow on interannual to interdecadal time-scales could be interpreted in terms of influence of the Gulf-Stream oceanic front on atmospheric circulation

Influence of ground surface characteristics on the mean radiant temperature in urban areas

The effect of variations in land cover on mean radiant surface temperature (Tmrt) is explored through a simple scheme developed within the radiation model SOLWEIG. Outgoing longwave radiation is parameterised using surface temperature observations on a grass and an asphalt surface, whereas outgoing shortwave radiation is modelled through variations in albedo for the different surfaces. The influence of surface materials on Tmrt is small compared to the effects of shadowing. Nevertheless, altering ground surface materials could contribute to a reduction on Tmrt to reduce the radiant load during heat-wave episodes in locations where shadowing is not an option. Evaluation of the new scheme suggests that despite its simplicity it can simulate the outgoing fluxes well, especially during sunny conditions. However, it underestimates at night and in shadowed locations. One grass surface used to develop the parameterisation, with very different characteristics compared to an evaluation grass site, caused Tmrt to be underestimated. The implications of using high resolution (e.g. 15 minutes) temporal forcing data under partly cloudy conditions are demonstrated even for fairly proximal sites

Misregulation of the LOB domain gene DDA1 suggests possible functions in auxin signalling and photomorphogenesis

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene family encodes plant-specific transcription factors. In this report, the LBD gene DOWN IN DARK AND AUXIN1 (DDA1), which is closely related to LATERAL ORGAN BOUNDARIES (LOB) and ASYMMETRIC LEAVES2 (AS2), was characterized. DDA1 is expressed primarily in vascular tissues and its transcript levels were reduced by exposure to exogenous indole-3-acetic acid (IAA or auxin) and in response to dark exposure. Analysis of a T-DNA insertion line, dda1-1, in which the insertion resulted in misregulation of DDA1 transcripts in the presence of IAA and in the dark revealed possible functions in auxin response and photomorphogenesis. dda1-1 plants exhibited reduced sensitivity to auxin, produced fewer lateral roots, and displayed aberrant hypocotyl elongation in the dark. Phenotypes resulting from fusion of a transcriptional repression domain to DDA1 suggest that DDA1 may act as both a transcriptional activator and a transcriptional repressor depending on the context. These results indicate that DDA1 may function in both the auxin signalling and photomorphogenesis pathways

Light Plays an Essential Role in Intracellular Distribution of Auxin Efflux Carrier PIN2 in Arabidopsis thaliana

BACKGROUND: Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed. METHODOLOGY AND PRINCIPLE FINDINGS: Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process. CONCLUSIONS AND SIGNIFICANCE: Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment

Functional characterization of a gain-of-function mutant of AtMKK9 in Arabidopsis thaliana

The relatively small number of MAPKKs encoded in the Arabidopsis genome suggests that this particular class of kinases acts as a point of convergence within the plant's 'integration of external stimuli and their transduction to elicit biological responses. In an effort to gain information about the function of the MAPKK, AtMKK9 , in Arabidopsis, I have characterized several aspects of the phenotype of DEX:CA-MKK9-FLAG transgenic plants, which express an inducible constitutively active version of AtMKK9, CA-MKK9. I have found that CA-MKK9 expression can control the production of ethylene by activating a downstream MAPK, AtMPK6, which is known to promote the stabilization of ethylene biosynthesis enzymes. CA-MKK9 induction was correlated with an increase in AtMPK6 activity in planta, and was rapidly followed by production of a burst of ethylene in the induced plant tissues. I hypothesized that CA-MKK9 directly activates AtMPK6, and demonstrated that a recombinant version of CA-MKK9 was capable of phosphorylating AtMPK6 in vitro. In addition, the production of the hormone was abolished when C A - MKK9was expressed in a mpk6 knock-out background, thus proving that AtMPK6 is required for CA-MKK9 -mediated ethylene biosynthesis. I have also confirmed preliminary data from our laboratory suggesting that CA-MKK9 plays a role in oxidative programmed cell death. The necrotic lesions induced by CA-MKK9 were still observed in the mpk6 background, indicating that programmed cell death was triggered by CA-MKK9 activity independently of AtMPK6 activity and of ethylene overproduction. In addition, in order to investigate short-term transcriptional events triggered by CA-MKK9, I attempted to capture the transcriptional profile of DEX:CA-MKK9-FLAG plants using two-channel oligonucleotide microarrays. The CA-MKK9 - affected genes included a number of genes involved in the octadecanoid pathway, and their promoters were enriched in ABRE-like elements. However, my attempts to validate the microarray results using additional biological replicates and quantitative real-time PCR revealed that the majority of these early-response microarray results were apparently false positives, indicating that the microarray experiment was probably inappropriately constructed for capturing early transcriptional responses to CA-MKK9 using the dexamethasone-inducible system.Science, Faculty ofBotany, Department ofGraduat