title: Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models.
abstract: Rational metabolic engineering methods are increasingly employed in designing the commercially viable processes for the production of chemicals relevant to pharmaceutical, biotechnology, and food and beverage industries. With the growing availability of omics data and of methodologies capable to integrate the available data into models, mathematical modeling and computational analysis are becoming important in designing recombinant cellular organisms and optimizing cell performance with respect to desired criteria. In this contribution, we used the computational framework ORACLE (Optimization and Risk Analysis of Complex Living Entities) to analyze the physiology of recombinant Escherichia coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO. The framework allowed us to integrate data across multiple levels and to construct a population of large-scale kinetic models despite the lack of available information about kinetic properties of every enzyme in the metabolic pathways. We analyzed these models and we found that the enzymes that primarily control the fluxes leading to BDO production are part of central glycolysis, the lower branch of tricarboxylic acid (TCA) cycle and the novel BDO production route. Interestingly, among the enzymes between the glucose uptake and the BDO pathway, the enzymes belonging to the lower branch of TCA cycle have been identified as the most important for improving BDO production and yield. We also quantified the effects of changes of the target enzymes on other intracellular states like energy charge, cofactor levels, redox state, cellular growth, and byproduct formation. Independent earlier experiments on this strain confirmed that the computationally obtained conclusions are consistent with the experimentally tested designs, and the findings of the present studies can provide guidance for future work on strain improvement. Overall, these studies demonstrate the potential and effectiveness of ORACLE for the accelerated design of microbial cell factories.
authors: Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
authors: Stefano
authors: S
authors: Andreozzi
authors: Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
authors: Anirikh
authors: A
authors: Chakrabarti
authors: Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
authors: Keng Cher
authors: KC
authors: Soh
authors: Genomatica, Inc., San Diego, CA, USA.
authors: Anthony
authors: A
authors: Burgard
authors: Genomatica, Inc., San Diego, CA, USA.
authors: Tae Hoon
authors: TH
authors: Yang
authors: Genomatica, Inc., San Diego, CA, USA.
authors: Stephen
authors: S
authors: Van Dien
authors: Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
authors: Ljubisa
authors: L
authors: Miskovic
authors: Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland. Electronic address: vassily.hatzimanikatis@epfl.ch.
authors: Vassily
authors: V
authors: Hatzimanikatis
mesh_terms: D002072:Butylene Glycols / Q000378:metabolism*; D002952:Citric Acid Cycle / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism*
chemical_list: D002072:Butylene Glycols; C039681:1,4-butanediol
keywords: 1,4-butanediol; Flux balance analysis; Kinetic models; Metabolic control analysis; ORACLE; Thermodynamics; Uncertainty