Engineering of synthetic biodegradation pathway towards environmental pollutant 1,2,3-trichloropropane

This dissertation describes the application of protein engineering and metabolic engineering approaches for rational redesign of metabolic pathway for biodegradation of important environmental pollutant 1,2,3-trichloropropane (TCP) utilizing both computational and experimental approaches. A yeast surface display based assay was developed for the limiting enzyme in the pathway - haloalkane dehalogeanse DhaA, which enables high-throughput screening for designed mutants and various strategies were attempted enzyme tunnel engineering on haloalkane dehalogenase DhaA to obtain new starting point towards TCP biodegradation including slot tunnel redesign, de novo tunnel design and chimeric protein design with preserved tunnels and generated combinatorial design library subject to high-throuput screening, but unfortunately no design with both decent expression and enhanced activity was detected. Apart from engineering, the TCP degradation pathway was optimized by metabolic engineering. A phosphorylation- and optically-responsive metabolon for the biodegradation of the environmental pollutant 1,2,3-trichloropropane (TCP) was constructed. The pathway efficiency improvement caused by co-localization in response to stimulus was demonstrated. The design method is modular and generalizable, and could enable spatio-temporal control over a wide variety of synthetic biotransformations. To summarize, this dissertation presents an innovative concept for rational engineering of a synthetic biodegradation pathway for pollutant compound 1,2,3-trichloropropane and explores design approaches of protein engineering and metabolic engineering.Ph.D.Includes bibliographical referencesby Lu Yan