Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems

SIMPLE SUMMARY: The limit screening method (LMS method) and product-tolerance engineering was proposed and applied. AcrB, flgFG, motB and ndk were found to be associated with an enhanced tolerance of E. coli to pinene. Using the I-SceI cleavage system, the promoters of acrB, flgFG, and ndk genes were replaced with P37, and pinene production was eventually increased 2.1 times. In general, a combination of shotgun, product-tolerance, and I-SceI cleavage systems provides a more convenient and efficient way of enhancing the synthesis of target products in a host strain. ABSTRACT: Tolerance breeding through genetic engineering, sequence and omics analyses, and gene identification processes are widely used to synthesize biofuels. The majority of related mechanisms have been shown to yield endogenous genes with high expression. However, the process was time-consuming and labor-intensive, meaning there is a need to address the problems associated with the low-throughput screening method and significant time and money consumption. In this study, a combination of the limit screening method (LMS method) and product-tolerance engineering was proposed and applied. The Escherichia coli MG1655 genomic DNA library was constructed using the shotgun method. Then, the cultures were incubated at concentrations of 0.25%, 0.5%, 0.75% and 1.0% of pinene with different inhibitory effects. Finally, the genes acrB, flgFG, motB and ndk were found to be associated with the enhanced tolerance of E. coli to pinene. Using the I-SceI cleavage system, the promoters of acrB, flgFG and ndk genes were replaced with P37. The final strain increased the production of pinene from glucose by 2.1 times.