| Oleanane-and Ursane-type represent the two major classes of pentacyclic triterpenoids.Ursolic acid is a well known Ursane-type terpene acid that occurs as free acid in certain plants,and hederagenin is known as the oxygenated Ursane derivative of the aglycone tripterpene.Both of the two compounds can exhibit important multiple pharmacological activities in the field of health care,including anti-tumor,anti-inflammatory,anti-bacterial,anti-HIV,as well as hepatoprotective.Currently the main approaches to obtain these chemicals are via direct extraction from plants or fully chemical synthesis,which have to face the drawbacks of low yield of products,low economic efficiency,hazardous pollution,waste of plant resources and so on.The last decade has seen a dramatic increase in the utilization of the metabolic engineering methods to achieve the biosynthesis of natural products,including customize the key enzymes,systematic design and modular rearrangement of cellular metabolic pathways,and assembly and optimization of cytoplasmic organoids of chassis cells.This trend has to a significant degree been fueled by advances in successfully heterologous synthesis of several star natural products,such as Artemisinin,Paclitaxel,Morphine,and Lycopene.The a-amyrin synthases(aAS)is function as the key enzyme for the biosynthesis of ursolic acid.For now,all of the identified a-amyrin synthases have been prove to be bifunctional enzymes,which catalyze 2,3-epoxide squalene to produce amyrin with both of?-and ?-stereo isomers.Given a-amyrin is the only desired substrate for the production of ursolic acid,the key point of the present work is screen of different candidates of aAS to promote catalysis activate and stereoselective specificity.First of all,we separately cloned the aAS gene from Eriobotrya japonica?Olea europaea?and Centella asiatica into a low-copy number expression vector,then transferred these plasmids into an engineered Saccharomyces cerevisiae strain BY-T3,a mutant has been increased the supply of the precursor 2,3 oxidosqualene,to test their capabilities for a-amyrin production.The results showed that the amyrin synthase from E.japonica exhibited the best a-amyrin production rate of 79.3%.Then,the gene of a-amyrin synthase derived from E.japonica was codon optimized,and co-expressed in strain BY-T3 with CYP15(CYP716A15)and SvvCPR from Vitis vinifera,batch cultural of this strain with defined medium for 5 days and the final yield titer for ursolic acid can achieved 13.4 mg/L.Hederagenin synthase considered to be a cytochromes P450 enzyme that could be able to catalyze C-23 hydroxylation of oleanolic acid to produce hederagenin.To construction of the cell plant of hederagenin,identification and characterization of the potential hederagenin synthase is another key point to this research.Here,we identified the homologs of CYP72A68v2 through BLAST search from the genomic databases of Malus x domestica?Vitis vinifera and centella asiatica.Then cloned and integrated these genes to the Saccharomyces cerevisiae BY-OA strain which in-house generated by our laboratory.MdMA02 gene from Malus x domestica,which demonstrated can achieved specific hydroxylation C-23 in Oleanic acid to generated hederagenin.The following batch cultural and high density fermentation experiments showed that the final production titers for hederagenin after 5 days can reach 0.2 mg/L and 101 mg/L,respectively.These results provide the basis for the microbial fermentation production of hederagenin from oleanane-type compounds.In summary,we reported constructed a preliminary ursolic acid and hederagenin cell factories by using synthetic biology approaches,which to our knowledge,have not yet been reported successfully achieved in yeast host before.Our work provides the basis for recombinant microorganism fermentation instead of plant extraction and chemical synthesis of pentacyclic triterpenoids. |
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