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