Development of Crispr Based Genome Edition and Pathway Optimization Techniques for Metabolic Engineering Applications

Microbial genome edition is to modify chromosome in way of deletion, insertion and replacement, which is one of the most important technique in Synthetic Biology and Metabolic Engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic edition methods. In this research, we perused the goal of development of the easiest method for Escherichia coli genome edition and technique for multiplex optimization of native metabolic pathway by chromosomal editing. For this purpose, we designed modular plasmid assembly strategy, compared effect of different length of homologous region for recombination, and tested different sets of recombinases. The genome editing technique we developed only requires the work of one plasmid construction and one transformation of practice to edit a genomic locus within 3 days with minimal lab work. By further development, multiple loci were able to be edited simultaneously. Base on this, regulation sequences of several genes in E. coli MEP pathway were disturbed for various expression levels. Thus a metabolic pathway library with different expression of each gene were obtained. And strains with highest production of β-carotene which represented optimized MEP pathway could be selected.  In this article, we described the process of method development.