Study On The Biocatalytic Modification Of The C24-C25 Double Bond On PPT-type Ginsenosides As A Critical Step Prior To Pharmace Utical Preparations

Objective:Using biotransformation method for efficient and targeted catalysis of hydration by C24-C25 double bonds of PPT-type ginsenosides to generate rare 25-OH derivatives,and fungal samples were selected at different stages for RNA-seq,further the differentially expressed genes/transcripts were systematically analyzed.Combined with the quantitative analysis technology of transformation products,potential key enzymes involved in the hydration reaction of ginsenosides were discovered,which provided a theoretical basis for the development of targeted catalytic engineering bacteria.Methods:Based on the findings of previous research,taking PPT-type ginsenosides Rg1 as a substrate,a single factor investigation method was used to screen the best inducing factor and substrate concentration for fungal D biotransformation to PPT-type ginsenosides.Using HPLC-IT-TOF-MS/MS and HPLC-QQQ-MS/MS techniques were analyzed the biological catalysis of PPT Ginsenoside qualitative and quantitative,and used the preparative liquid to purify the target product,the chemical structure of the product verified by ¹³C-NMR technology,Finally,the transformation pathway of ginsenoside structure-modified intermediates and products was determined by time course experiments,and calculated the conversion rate of the target product.Furthermore,the full-length transcriptome sequencing technology was used to analyze the differentially expressed genes/transcripts of fungus D at different induction stages to find candidate hydratase genes.Results:The best inducing factor for fungal D to transform PPT-type ginsenosides is FeSO4,Optimum substrate concentration is 1.2g/L,The final composition of the transformation medium is:20g/L glucose,5g/L ammonium sulfate,and 1mM FeSO4,Under these conditions,different PPT-type ginsenosides were transformed,and the results showed thatthe transformation pathway of ginsenoside Rg1 is:Rg1?20?S/R?-Rh1?25-OH-?S/R?-Rh1,the conversion rates of the products are:7.3%20?S?-Rh1,6.7%20?R?-Rh,43.2%25-OH-20?S?-Rh1 and 39.3%25-OH-20?R?-Rh1;the transformation pathway of ginsenoside Re is:Re?20?S/R?-Rg2?20?S/R?-Rf2,conversion rates are:8.8%20?S?-Rg2,5.8%20?R?-Rg2,46.8%20?S?-Rf2,36.3%20?R?-Rf2;the transformation pathway of ginsenoside Rf is:Rf?25-OH-Rf,Conversion products 25-OH-Rf conversion rate of 85.5%;the transformation pathway of ginsenoside Rg2 is;20?S/R?-Rg2?20?S/R?-Rf2,conversion rates are:83.8%20?S?-Rf2,53.8%20?R?-Rf;the transformation pathway of ginsenoside Rh1 is;20?S/R?-Rh1?25-OH-20?S/R?-Rh1,conversion rates are:70.2%20?S?-Rh1,46.2%20?R?-Rh1;Ginsenoside20?S?-Rf2 and 25-OH-20?S/R?-Rf are three new compounds that have been isolated and identified for the first time.By analyzing the changes in the content of the conversion products of fungal D to different PPT saponins,we summarize the characteristics of the biocatalytic system:?1?The catalytic system can specifically C24-C25 double bond of hydrated PPT-type saponin,and has no effect on PPD-type ginsenosides;?2?The glycosyl group at position 20 has a steric hindrance to this hydration reaction,which requires hydrolysis of glycosidic bonds before proceeding;?3?The more polar saponin has a higher conversion rate of its 25-OH derivative;?4?The saponin of the 20-S configuration has a higher conversion rate of its 25-OH derivative.Differential transcriptomics analysis showed that compared with the blank culture,There are three main hydratases in the expression changes of fungal D transcripts induced by the addition of saponin substrates.Among them,consistent with the change trend of the content of 25-OH derivatives is phosphopyruvate hydratase?E.C.4.2.1.11?;There are mainly three glycosidic bond hydrolase expressions occurring in the transcript,after the induction with PPT-type ginsenosides,NAD-dependent epimerase/dehydratase is the only hydrolase that shows up-regulated expression.The upward trend of the change curve is consistent with the process of ginsenoside Re and Rg1 conversion to Rg2 and Rh1.Therefore,we speculate that this hydrolase may be the key enzyme for hydrolysis of glycosidic bond at C20 of PPT-type ginsenosides.Conclusion:Fungus D was a strain with development potential,which can produce different catalytic reactions under suitable conditions.Found in this paper,it was found that fungus D can hydrate the PPT-type ginsenoside C24-C25 double bond to form 25-OH derivatives.By optimizing the conditions,the efficiency and selectivity of the reaction can be greatly improved,the occurrence of side reactions can be reduced,and fermentation products with simple chemical background can be generated,which was beneficial to the separation and purification of the target product.This reaction can be used as a powerful method for the preparation of ginsenoside series 25-OH derivatives and the discovery of new compounds.Simultaneously,the changes of enzyme gene expression in the catalytic process were tracked by transcriptome technique,which greatly improved the success rate of target enzyme discovery,and provided a lot of theoretical basis for the subsequent elucidation of substrate selectivity and catalytic mechanism of the reaction.