Model-guided metabolic engineering of Pseudomonas taiwanensis VLB120 for the production of methyl ketones

Aliphatic methyl ketones are discussed as promising novel diesel blendstocks because of their favorable cetane numbers. To achieve sustainable production of these compounds, bio-based production in engineered microbes is followed and successful synthesis in Escherichia coli1,2,3 and Pseudomonas putida4 has recently been shown. In this presentation, we report on the metabolic engineering of Pseudomonas taiwanensis VLB1205 for the production of saturated and monounsaturated medium chain methyl ketones (C11, C13, C15, C17). Major arguments for the use of this microbe are its metabolic versatility, high tolerance of organic solvents5 and ease of cultivation. P. taiwanensis VLB120 can grow on various carbon sources besides glucose such as glycerol, an important by-product of biodiesel production, as well as on major components of biomass hydrolysate such as xylose, organic acids and aromatic compounds, e.g., 4-hydroxybenzoate4. Further, its superior redox cofactor regeneration capability6 might benefit the synthesis of the reduced, aliphatic target compounds. The transformation of P. taiwanensis VLB120 into a microbial cell factory for methyl ketone production was achieved by: (i) overproduction of the fatty acyl-CoA synthetase FadB to increase acyl-CoA availability, (ii) oxidation of acyl-CoA to a trans-2-enoyl-CoA by a heterologously expressed acyl-CoA oxidase from Micrococcus luteus, (iii) conversion of this intermediate to β-hydroxyacyl-CoA and further oxidation to a β -ketoacyl-CoA by overexpression of the native fadB gene, (iv) increased thioesterase activity by overexpression of fadM to form free β -keto acids, which spontaneously decarboxylate to methyl ketones. The 1st generation production strain yielded 550 mg L-1aq methyl ketones in a batch fermentation with in situ product extraction into a second organic layer of n-decane. Further strain optimization was guided by metabolic modeling, which suggested an additional deletion of the acyl-CoA thioesterase II (tesB). TesB hydrolyzes acyl-CoA to free fatty acids, hence, reverses the desired FadD reaction. In a simple batch fermentation, the proposed gene deletion resulted in a 2.5-fold increased product titer of 1.4 g L-1aq while 9.4 g L-1aq were reached in fed-batch cultivations. Additional, successful strategies tested in parallel were the deletion of the pha operon, responsible for polyhydroxyalkanoate synthesis and deletion of a fadA homologue in the 1st generation production strain, with the later resulting in an even 4-fold improvement of the product titer. While the production of 9.4 g L-1aq is already the highest reported titer of recombinantly produced methyl ketones so far, consolidation of all successfully tested engineering strategies holds great promise to significantly boost methyl ketone production in P. taiwanensis VLB120 to even higher titers. Overall, the results of this study underline the high potential of P. taiwanensis VLB120 for the production of methyl ketones and highlight model-guided metabolic engineering as a means to rationalize and accelerate strain optimization efforts. 1Dong et al. 2018: doi:10.1101/496497 2Goh et al. 2012: doi: 10.1128/AEM.06785-11 3Goh et al. 2014: doi: 10.1016/j.ymben.2014.09.003. 4Goh et al. 2018: doi: 10.1002/bit.26558. 5Rühl et al. 2009: doi: 10.1128/AEM.00225-09 6Blank et al. 2008: doi: 10.1111/j.1742-4658.2008.06648.x

The impact of emerging sustainable technologies on existing electrical infrastructure in Ontario

Sustainable energy sources are urgently required, as traditional non-renewable energy sources are increasing in scarcity and subsequently in cost. Significant innovation and investment is required to incorporate newly developed sustainable energy technologies into the existing energy infrastructure network. This presentation will review how emerging sustainable technologies are interacting with existing energy infrastructure. Specifically it will review the existing electrical grid in Ontario, Canada, and the impact of sustainable technologies such as electric cars and distributed generation. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3107

Immunization coverage and risk factors for failure to immunize within the Expanded Programme on Immunization in Kenya after introduction of new Haemophilus influenzae type b and hepatitis b virus antigens

Background: Kenya introduced a pentavalent vaccine including the DTP, Haemophilus influenzae type b and hepatitis b virus antigens in Nov 2001 and strengthened immunization services. We estimated immunization coverage before and after introduction, timeliness of vaccination and risk factors for failure to immunize in Kilifi district, Kenya. Methods: In Nov 2002 we performed WHO cluster-sample surveys of > 200 children scheduled for vaccination before or after introduction of pentavalent vaccine. In Mar 2004 we conducted a simple random sample (SRS) survey of 204 children aged 9 - 23 months. Coverage was estimated by inverse Kaplan-Meier survival analysis of vaccine- card and mothers' recall data and corroborated by reviewing administrative records from national and provincial vaccine stores. The contribution to timely immunization of distance from clinic, seasonal rainfall, mother's age, and family size was estimated by a proportional hazards model. Results: Immunization coverage for three DTP and pentavalent doses was 100% before and 91% after pentavalent vaccine introduction, respectively. By SRS survey, coverage was 88% for three pentavalent doses. The median age at first, second and third vaccine dose was 8, 13 and 18 weeks. Vials dispatched to Kilifi District during 2001 - 2003 would provide three immunizations for 92% of the birth cohort. Immunization rate ratios were reduced with every kilometre of distance from home to vaccine clinic (HR 0.95, CI 0.91 - 1.00), rainy seasons ( HR 0.73, 95% CI 0.61 - 0.89) and family size, increasing progressively up to 4 children ( HR 0.55, 95% CI 0.41 - 0.73). Conclusion: Vaccine coverage was high before and after introduction of pentavalent vaccine, but most doses were given late. Coverage is limited by seasonal factors and family siz

