Cofactor engineering for enhancing the flux of metabolic pathways.

The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering

Microbial production of 1-octanol: a naturally excreted biofuel with diesel-like properties

The development of sustainable, bio-based technologies to convert solar energy and carbon dioxide into fuels is a grand challenge. A core part of this challenge is to produce a fuel that is compatible with the existing transportation infrastructure. This task is further compounded by the commercial desire to separate the fuel from the biotechnological host. Based on its fuel characteristics, 1-octanol was identified as an attractive metabolic target with diesel-like properties. We therefore engineered a synthetic pathway specifically for the biosynthesis of 1-octanol in Escherichia coli BL21(DE3) by over-expression of three enzymes (thioesterase, carboxylic acid reductase and aldehyde reductase) and one maturation factor (phosphopantetheinyl transferase). Induction of this pathway in a shake flask resulted in 4.4 mg 1-octanol L(-1) h(-1) which exceeded the productivity of previously engineered strains. Furthermore, the majority (73%) of the fatty alcohol was localised within the media without the addition of detergent or solvent overlay. The deletion of acrA reduced the production and excretion of 1-octanol by 3-fold relative to the wild-type, suggesting that the AcrAB-TolC complex may be responsible for the majority of product efflux. This study presents 1-octanol as a potential fuel target that can be synthesised and naturally accumulated within the media using engineered microbes

Modulating the import of medium-chain alkanes in E. coli through tuned expression of FadL

BACKGROUND: In recent years, there have been intensive efforts to develop synthetic microbial platforms for the production, biosensing and bio-remediation of fossil fuel constituents such as alkanes. Building predictable engineered systems for these applications will require the ability to tightly control and modulate the rate of import of alkanes into the host cell. The native components responsible for the import of alkanes within these systems have yet to be elucidated. To shed further insights on this, we used the AlkBGT alkane monooxygenase complex from Pseudomonas putida GPo1 as a reporter system for assessing alkane import in Escherichia coli. Two native E. coli transporters, FadL and OmpW, were evaluated for octane import given their proven functionality in the uptake of fatty acids along with their structural similarity to the P. putida GPo1 alkane importer, AlkL. RESULTS: Octane import was removed with deletion of fadL, but was restored by complementation with a fadL-encoding plasmid. Furthermore, tuned overexpression of FadL increased the rate of alkane import by up to 4.5- fold. A FadL deletion strain displayed a small but significant degree of tolerance toward hexane and octane relative to the wild type, while the responsiveness of the well-known alkane biosensor, AlkS, toward octane and decane was strongly reduced by 2.7- and 2.9-fold, respectively. CONCLUSIONS: We unequivocally show for the first time that FadL serves as the major route for medium-chain alkane import in E. coli. The experimental approaches used within this study, which include an enzyme-based reporter system and a fluorescent alkane biosensor for quantification and real-time monitoring of alkane import, could be employed as part of an engineering toolkit for optimizing biological systems that depend on the uptake of alkanes. Thus, the findings will be particularly useful for biological applications such as bioremediation and biomanufacturing

A microbial platform for renewable propane synthesis based on a fermentative butanol pathway

Background Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli. Results We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L). Conclusions This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde

Screening of various botanical extracts for antioxidant activity using DPPH free radical method

Aiming at the exploration of herbal use by society, crude extracts of the seeds of some commonly used medicinal plants (Vitis vinifera, Tamarindus indica and Glycin max) were screened for their free radical scavenging  properties using ascorbic acid as standard antioxidant. Free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. The overall antioxidant activity of grape seeds (Vitis vinifera) was the strongest, followed in descending order by soybean (Glycin max) and tamarind (Tamarindus indica). The seeds extract of Vitis vinifera, Glycin max and Tamarindus indica showed 85.61%, 83.45% and 79.26%, DPPH  scavenging activity respectively.Key words: Antioxidant activity, DPPH, free-radical, Vitis vinifera, Glycin max, Tamarindus indica

In vivo Evaluation of a Cosmetic Emulsion Containing Soybean Extract for Anti-Aging

Purpose: To develop and assess the anti-aging potential of a cosmetic W/O emulsion containing an extract of soybean, Glycine max (L.) Merr. Fabaceae.Methods: This single-blind placebo-controlled study was performed in 11 healthy male human volunteers. A formulation comprising of 4 % of concentrated extract of soybean was prepared by loading the extract in the internal aqueous phase of the emulsion. A control (base), consisting of theemulsion without the extract, was also prepared. Both formulations were applied to the cheeks of all volunteers for 12 weeks and their effect on different skin parameters, i.e., moisture contents, elasticity and surface evaluation of living skin (SELS) were assessed.Results: The formulation containing 4 % soybean extract showed significant (p ≤ 0.05) effects on skin elasticity and moisture contents but the base showed insignificant effect (p ≤ 0.05). There was significant (p . 0.05) decline in SELS, i.e., SEsc (skin scaliness, from 1.73 } 0.05 to 1.66 } 0.06), SEw (skin wrinkles, from 71.74 ± 1.52 to 68.51 ± 1.64), SEsm (skin smoothness, from 109.01 ± 4.77 to 102.03 ± 4.23), and SEr (skin roughness, from 4.04 ±0.09 to 3.82± 0.08) parameters after applicationof the extract formulation for 12 weeks.Conclusion: Topical application of the cosmetic emulsion containing soybean extract exerts potential skin anti-aging effects.Keywords: Glycine max, Soybean, Anti-aging, Skin elasticity, Cosmetic emulsion, Surface evaluation of living skin (SELS), Skin moisture conten

