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Open Access Methodology article

Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

Nuno Carinhas1, Vicente Bernal2, Ana P Teixeira1, Manuel JT Carrondo13, Paula M Alves1 and Rui Oliveira3*

Author Affiliations

1 Instituto de Tecnologia Quimica e Biológica-Universidade Nova de Lisboa/Instituto de Biologia Experimental e Tecnológica (ITQB-UNL/IBET), Apartado 12, P-2781-901 Oeiras, Portugal

2 Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, E-30100 Murcia, Spain

3 REQUIMTE, Systems Biology&Engineering Group, Chemistry Department, Universidade Nova de Lisboa, P-2829-516 Caparica, Portugal

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BMC Systems Biology 2011, 5:34  doi:10.1186/1752-0509-5-34

Published: 25 February 2011

Abstract

Background

Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.

Results

A hybrid framework is presented combining classical metabolic flux analysis with projection to latent structures to further link estimated metabolic fluxes with measured productivities. We first explore the functional metabolic decomposition of a baculovirus-producing insect cell line from experimental data, highlighting the TCA cycle and mitochondrial respiration as pathways strongly associated with viral replication. To reduce uncertainty in metabolic target identification, a Monte Carlo sampling method was used to select meaningful associations with the target, from which 66% of the estimated fluxome had to be screened out due to weak correlations and/or high estimation errors. The proposed hybrid model was then validated using a subset of preliminary experiments to pinpoint the same determinant pathways, while predicting the productivity of independent cultures.

Conclusions

Overall, the results indicate our hybrid metabolic flux analysis framework is an advantageous tool for metabolic identification and quantification in incomplete or ill-defined metabolic networks. As experimental and computational solutions for constructing comprehensive global cellular models are in development, the contribution of hybrid metabolic flux analysis should constitute a valuable complement to current computational platforms in bridging the metabolic state with improved cell culture performance.