Fig.6 UA derivatives biotransformed by fungi
Efficient synthesis of UA and its derivatives in engineered microbial cell factories
As mentioned above, traditional extraction methods are increasingly unfavorable for large-scale production of UA. Furthermore, complex structure and oxidation position, as well as the optical purity of the final product all increase the difficulty for its chemical synthesis. Green biosynthesis of valuable compounds by microbial cell factories may reduce the limitations of traditional extraction methods from plants as it is not restricted by the natural climate and is suitable for large-scale industrial production under high density fermentation. More importantly, some terpenoids are synthesized efficiently in microorganisms with various regulatory strategies, such as excavation and modification of key enzymes 18,64, optimization of endogenous metabolic pathways 65-67, inhibition of branching pathways 68-70. Therefore, microbial engineering also has a wide application prospect in the green synthesis of UA and its derivatives.
5.1 The biosynthesis pathway of UA
In recent years, synthetic pathway of UA has been well explored in plants, which is roughly divided into three modules: synthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), synthesis of α-amyrin and oxidation of α-amyrin. In plant organelles, IPP and DMAPP are synthesized by methyl erythritol-4-phosphate (MEP) pathway in plastids and mevalonate (MVA) pathway in cytoplasm. Afterwards, two IPP molecules are combined with one molecule of DMAPP end-to-end, and condense to farnesyl diphosphate (FPP). Two FPP molecules bind together to form the triterpenoid precursor, squalene followed by oxidiation to form 2,3-oxidosqualene, which subsequently undergoes cyclization into α-amyrin, the direct precursor of UA. Eventually, α-amyrin is oxidized at C-28 to form UA through a three-step oxidation reaction by employing the action of Cytochrome P450 (CYP450) monooxygenase and its redox chaperone Cytochrome P450 Reductase (CPR). In addition to the common rate-limiting enzymes for triterpenoid, α-amyrin synthase (αAS) and CYP450 greatly affect the synthesis of UA (Fig. 7A ).