Development of improved host-vector system for enhanced production of santalene isoforms in Azospirillum brasilense

Azospirillum brasilense is a non-photosynthetic bacterium that possesses the ability to produce carotenoids, in which squalene is a critical intermediate of the carotenoid biosynthetic pathway. Farnesyl pyrophosphate (FPP) serves as a precursor for the production of sesquiterpenoids; hence, its availability is a major bottleneck in the microbial biosynthesis of several major plant sesquiterpenes. We improved the utility of A. brasilense Cd for the heterologous production of sesquiterpenes by enhancing intracellular availability of FPP and disrupted the chromosomal hpnC gene to block the squalene biosynthetic pathway to decrease a competing sink for FPP, along with the introduction of the heterologous Escherichia coli ispA gene, together with additional copies of the native A. brasilense dxps and crtE genes. These genetic manipulations, along with the expression of codon-harmonized santalene synthase from Santalum album, enabled an increase in the production of α -santalene by 50-fold, α-exo -bergamotene by 20-fold, and β -santalene by 10-fold in A. brasilense Cd. Additionally, we developed a fructose-inducible santalene synthase expression system, which may be more efficient and economical than the isopropyl β-D-1-thiogalactopyranoside-inducible system. Finally, by cloning and expressing genes encoding cytochrome P450 monooxygenase and its cognate NADPH-cytochrome P450 reductase, we have shown the potential of A. brasilense to produce santalols, the most desired ingredient of sandalwood oil.