Improving biosynthetic production of pinene through plasmid recombination elimination and pathway optimization.

Pinene is a monoterpene with wide industrial applications, especially as a promising high energy-density jet fuel. Traditional production of pinene on an industrial scale is material consumptive and has a low yield. As an alternative, microbial organisms have been engineered though advanced synthetic biological techniques to produce a variety of heterologous products, including pinene. Here, we investigated the stability of genetic circuits encoding the pinene producing pathway during fermentation and its relationship to the pinene titer. By replacing scar sequences in the genetic elements and modifying the genome of E. coli strain MG1655, plasmid loss caused by serious metabolic burden was eliminated, generating a remarkable increase in the pinene titer. Furthermore, the heterologous mevalonate pathway was analyzed by overexpression of enzymes and intermediates monitoring. Optimized pathway plasmids and strains were combined to increase the pinene titer to 104.6 mg/L.