Systematic Optimization of Limonene Production in Engineered Escherichia coli

Limonene, a cyclic monoterpene, is widely used in food and cosmetics industries as well as in agriculture. In the work described herein, employing a systematic optimization strategy, we constructed an efficient platform for producing limonene via the heterologous mevalonate pathway in Escherichia coli. By site-directed mutation of EfMvaS and tuning the initial translation of EfMvaE and EfMvaSA110G through ribosome binding site engineering, the upstream module for overproducing mevalonate was obtained. Expression of MmMK with ScPMK, ScPMD, and ScIDI under FAB80 promoter resulted in an efficient midstream module to produce 181.73 mg/L of limonene. Subsequently, coexpression of SlNPPS and MsLS in the downstream module led to a great improvement of limonene production to 694.61 mg/L. Finally, metabolically engineered strain ELIM78 produced 1.29 g/L of limonene in 84 h by fed-batch fermentation in a shake-flask. This is the first report on limonene biosynthesis in E. coli using neryl pyrophosphate synthase, which has promising potential for producing other monoterpenes.