Sesquiterpene Synthase Engineering and Targeted Engineering of α‑Santalene Overproduction in Escherichia coli

As a natural sesquiterpene compound with numerous biological activities, α-santalene has extensive applications in the cosmetic and pharmaceutical industries. Although several α-santalene-producing microbial strains have been constructed, low productivity still hampers large-scale fermentation. Herein, we present a case of engineered sesquiterpene biosynthesis where the insufficient downstream pathway capacity limited high-level α-santalene production in Escherichia coli. The initial strain was constructed, and it produced 6.4 mg/L α-santalene. To increase α-santalene biosynthesis, we amplified the flux toward farnesyl diphosphate (FPP) precursor by screening and choosing the right FPP synthase and reprogrammed the rate-limiting downstream pathway by generating mutations in santalene synthase (Clausena lansium; ClSS). Santalene synthase was engineered by site-directed mutagenesis, resulting in the improved soluble expression of ClSS and an α-santalene titer of 887.5 mg/L; the α-santalene titer reached 1078.8 mg/L after adding a fusion tag to ClSS. The most productive pathway, which included combining precursor flux amplification and mutant synthases, conferred an approximate 169-fold increase in α-santalene levels. Maximum titers of 1272 and 2916 mg/L were achieved under shake flask and fed-batch fermentation, respectively, and were among the highest levels reported using E. coli as the host.