Fig. 7 Biosynthesis of UA (A) Biosynthesis pathway; (B)
The electron transfer between CYP450 and CPR
It is generally believed that cyclization of 2,3-oxidosqualene is
carried out by the Oxidosqualene Cyclase (OSC) by employing protonation,
cascade cyclization of polyene addition, hydride and/or methyl
translocation, and unwinding 71. αASs are
multifunctional enzymes, which catalyze 2,3-oxidosqualene to several
pentacyclic triterpenoids like α-amyrin, β-amyrin and lupeol etc72,73. However, product isomerization limits their
catalytic ability and ultimately affects the downstream synthesis of UA.
CYP450s are most versatile proteases found in nature, the first one of
which was discovered in the rat liver microsomes in 195874. CYP450s contain the heme, which primarily binds to
the cytoplasmic surface of Endoplasmic Reticulum (ER) and catalyze a
series of oxidation reactions like oxidation, hydroxylation,
dealkylation and breakage of carbon-carbon bonds 75.
Mainly, CYP450s catalysis involves the introduction of oxygen into
inactive carbon-hydrogen bonds 76, while the
corresponding reduction process is assisted by CPR. CYP450s utilize two
electrons from NADH or NADPH transferred by CPR to the heme center of
iron to activate the oxygen molecules and further catalyze the
substrates to form functional groups.
5.2 De novo synthesis of
UA in engineered
microbial cell factories
With strong resistance to low pH, osmotic stress and environmental
factors, engineered microbial cell factories such as S.
cerevisiae is widely used to biosynthesize the valuable natural
products on industrial scale. What’s more, S. cerevisiae has
proven to be an enormously suitable candidate for synthesis of complex
terpenoids due to its endogenous MVA pathway, which provides sufficient
precursors to synthesize the terpenoids to meet subsequent demand65. Terpene skeleton is modified by CYP450 in a better
way due to presence of protein modification system, biofilm system and
redox system in S. cerevisiae 18. With clear
genetic background and suitable biological safety, various
gene manipulation and genetic
modification methods have been widely
employed in S. cerevisiaefor the green synthesis of terpenoids.
As early as 2012, α-amyrin has been synthesized in microbial cell
factories 77, but the UA synthesis was inadequate. It
is generally accepted that UA biosynthesis in S. cerevisiae is
limited due to the accumulation of α-amyrin and the oxidation ability of
CYP450 78. These limitations led researchers to adopt
metabolic engineering and synthetic biology approaches to synthesize UA
more efficiently in S. cerevisiae . Dai et al. introduced
CYP716A15 and CPR from Vitis vinifera and αAS fromEriobotrya japonica into S. cerevisiae . With ERG1, ERG 9
and ERG 20 overexpressed to enhance the precursor production, they
subsequently got a final UA yield of 1.76 mg/L/OD60075. In another study, Lu et al. heterologously
expressed CYP716A12 from Medicago Sativa , αAS fromCatharanthus roseus , ERG1 from Candida albicans , and CPR
from Arabidopsis thaliana in Saccharomyces cerevisiae . By
optimization of fermentation conditions and overexpression of tHMG1,
ERG9, ERG20 to strengthen MVA pathway, the yield of UA in shake flask
culture reached 25.85mg/L 79.
5.3
Optimization strategies to enhance the microbial synthesis of UA
Biosynthesis of UA in microbial cell factories provides an environment
friendly platform for synthesis of specialized terpenoids, while the
bioproduction of UA still needs to be improved as compared to its
counterpart triterpenes. Therefore, multiple optimization strategies are
still needed to further develop the production potential of S.
cerevisiae. On the basis of UA’s
synthetic pathway, we propose three strategies to enhance the green
biosynthesis of UA at industrial scale.
5.3.1Strengthening precursors
supplementation
Microbial biosynthesis of natural products is improved by adopting
effective metabolic engineering approaches to enhance the precursors
supply and reducing their unnecessary consumption. Common strategy
includes the overexpression of MVA-related (e.g. ERG9, ERG1and ERG20)
genes and tHMG1 (the truncated version of HMG1) gene. Lu et al.enhanced the supply of 2,3-oxidosqualene as mentioned above, resulting
in 25.99 mg/L α-amyrin in shake flask culture 79.
Enhancement of precursor supply is also achieved by adopting the
traditional metabolic engineering approaches to overexpress the key
genes, utilization of balanced pathways and downregulation of
competitive pathways 80. In another study, researchers
balanced the metabolic pathway and achieved transcriptional regulation
of aligned oleanane-type triterpenoids by overexpression of UPC2-1, a
global transcription factor for ergosterol synthesis in yeast81. In addition, they reconstructed the promoter at
the binding site of UPC2 and the galactose regulatory network to promote
gene expression, resulting in a 65 and 6.8-fold increase in β-amyrin and
oleanolic acid, respectively 82. This also provides a
new idea for the synthesis and regulation of both α-amyrin and UA in
yeast.
Furthermore, the catalytic potential of αAS also plays a decisive role
in the synthesis of α-amyrin and UA. A highly active αAS, Md OSC1
from Malus domestica , have been identified through bioinformatics
screening and phylogenetic analysis 37. Md OSC1
expression combined with overexpression of genes related to the MVA
pathway in S. cerevisiae yielded an α-amyrin titer of
11.97\(\pm\)0.61 mg/L 83. Furthermore, the yield of
α-amyrin was increased to 11-fold higher than that of the control with
the triple mutant Md OSC1N11T/P250H/P373A by
remodeling Md OSC1 84. Owing to these strategy,
UA biosynthesis will be guaranteed by enhancing the supply of its
precursors.
5.3.2Enhancing the coupling
efficiency of CPR with CYP450
Lower microbial production of UA as compared to α-amyrin depicts the
inefficient oxidation process which in turn depends upon both the
catalytic efficiency of CYP450 as well as electron transfer between
CYP450 and CPR 80. In this regard, it is essential to
excavate and characterize CYP450 and CPR with high catalytic efficiency.
By means of gene linkage, genome sequencing and transcriptome
sequencing, many CYP450s with the ability to oxidize C-28 of α-amyrin
have been identified from Arabidopsis thaliana , Medicago
truncatula , Barbarea vulgaris and other plants. Combining
different sources of CYP with CPR and finding the best combination is a
common way to improve its oxidation ability (Table 3 ).
Table 3. Oxidation of C-28 position of α-amyrin