From In Silico Prediction To In Vivo Validation: Advancing Biohydrogen Production Through Metabolic Engineering Of E. Coli MG1655

Abstract

Metabolic engineering and synthetic biology advanced in recent times resulting in plenty of methods and tools for genetic manipulation rationally. This study explains the application of Genome-scale metabolic model and its experimental validation using biohydrogen production as a case study. The iJO1366 GEM was explored to identify the probable targets for the metabolic engineering Escherichia coli MG1655 strain by simulating its anaerobic growth in three different carbon sources demonstrating its versatility to predict accurate phenotypes. Moreover, availability of advanced engineering tools such as CRISPR has aided the field of metabolic engineering increasing the efficiency of genetic intervention. Therefore, this work also demonstrates the application of CRISPR-Cas9 technology and its modification to ease it’s the process of screening out successful mutants. Developed methodology follows primer based mutant screening and avoiding the need of sequencing process which saves time and resources. Utilizing this knowledge, this study uses E. coli as a chassis to illustrate ΔhybC mutant construction and its application for biohydrogen production using different carbon substrates emphasising the value of substrate based on the growth phase of the cell.