Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli

Abstract

Limonene is an important monoterpene used as a chemical commodity and precursor for producing biofuels, flavor and medicinal compounds. In this paper, we engineered Escherichia coli by embedding two exogenous genes encoding a limonene synthase (LS) and a geranyl diphosphate synthase (GPPS) for production of limonene. Out of 12 E. coli strains transformed with various plasmids, the best one with p15T7-ls-gpps produced limonene with a titer of 4.87 mg/L. In order to enhance the limonene production, two rate-limiting enzymes in the endogenous MEP pathway of E. coli, 1-deoxy-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate isomerase (IDI), were overexpressed consecutively on vector pET21a+, resulting in a production of 17.4 mglimonene/L at 48 h. After the preliminary optimization of the medium in a two-phase culture system composed of n-hexadecane (1/50, Vorg/Vaq), the final production of limonene was raised up to 35.8 mg/L, representing approximately a 7-fold improvement compared to the initial titer.