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.IMPORTANCESandalwood oil is among the most valuable natural fragrances and is also known for its diverse biological activities. The biosynthesis of santalene, a major constituent of sandalwood oil, originates from the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway. This pathway is naturally present in Azospirillum brasilense, a plant growth-promoting rhizobacterium, making it a suitable host for the heterologous production of sesquiterpenes. This study has shown the possibility of using A. brasilense as a promising host-vector system for enhancing the production of santalene isomers by several fold. It has also shown the superiority of a fructose-inducible system over the isopropyl β-D-1-thiogalactopyranoside-inducible system for the industrial production of high-value secondary metabolites in this bacterium.