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Redirector: designing cell factories by reconstructing the metabolic objective.

ABSTRACT: Advances in computational metabolic optimization are required to realize the full potential of new in vivo metabolic engineering technologies by bridging the gap between computational design and strain development. We present Redirector, a new Flux Balance Analysis-based framework for identifying engineering targets to optimize metabolite production in complex pathways. Previous optimization frameworks have modeled metabolic alterations as directly controlling fluxes by setting particular flux bounds. Redirector develops a more biologically relevant approach, modeling metabolic alterations as changes in the balance of metabolic objectives in the system. This framework iteratively selects enzyme targets, adds the associated reaction fluxes to the metabolic objective, thereby incentivizing flux towards the production of a metabolite of interest. These adjustments to the objective act in competition with cellular growth and represent up-regulation and down-regulation of enzyme mediated reactions. Using the iAF1260 E. coli metabolic network model for optimization of fatty acid production as a test case, Redirector generates designs with as many as 39 simultaneous and 111 unique engineering targets. These designs discover proven in vivo targets, novel supporting pathways and relevant interdependencies, many of which cannot be predicted by other methods. Redirector is available as open and free software, scalable to computational resources, and powerful enough to find all known enzyme targets for fatty acid production.

SUBMITTER: Rockwell G

PROVIDER: S-EPMC3547792 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.

Project description:BackgroundWhen a new drug is launched onto the market, information about the new manufactured product is contained in its monograph and evaluation report published by national drug agencies. Health professionals need to be able to determine rapidly and easily whether the new manufactured product is potentially useful for their practice. There is therefore a need to identify the best way to group together and visualize the main items of information describing the nature and potential impact of the new drug. The objective of this study was to identify these items of information and to bring them together in a model that could serve as the standard for presenting the main features of new manufactured product.MethodsWe developed a preliminary conceptual model of pharmaceutical innovations, based on the knowledge of the authors. We then refined this model, using a random sample of 40 new manufactured drugs recently approved by the national drug regulatory authorities in France and covering a broad spectrum of innovations and therapeutic areas. Finally, we used another sample of 20 new manufactured drugs to determine whether the model was sufficiently comprehensive.ResultsThe results of our modeling led to three sub models described as conceptual maps representingi) the medical context for use of the new drug (indications, type of effect, therapeutical arsenal for the same indications), ii) the nature of the novelty of the new drug (new molecule, new mechanism of action, new combination, new dosage, etc.), and iii) the impact of the drug in terms of efficacy, safety and ease of use, compared with other drugs with the same indications.ConclusionsOur model can help to standardize information about new drugs released onto the market. It is potentially useful to the pharmaceutical industry, medical journals, editors of drug databases and medical software, and national or international drug regulation agencies, as a means of describing the main properties of new pharmaceutical products. It could also used as a guide for the writing of comprehensive and objective texts summarizing the nature and interest of new manufactured product.

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Redirector: designing cell factories by reconstructing the metabolic objective.

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