Development of a genome engineering tool for insertion of pathway-sized DNAs in Escherichia coli

Background

The approach of metabolic engineering enables reprogramming of the producer cell to overproduce products of interest. It usually requires simultaneous manipulation of many genes to achieve the engineering purpose. To circumvent the plasmid-incurred problems, the recombineering technology mainly involving λ Red has been developed for genomic insertion of target genes. However, these λ Red-dependent recombination methods are generally afflicted by insertion of a large size DNA construct. This issue was addressed by development of a genome engineering tool by combination of phage integrases with CRISPR-λ (PIC herein).

Methods

As a proof of concept, PIC was applied for engineering of metabolic pathways leading to pyridine nucleotides. This was carried out by assembly of a heterologous operon (13.9 kb) and an artificial operon (8.2 kb) composed of endogenous genes. Aided by phage integrases, the two assembled DNA constructs were sequentially integrated into the prophage attB sites of Escherichia coli at high efficiency. The kanamycin cassette from the Keio strain collection was leveraged by CRISPR-λ to eliminate the undesired gene in E. coli by knock-in of the attB site which was utilized for iterative integration of the DNA construct.

Significant findings

The engineered strain consequently overproduced uracil. It resulted in a 160-fold increase in uracil over that of the parent strain. This evidently suggests that the producer strain harbors reprogrammed metabolic pathways in favor of the uracil synthesis. Overall, the result indicates that the developed system is useful for metabolic engineering of E. coli by genomic insertion of pathway-sized DNA cargos.