A synthetic biology and green bioprocess approach to recreate agarwood sesquiterpenoid mixtures

Certain endangered Thymelaeaceous trees are major sources of the fragrant and highly valued resinous agarwood, comprised of hundreds of oxygenated sesquiterpenoids (STPs). Despite growing pressure on natural agarwood sources, the chemical complexity of STPs severely limits their synthetic production. Here, we catalogued the chemical diversity in 58 agarwood samples by two-dimensional gas chromatography-mass spectrometry and used synthetic biology to produce subsets of the complex STP mixtures found in agarwood. We improved STP yields in the unicellular alga Chlamydomonas reinhardtii by 25-fold through combinatorial engineering to biosynthesise nine macrocyclic STP backbones found in agarwood. A bioprocess following green-chemistry principles was developed that exploits ‘milking’ of STPs without cell lysis, solvent-solvent STP extraction, solvent-STP nanofiltration, and bulk STP oxy-functionalisation to obtain terpene mixtures like those of agarwood. This green bioprocess allows total solvent recycling and continuous production. Our synthetic biology and green bioprocess approach enables a more sustainable synthesis of complex, fragrant terpenes as an alternative to their exploitation from natural sources.