Establishment And Application Of Multiplex Iterativeplasmid Engineering And CRISPR-Cas9 Mediated Genome Editing

A major challenge in metabolic engineering and synthetic biology is the construction of synthetic metabolic pathways with optimal functions. Here, we first present Multiplex Iterative Plasmid Engineering(MIPE) which is a highly efficient method for combinatorial optimization of metabolic pathways constructed on plasmids. MIPE uses multiplex ssDNA mediated recombineering for the introduction of mutations, allowing it to target several sites simultaneously and generate plasmid libraries of up to 107 sequences in one reaction. To improve ssDNA recombineering and MIPE mutagenesis efficiency, we used the strategy of ssDNA and plasmid cotransformation and developed Restriction Digestion mediated Co-Selection(RD CoS) method. To demonstrate this approach, we applied MIPE to fine-tune gene expression level in the 5-gene riboflavin biosynthetic pathway and successfully isolated a clone with 2.67-fold improved production in less than a week. Then, we used MIPE to simultaneously target 23 codons scattered along the 750 bp red fluorescent protein sequence, yielding 31% average mutation efficiency for each targeted codon.Then, we established a CRISPR-Cas9 based system for rapid, efficient and iterative genome editing of Escherichia coli, providing a valuable tool for the optimization of metabolic pathways on the chromosome with superior speed and ease. This system enabled us to introduce various types of genomic modifications, including deletion and insertion, with near 100% editing efficiency and to introduce three mutations simultaneously. In addition, we constructed a CIRPSR-Cas9 mediated inducible plasmid curing system for eliminating gRNA plasmid from the cells, allowing iterative genome editing within two days per cycle. We also found that cells with functional mismatch respire(MMR) system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential in metabolic engineering, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate(MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorial exploring the metabolic landscape. Our best producer contained 15 targeted mutations and produced 2.0 g/L β-carotene in fed-batch fermentation.