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.
5.3.3Optimization of
subcellular structure
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.
5.4UA derivatives decorated
by engineered microorganisms
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.