Further developments towards a genome-scale metabolic model of yeast

BACKGROUND: To date, several genome-scale network reconstructions have been used to describe the metabolism of the yeast Saccharomyces cerevisiae, each differing in scope and content. The recent community-driven reconstruction, while rigorously evidenced and well annotated, under-represented metabolite transport, lipid metabolism and other pathways, and was not amenable to constraint-based analyses because of lack of pathway connectivity. RESULTS: We have expanded the yeast network reconstruction to incorporate many new reactions from the literature and represented these in a well-annotated and standards-compliant manner. The new reconstruction comprises 1102 unique metabolic reactions involving 924 unique metabolites - significantly larger in scope than any previous reconstruction. The representation of lipid metabolism in particular has improved, with 234 out of 268 enzymes linked to lipid metabolism now present in at least one reaction. Connectivity is emphatically improved, with more than 90% of metabolites now reachable from the growth medium constituents. The present updates allow constraint-based analyses to be performed; viability predictions of single knockouts are comparable to results from in vivo experiments and to those of previous reconstructions. CONCLUSIONS: We report the development of the most complete reconstruction of yeast metabolism to date that is based upon reliable literature evidence and richly annotated according to MIRIAM standards. The reconstruction is available in the Systems Biology Markup Language (SBML) and via a publicly accessible database http://www.comp-sys-bio.org/yeastnet/

Scaled deployment of Wolbachia to protect the community from dengue and other Aedes transmitted arboviruses.

Background: A number of new technologies are under development for the control of mosquito transmitted viruses, such as dengue, chikungunya and Zika that all require the release of modified mosquitoes into the environment. None of these technologies has been able to demonstrate evidence that they can be implemented at a scale beyond small pilots. Here we report the first successful citywide scaled deployment of Wolbachia in the northern Australian city of Townsville. Methods: The wMel strain of Wolbachia was backcrossed into a local Aedes aegypti genotype and mass reared mosquitoes were deployed as eggs using mosquito release containers (MRCs). In initial stages these releases were undertaken by program staff but in later stages this was replaced by direct community release including the development of a school program that saw children undertake releases. Mosquito monitoring was undertaken with Biogents Sentinel (BGS) traps and individual mosquitoes were screened for the presence of Wolbachia with a Taqman qPCR or LAMP diagnostic assay. Dengue case notifications from Queensland Health Communicable Disease Branch were used to track dengue cases in the city before and after release. Results: Wolbachia was successfully established into local Ae. aegypti mosquitoes across 66 km 2 in four stages over 28 months with full community support.  A feature of the program was the development of a scaled approach to community engagement. Wolbachia frequencies have remained stable since deployment and to date no local dengue transmission has been confirmed in any area of Townsville after Wolbachia has established, despite local transmission events every year for the prior 13 years and an epidemiological context of increasing imported cases. Conclusion: Deployment of Wolbachia into Ae. aegypti populations can be readily scaled to areas of ~60km 2 quickly and cost effectively and appears in this context to be effective at stopping local dengue transmission

Toward community standards and software for whole-cell modeling

Whole-cell (WC) modeling is a promising tool for biological research, bioengineering, and medicine. However, substantial work remains to create accurate, comprehensive models of complex cells. Methods: We organized the 2015 Whole-Cell Modeling Summer School to teach WC modeling and evaluate the need for new WC modeling standards and software by recoding a recently published WC model in SBML. Results: Our analysis revealed several challenges to representing WC models using the current standards. Conclusion: We, therefore, propose several new WC modeling standards, software, and databases. Significance:We anticipate that these new standards and software will enable more comprehensive models

