In addition, exact mechanism of action of CYP450 and CPR is not clear yet and there is a need to explore the ultimate cause that affects the electron transfer efficiency between them. Analysis of the protein structure of CYP450 helps to elucidate the underlying causes of electron transfer from CPR to CYP450. In addition, with the advent of protein engineering along with high throughput screening, rational design and modification of CYP450 and CPR have attracted much attention, which with optimized structures have also become common means to solve the problem of insufficient oxidation ⁸⁰.

S. cerevisiae harbors diverse organelles with different structures and specialized functions. Rational design and harnessing of these organelles to produce valuable compounds is of great application prospect in terpenoid synthesis. Heterologous FPP synthase and sesquiterpene synthase to mitochondria by fusing the mitochondrial targeting signal peptide of yeast COX4, resulting in a 20-fold increase in sophoradiene production ⁹⁸. ER can also be subjected to morphological modifications such as increasing the membrane area to harbor more enzymes to enhance the catalysis processes. As many endogenous or exogenous proteins are located in ER membrane, like CYP450, the area of ER membrane have been significantly increased by knocking out the PAH1 by CRISPR/cas9 in S. cerevisiae to enhance the activity of enzymes related to terpene synthesis for efficient biosynthesis of terpenoids ⁹⁹. This engineering strategy increased the yield of β-amyrin, artemisinic acid, alfalfa acid and its glycosylated derivatives by 8-fold, 2-fold, 6-fold and 16-fold respectively, which showed great potential of microbial cell factories to enhance the yield of terpenoids.

In addition, mass transfer efficiency can significantly boost the synthetic capacity of microorganisms ⁶⁵. The highest yield of α-amyrin was obtained in engineered S. cerevisiae by expanding the storage pool, where DGA1 (Diacylglycerol acyltransferase) was overexpressed to enhance the intracellular storage capacity⁸⁴. Draw lessons from it, UA biosynthesis can also be improved by modifying chemical mass transfer such as the translocation of its synthesis and the condition of transportation.

By combining all these strategies including the precursor supplementation, enhancing the coupling efficiency of CPR with CYP450 and optimization of subcellular structure, de novo biosynthesis of UA will be significantly enhanced in microbial cell factories. These studies fully demonstrated the potential of utilizing S. cerevisiae cell factories to synthesize UA efficiently, providing an effective means to replace traditional extraction and chemical synthesis.

Biosynthesis of UA derivatives by construction of metabolic pathways in engineered microbial cell factories have been promoted along with the development of de novo microbial synthesis of UA. In recent years, biosynthesis of UA derivatives mainly involved in hydroxylation on C-2α and glycosylation.

For C-2α hydroxylation of UA, sequence analysis of RNA of Avicennia marina leaves revealed the functional CYP716C53, which catalyzed the C-2α hydroxylation of UA to yield Corosolic acid (Fig. 8 ) ¹⁰⁰. It’s a triterpenoid compound which has attracted commercial and research interest for unique anti-diabetic properties ¹⁰¹. In addition, the heterologous expression of Lagerstroemia speciose -CYP716C55 in S. cerevisiae also led to C-2α hydroxylation of UA ⁹⁶. Moreover, CYP716C49 was identified in Crataegus pinnatifida and its expression along with the αAS from E. japonica as well as CYP716A15 and CPR fromV. vinifera in microbial cell factories increased the production titer of Corosolic acid to 141.0 mg/L ⁷⁵.