Computer-Assisted Engineering of Synthetic Pathway for Biodegradation of Anthropogenic Pollutant under in vitro and in vivo Condition
Synthetic Biology Engineering Evolution Design SEED
2015
2015 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Poster Session A
Thursday, June 11, 2015 - 5:30pm to 7:00pm
Metabolic engineering has recently attracted attention of scientific community and industry due to its potential in biosynthesis of value added chemicals or biodegradation of toxic compounds. However, complexity of life even in the form of the simplest microbial cells makes the rational engineering of metabolic routes a challenging task. Recent progress in analytical methods, synthetic biology tools and computer modelling enables detailed studies of entire multi-enzyme reactions under in vitro and in silico conditions. Knowledge obtained under these simplified conditions can be employed to dissect limitations of a metabolic pathway and optimize its performance in a suitable host organism.
We applied such approach for engineering of our model system - synthetic catabolic pathway for biodegradation of toxic environmental pollutant of anthropogenic origin 1,2,3-trichloropropane.1 The function of the pathway consisting of three enzymes from two different microorganisms was initially studied in vitro.2 Activity of the enzyme catalyzing the first reaction step was improved by computer-assisted protein engineering.3 Kinetic parameters of individual enzymes were used for construction of the kinetic model describing the five-step conversion of 1,2,3-trichloropropane to the final product glycerol.4 The model was employed in developed mathematical algorithm and was applied for study of a dynamic behaviour of in vitro three-enzyme system and for its optimization.
Validated mathematical model was subsequently utilized also for rational design of the pathway in vivo in the heterologous host Escherichia coli.5 Based on in silico simulations, we selected appropriate combinations of vectors leading to optimized expression pattern of three enzymes. This resulted in improved viability of engineered host organism in the presence of toxic substrate. Eventually, we also identified a remaining bottleneck of the pathway with help of recorded time-courses of in vivo TCP conversions and proposed engineering steps to further improve efficiency of the pathway. Our study presents a new concept for engineering of a synthetic metabolic pathway for degradation of toxic compound in a heterologous host.
This work was financially supported by the grants P503/12/0572 from the Czech Grant Agency and CZ.1.05/1.1.00/02.0123 from the European Regional Development Fund.
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