Formulation, stabilisation and encapsulation of bacteriophage for phage therapy

Against a backdrop of global antibiotic resistance and increasing awareness of the importance of the human microbiota, there has been resurgent interest in the potential use of bacteriophages for therapeutic purposes, known as phage therapy. A number of phage therapy phase I and II clinical trials have concluded, and shown phages don’t present significant adverse safety concerns. These clinical trials used simple phage suspensions without any formulation and phage stability was of secondary concern. Phages have a limited stability in solution, and undergo a significant drop in phage titre during processing and storage which is unacceptable if phages are to become regulated pharmaceuticals, where stable dosage and well defined pharmacokinetics and pharmacodynamics are de rigueur. Animal studies have shown that the efficacy of phage therapy outcomes depend on the phage concentration (i.e. the dose) delivered at the site of infection, and their ability to target and kill bacteria, arresting bacterial growth and clearing the infection. In addition, in vitro and animal studies have shown the importance of using phage cocktails rather than single phage preparations to achieve better therapy outcomes. The in vivo reduction of phage concentration due to interactions with host antibodies or other clearance mechanisms may necessitate repeated dosing of phages, or sustained release approaches. Modelling of phage-bacterium population dynamics reinforces these points. Surprisingly little attention has been devoted to the effect of formulation on phage therapy outcomes, given the need for phage cocktails, where each phage within a cocktail may require significantly different formulation to retain a high enough infective dose. This review firstly looks at the clinical needs and challenges (informed through a review of key animal studies evaluating phage therapy) associated with treatment of acute and chronic infections and the drivers for phage encapsulation. An important driver for formulation and encapsulation is shelf life and storage of phage to ensure reproducible dosages. Other drivers include formulation of phage for encapsulation in micro- and nanoparticles for effective delivery, encapsulation in stimuli responsive systems for triggered controlled or sustained release at the targeted site of infection. Encapsulation of phage (e.g. in liposomes) may also be used to increase the circulation time of phage for treating systemic infections, for prophylactic treatment or to treat intracellular infections. We then proceed to document approaches used in the published literature on the formulation and stabilisation of phage for storage and encapsulation of bacteriophage in micro- and nanostructured materials using freeze drying (lyophilization), spray drying, in emulsions e.g. ointments, polymeric microparticles, nanoparticles and liposomes. As phage therapy moves forward towards Phase III clinical trials, the review concludes by looking at promising new approaches for micro- and nanoencapsulation of phages and how these may address gaps in the field

Kinetic modelling of metabolic pathways: Application to serine biosynthesis

In this chapter, we describe the steps needed to create a kinetic model of a metabolic pathway using kinetic data from both experimental measurements and literature review. Our methodology is presented by using the example of serine biosynthesis in E. coli

Kinetic modelling of metabolic pathways

We show how to build a kinetic model of a metabolic pathway. We provide the example of building a model of glycerol synthesis using Copasi. However, the techniques required remain the same for any pathway, using any software

Letting the flux define the kinetics: using a single steady state to predict network behaviour under diverse stress conditions

Motivation The understanding of metabolic interactions has grown rapidly in recent years with metabolic network reconstructions constantly increasing in scope and quality. These networks can be manipulated into models which allow for fluxes to be estimated using methods such as flux balance analysis. However, the solution space of fluxes is large, and the inability to provide dynamic behaviour of the system can make understanding it under changing conditions difficult. It is under these circumstances where kinetic models can prove more valuable. However, collecting kinetic information for large scale networks is time consuming and expensive, and existing data is difficult to find and collate. Results We develop and validate a novel methodology which uses the flux information of one steady state to define the kinetics of the system and produce a reasonable approximation of the steady state behaviour of the real system. We show that first approximation models, in the first instance, show a poor ability to extrapolate beyond the initial conditions. However, metabolic control analysis directs our attention towards the most important reactions of the network. When experimentally-elucidated kinetics are used for those reactions, and the remainder of the network modelled using empirical rate laws that are inspired in enzyme kinetics, predictability improves dramatically

RobOKoD: microbial strain design for (over)production of target compounds

Sustainable production of target compounds such as biofuels and high-value chemicals for pharmaceutical, agrochemical, and chemical industries is becoming an increasing priority given their current dependency upon diminishing petrochemical resources. Designing these strains is difficult, with current methods focusing primarily on knocking-out genes, dismissing other vital steps of strain design including the overexpression and dampening of genes. The design predictions from current methods also do not translate well-into successful strains in the laboratory. Here, we introduce RobOKoD (Robust, Overexpression, Knockout and Dampening), a method for predicting strain designs for overproduction of targets. The method uses flux variability analysis to profile each reaction within the system under differing production percentages of target-compound and biomass. Using these profiles, reactions are identified as potential knockout, overexpression, or dampening targets. The identified reactions are ranked according to their suitability, providing flexibility in strain design for users. The software was tested by designing a butanol-producing Escherichia coli strain, and was compared against the popular OptKnock and RobustKnock methods. RobOKoD shows favorable design predictions, when predictions from these methods are compared to a successful butanol-producing experimentally-validated strain. Overall RobOKoD provides users with rankings of predicted beneficial genetic interventions with which to support optimized strain design