Predictors of children's secondhand smoke exposure at home: a systematic review and narrative synthesis of the evidence

BACKGROUND: Children's exposure to secondhand smoke (SHS) has been causally linked to a number of childhood morbidities and mortalities. Over 50% of UK children whose parents are smokers are regularly exposed to SHS at home. No previous review has identified the factors associated with children's SHS exposure in the home. AIM: To identify by systematic review, the factors which are associated with children's SHS exposure in the home, determined by parent or child reports and/or biochemically validated measures including cotinine, carbon monoxide or home air particulate matter. METHODS: Electronic searches of MEDLINE, EMBASE, PsychINFO, CINAHL and Web of Knowledge to July 2014, and hand searches of reference lists from publications included in the review were conducted. FINDINGS: Forty one studies were included in the review. Parental smoking, low socioeconomic status and being less educated were all frequently and consistently found to be independently associated with children's SHS exposure in the home. Children whose parents held more negative attitudes towards SHS were less likely to be exposed. Associations were strongest for parental cigarette smoking status; compared to children of non-smokers, those whose mothers or both parents smoked were between two and 13 times more likely to be exposed to SHS. CONCLUSION: Multiple factors are associated with child SHS exposure in the home; the best way to reduce child SHS exposure in the home is for smoking parents to quit. If parents are unable or unwilling to stop smoking, they should instigate smoke-free homes. Interventions targeted towards the socially disadvantaged parents aiming to change attitudes to smoking in the presence of children and providing practical support to help parents smoke outside the home may be beneficial

Swift trust and commitment: the missing links for humanitarian supply chain coordination?

Coordination among actors in a humanitarian relief supply chain decides whether a relief operation can be or successful or not. In humanitarian supply chains, due to the urgency and importance of the situation combined with scarce resources, actors have to coordinate and trust each other in order to achieve joint goals. This paper investigated empirically the role of swift trust as mediating variable for achieving supply chain coordination. Based on commitment-trust theory we explore enablers of swift-trust and how swift trust translates into coordination through commitment. Based on a path analytic model we test data from the National Disaster Management Authority of India. Our study is the first testing commitment-trust theory (CTT) in the humanitarian context, highlighting the importance of swift trust and commitment for much thought after coordination. Furthermore, the study shows that information sharing and behavioral uncertainty reduction act as enablers for swift trust. The study findings offer practical guidance and suggest that swift trust is a missing link for the success of humanitarian supply chains

The HAPPY (Healthy and Active Parenting Programmme for early Years) feasibility randomised control trial: acceptability and feasibility of an intervention to reduce infant obesity.

The prevalence of infant obesity is increasing, but there is a lack of evidence-based approaches to prevent obesity at this age. This study tested the acceptability and feasibility of evaluating a theory-based intervention aimed at reducing risk of obesity in infants of overweight/obese women during and after pregnancy: the Healthy and Active Parenting Programme for Early Years (HAPPY).A feasibility randomised controlled trial was conducted in Bradford, England. One hundred twenty overweight/obese pregnant women (Body Mass Index [BMI] ≥25 kg/m(2)) were recruited between 10-26 weeks gestation. Consenting women were randomly allocated to HAPPY (6 antenatal, 6 postnatal sessions: N = 59) or usual care (N = 61). Appropriate outcome measures for a full trial were explored, including: infant's length and weight, woman's BMI, physical activity and dietary intake of the women and infants. Health economic data were collected. Measurement occurred before randomisation and when the infant was aged 6 months and 12 months. Feasibility outcomes were: recruitment/attrition rates, and acceptability of: randomisation, measurement, and intervention. Intra-class correlations for infant weight were calculated. Fidelity was assessed through observations and facilitator feedback. Focus groups and semi-structured interviews explored acceptability of methods, implementation, and intervention content.Recruitment targets were met (~20 women/month) with a recruitment rate of 30 % of eligible women (120/396). There was 30 % attrition at 12 months; 66 % of recruited women failed to attend intervention sessions, but those who attended the first session were likely to continue to attend (mean 9.4/12 sessions, range 1-12). Reaction to intervention content was positive, and fidelity was high. Group clustering was minimal; an adjusted effect size of -0.25 standard deviation scores for infant weight at 12 months (95 % CI: -0.16-0.65) favouring the intervention was observed using intention to treat analyses. No adverse events were reported.The HAPPY intervention appeared feasible and acceptable to participants who attended and those delivering it, however attendance was low; adaptations to increase initial attendance are recommended. Whilst the study was not powered to detect a definitive effect, our results suggest a potential to reduce risk of infant obesity. The evidence reported provides valuable lessons to inform progression to a definitive trial.Current Controlled Trials ISRCTN56735429