Construction Of Cell Factories For Production Of Pentacyclic Triterpenoids In Saccharomyces Cerevisiae

Ursolic acid is an important ursane-type pentacyclic triterpenoid,which has anti-cancer,anti-diabetic,anti-ulcer and hypolipidemic properties.Betulinic acid,a representative of lupane-type pentacyclic triterpenoids,not only has a higher choice anti-melanoma activity,but also has excellent anti-HIV activity.Morolic acid belongs to the oleanane-type pentacyclic triterpenoid,with cytotoxicity,anti-HIV,anti-HSV,anti-inflammatory and anti-diabetic characteristics.However,these compounds have complex structures and are difficult to achieve chemical synthesis.At present,it is mainly prepared by extracting from plants.Based on the strategy of synthetic biology,this study used CRISPR/Cas9 technology to construct Saccharomyces cerevisiae cell factories for producing ursolic acid,betulinic acid and morolic acid,and established triterpenoid-producing platforms,which provides basis for engineered microbial fermentation,with potential to replace plant extraction with pentacyclic triterpenoids.The main findings are as follows:（1）In order to realize the biosynthesis of ursolic acid in S.cerevisiae,this study first integrated 4 different sources ofα-amyrin synthases（αAS）.The results showed that the MdαAS from Malus domestica had the highest activity,and it was obtained prior to ursolic acid production.The precursorα-amyrin chassis cells had a yield of 101.4±3.2 mg·L^-1.On this basis,the cytochrome P450 reductase（JcCPR）derived from Jatropha curcas and 4different sources of cytochrome P450（CYP450）oxidases combination,the highest catalytic activity of CYP716AL1 derived from Catharanthus roseus was obtained.In order to further increase the production of ursolic acid,CYP716AL1 was combined with 4 different sources of CPRs to obtain At CPR from Arabidopsis thaliana with higher electron transfer efficiency.The output of ursolic acid reached 41.4±1.2 mg·L^-1.（2）In order to construct the synthetic pathway of betulinic acid in S.cerevisiae,this study first introduced 4 different sources of lupeol synthases（LUS）.The results showed that chassis cells of Oe LUS from Olea europaea had the highest activity,with produced a yield of91.6±2.6 mg·L^-1 betulinic acid precursor,lupeol.Then JcCPR was adapted to CYP450oxidases from 4 different sources,and the Vitis vinifera-derived CYP716A15 with the highest catalytic activity was selected.In order to further increase the production of betulinic acid by adapting CPRs from 4 different sources to CYP716A15,the best Lj CPR from Lotus japonicus was obtained,and the yield of betulinic acid reached 16.5±0.3 mg·L^-1.（3）In order to obtain the engineered strain of S.cerevisiae that produces morolic acid,this study firstly expressed 4 different sources of germanicol synthases（GES）heterologously.The results showed that the Md GES from Malus domestica had the highest activity,and the morolic acid-producing precursor was obtained.The yield of the chassis cells of germanicol was 68.3±2.1 mg·L^-1.Then,JcCPR was co-expressed with 4 CYP450 oxidases from different sources,and CYP716AL1 from Catharanthus roseus with the highest catalytic activity was screened.For further improvement in terms of morolic acid production,4 different sources of CPRs were screened to obtain At CPR from Arabidopsis thaliana that can supply electrons more efficiently.The morolic acid production reached 24.3±0.4 mg·L^-1.（4）Moreover,on the basis of previous work,the above-mentioned engineered strains mevalonate（MVA）pathway was optimized through metabolic engineering strategies,that is,the farnesyl pyrophosphate synthase（ERG20）and squalene epoxidase（ERG1）were overexpressed.Finally,the obtained cell factories of S.cerevisiae produced ursolic acid,betulinic acid and morolic acid yields reaching 62.5±1.5,26.7±0.9 and 34.1±1.0 mg·L^-1,respectively.