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Identification of metabolic engineering targets through analysis of optimal and sub-optimal routes.

ABSTRACT: Identification of optimal genetic manipulation strategies for redirecting substrate uptake towards a desired product is a challenging task owing to the complexity of metabolic networks, esp. in terms of large number of routes leading to the desired product. Algorithms that can exploit the whole range of optimal and suboptimal routes for product formation while respecting the biological objective of the cell are therefore much needed. Towards addressing this need, we here introduce the notion of structural flux, which is derived from the enumeration of all pathways in the metabolic network in question and accounts for the contribution towards a given biological objective function. We show that the theoretically estimated structural fluxes are good predictors of experimentally measured intra-cellular fluxes in two model organisms, namely, Escherichia coli and Saccharomyces cerevisiae. For a small number of fluxes for which the predictions were poor, the corresponding enzyme-coding transcripts were also found to be distinctly regulated, showing the ability of structural fluxes in capturing the underlying regulatory principles. Exploiting the observed correspondence between in vivo fluxes and structural fluxes, we propose an in silico metabolic engineering approach, iStruF, which enables the identification of gene deletion strategies that couple the cellular biological objective with the product flux while considering optimal as well as sub-optimal routes and their efficiency.

SUBMITTER: Soons ZI

PROVIDER: S-EPMC3633962 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundComputational modeling and analysis of metabolic networks has been successful in metabolic engineering of microbial strains for valuable biochemical production. Limitations of currently available computational methods for metabolic engineering are that they are often based on reaction deletions rather than gene deletions and do not consider the regulatory networks that control metabolism. Due to the presence of multi-functional enzymes and isozymes, computational designs based on reaction deletions can sometimes result in strategies that are genetically complicated or infeasible. Additionally, strains might not be able to grow initially due to regulatory restrictions. To overcome these limitations, we have developed a new approach (OptORF) for identifying metabolic engineering strategies based on gene deletion and overexpression.ResultsHere we propose an effective method to systematically integrate transcriptional regulatory networks and metabolic networks. This allows for the formulation of linear optimization problems that search for metabolic and/or regulatory perturbations that couple biomass and biochemical production, thus proposing adaptive evolutionary strain designs. Using genome-scale models of Escherichia coli, we have implemented the OptORF algorithm (which considers gene deletions and transcriptional regulation) and compared its metabolic engineering strategies for ethanol production to those found using OptKnock (which considers reaction deletions). Our results found that the reaction-based strategies often require more gene deletions to remove the identified reactions (2 more genes than reactions), and result in lethal growth phenotypes when transcriptional regulation is considered (162 out of 200 cases). Finally, we present metabolic engineering strategies for producing ethanol and higher alcohols (e.g. isobutanol) in E. coli using our OptORF approach. We have found common genetic modifications such as deletion of pgi and overexpression of edd, as well as chemical specific strategies for producing different alcohols.ConclusionsBy taking regulatory effects into account, OptORF can propose changes such as the overexpression of metabolic genes or deletion of transcriptional factors, in addition to the deletion of metabolic genes, that may lead to faster evolutionary trajectories. While biofuel production in E. coli is evaluated here, the developed OptORF approach is general and can be applied to optimize the production of different compounds in other biological systems.

Project description:Genome-scale metabolic models are valuable tools for the design of novel strains of industrial microorganisms, such as Komagataella phaffii (syn. Pichia pastoris). However, as is the case for many industrial microbes, there is no executable metabolic model for K. phaffiii that confirms to current standards by providing the metabolite and reactions IDs, to facilitate model extension and reuse, and gene-reaction associations to enable identification of targets for genetic manipulation. In order to remedy this deficiency, we decided to reconstruct the genome-scale metabolic model of K. phaffii by reconciling the extant models and performing extensive manual curation in order to construct an executable model (Kp.1.0) that conforms to current standards. We then used this model to study the effect of biomass composition on the predictive success of the model. Twelve different biomass compositions obtained from published empirical data obtained under a range of growth conditions were employed in this investigation. We found that the success of Kp1.0 in predicting both gene essentiality and growth characteristics was relatively unaffected by biomass composition. However, we found that biomass composition had a profound effect on the distribution of the fluxes involved in lipid, DNA, and steroid biosynthetic processes, cellular alcohol metabolic process, and oxidation-reduction process. Furthermore, we investigated the effect of biomass composition on the identification of suitable target genes for strain development. The analyses revealed that around 40% of the predictions of the effect of gene overexpression or deletion changed depending on the representation of biomass composition in the model. Considering the robustness of the in silico flux distributions to the changing biomass representations enables better interpretation of experimental results, reduces the risk of wrong target identification, and so both speeds and improves the process of directed strain development.

Project description:Our research aims to help industrial biotechnology develop a sustainable economy using green technology based on microorganisms and synthetic biology through two case studies that improve metabolic capacity in yeast models Yarrowia lipolytica (Y. lipolytica) and Saccharomyces cerevisiae (S. cerevisiae). We aim to increase the production capacity of beta-carotene (β-carotene) and succinic acid, which are among the highest market demands due to their versatile use in numerous consumer products. We performed simulations to identify in silico ranking of strains based on multiple objectives: the growth rate of yeast microorganisms, the number of used chromosomes, and the production capability of β-carotene (for Y. lipolytica) and succinate (for S. cerevisiae). Our multiobjective optimization methodology identified notable gene deletions by searching a vast solution space to highlight near-optimal strains on Pareto Fronts, balancing the above-cited three objectives. Moreover, preserving the metabolic constraints and the essential genes, this study produced robust results: seven significant strains of Y. lipolytica and seven strains of S. cerevisiae. We examined gene knockout to study the function of genes and pathways. In fact, by studying the frequently silenced genes, we found that when the GPH1 gene is knocked out in S. cerevisiae, the isocitrate lyase enzyme is activated, which converts the isocitrate into succinate. Our goals are to simplify and facilitate the in vitro processes. Hence, we present strains with the least possible number of knockout genes and solutions in which the genes are turned off on the same chromosome. Therefore, we present results where the constraints mentioned above are met, like the strains where only two genes are switched off and other strains where half of the knockout genes are on the same chromosome. This study offers solutions for developing an efficient in vitro mutagenesis for microorganisms and demonstrates the efficiency of multiobjective optimization in automatizing metabolic engineering processes.

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Identification of metabolic engineering targets through analysis of optimal and sub-optimal routes.

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