Sesquiterpene Synthase Engineering and Targeted Engineering of ?-Santalene Overproduction in Escherichia coli

As a natural sesquiterpene compound with numerous biological activities,alpha-santalene has extensive applications inthe cosmetic and pharmaceutical industries. Although several alpha-santalene-producing microbial strains have been constructed, lowproductivity still hampers large-scale fermentation. Herein, we present a case of engineered sesquiterpene biosynthesis where theinsufficient downstream pathway capacity limited high-level alpha-santalene production inEscherichia coli. The initial strain wasconstructed, and it produced 6.4 mg/L alpha-santalene. To increase alpha-santalene biosynthesis, we amplified theflux toward farnesyldiphosphate (FPP) precursor by screening and choosing the right FPP synthase and reprogrammed the rate-limiting downstreampathway by generating mutations in santalene synthase (Clausena lansium;ClSS). Santalene synthase was engineered by site-directedmutagenesis, resulting in the improved soluble expression ofClSS and an alpha-santalene titer of 887.5 mg/L; the alpha-santalene titerreached 1078.8 mg/L after adding a fusion tag toClSS. The most productive pathway, which included combining precursorfluxamplification and mutant synthases, conferred an approximate 169-fold increase in alpha-santalene levels. Maximum titers of 1272 and2916 mg/L were achieved under shakeflask and fed-batch fermentation, respectively, and were among the highest levels reportedusingE. colias the host.

Automated summary

This study presents the engineering of a biosynthetic pathway for alpha-santalene in Escherichia coli, resulting in high production levels of this natural compound. By amplifying the precursor flux and mutating the synthase enzyme, the titer of alpha-santalene was significantly increased, reaching one of the highest levels reported in E. coli as a host.