\u3cem\u3eHST\u3c/em\u3e Imaging of the Dusty Filaments and Nucleus Swirl in NGC4696 at the Centre of the Centaurus Cluster

Narrow-band HST imaging has resolved the detailed internal structure of the 10 kpc diameter H α+[N II] emission line nebulosity in NGC4696, the central galaxy in the nearby Centaurus cluster, showing that the dusty, molecular, filaments have a width of about 60 pc. Optical morphology and velocity measurements indicate that the filaments are dragged out by the bubbling action of the radio source as part of the active galactic nucleus feedback cycle. Using the drag force we find that the magnetic field in the filaments is in approximate pressure equipartition with the hot gas. The filamentary nature of the cold gas continues inwards, swirling around and within the Bondi accretion radius of the central black hole, revealing the magnetic nature of the gas flows in massive elliptical galaxies. HST imaging resolves the magnetic, dusty, molecular filaments at the centre of the Centaurus cluster to a swirl around and within the Bondi radius

Rabies Diagnosis for Developing Countries

A new diagnostic test for rabies in animals was evaluated in N'Djaména, capital of Chad. The test is based on a direct immuno-histochemical detection of rabies virus in brain tissue (dRIT) visible by normal light microscopy. Rabies detection by dRIT light microscopy is 10 times less expensive than fluorescence microscopy required for the current gold standard of rabies diagnosis. The test showed ideal results in fresh samples with 100% agreement with the gold standard and confirms the results of a first study in Tanzania. Thus, it has a significant potential for diagnosing rabies in low-income countries, and under field conditions where rabies diagnosis is unavailable for the moment. This new test opens up a great potential to train technical staff and to establish rabies diagnosis without delay in low-income countries with urban rabies

The importance of myo-inositol and D-chiro-inositol to support fertility and reproduction

This review details the physiologic roles of two insulin sensitizers, myo-inositol (MI) and D-chiro-inositol (DCI). In the human ovary, MI is a second messenger of follicle stimulating hormone (FSH) and DCI is an aromatase inhibitor. These activities allow a treatment for polycystic ovary syndrome (PCOS) to be defined based on the combined administration of MI and DCI, where the best MI:DCI ratio is 40:1. In addition, MI plays a pivotal role in the physiology of reproduction, and has beneficial effects on the development of oocytes, spermatozoa, and embryos. By contrast, DCI has little effect on spermatozoa, but high concentrations in the ovary can negatively affect the quality of oocytes and the blastocyst. Overall, the evidence in the literature supports the beneficial effects of MI in both female and male reproduction, warranting clinical use of MI in assisted reproductive treatment (ART).Cette revue détaille les rôles physiologiques de deux sensibilisateurs à l'insuline, le myo-inositol (MI) et le D-chiro-inositol (DCI). Dans l'ovaire humain, le MI est un second messager de l'hormone folliculostimulante (FSH) et le DCI est un inhibiteur de l'aromatase. Ces activités permettent de définir un traitement du syndrome des ovaires polykystiques (SOPK) basé sur l'administration combinée de MI et de DCI, où le meilleur rapport MI:DCI est de 40:1. En outre, le MI joue un rôle essentiel dans la physiologie de la reproduction et a des effets bénéfiques sur le développement des ovocytes, des spermatozoïdes et des embryons. En revanche, le DCI a peu d'effet sur les spermatozoïdes, mais des concentrations élevées dans l'ovaire peuvent avoir un effet négatif sur la qualité des ovocytes et du blastocyste. Dans l'ensemble, les données de la littérature confirment les effets bénéfiques du MI dans la reproduction féminine et masculine, ce qui justifie l'utilisation clinique du MI dans l'assistance médicale à la procréation

The coincidence of ecological opportunity with hybridization explains rapid adaptive radiation in Lake Mweru cichlid fishes

Abstract: The process of adaptive radiation was classically hypothesized to require isolation of a lineage from its source (no gene flow) and from related species (no competition). Alternatively, hybridization between species may generate genetic variation that facilitates adaptive radiation. Here we study haplochromine cichlid assemblages in two African Great Lakes to test these hypotheses. Greater biotic isolation (fewer lineages) predicts fewer constraints by competition and hence more ecological opportunity in Lake Bangweulu, whereas opportunity for hybridization predicts increased genetic potential in Lake Mweru. In Lake Bangweulu, we find no evidence for hybridization but also no adaptive radiation. We show that the Bangweulu lineages also colonized Lake Mweru, where they hybridized with Congolese lineages and then underwent multiple adaptive radiations that are strikingly complementary in ecology and morphology. Our data suggest that the presence of several related lineages does not necessarily prevent adaptive radiation, although it constrains the trajectories of morphological diversification. It might instead facilitate adaptive radiation when hybridization generates genetic variation, without which radiation may start much later, progress more slowly or never occur