Study On The Function Of P450 (CYP76AHS And CYP76AK1) In The Biosynthetic Pathway Of Tanshinone

Tanshinones are lipophilic compounds of Salvia miltiorrhiza（Danshen）and have significant effect on cardiovascular disease,they are difficult to obtain by chemical synthesis because of their complex structures.In order to meet the clinical needs,tanshinones are mainly gained by extraction and separation from the root of Salvia miltiorrhiza,which is not conducive to the sustainable development of salvia miltiorrhiza and resources.It is an efficient way to produce or improve the content of these compounds in plant or microorganisms by metabolic engineering or synthetic biology,which could relieve the pressure on resources and promote the sustainable development of Danshen.The elucidation of tanshinone biosynthesis is the basis of metabolic engineering,and it’s also the foundation of synthetic biology,which is important to the resource of traditional Chinese medicine.Great achievements have been made in the biosynthesis of tanshinones.Many genes involved in tanshinone biosynthesis have been cloned,including several diterpene synthases and two P450s genes.According to the structure of tanshinones,P450s were speculated to play important roles in the downstream biosynthesis of tanshinones.In this study,we combinded in vitro enzyme assay with synthetic biology to screen and analysis the functional P450s.The major results of the study were list as follows:1.Function of CYP76AH1 and CYP76AH3Preliminary studies showed that CYP76AH1 and CYP76AH3 were key enzymes involved in biosynthesis of tanshinones,whose gene sequences are highly homologous.CYP76AH1 could catalyze the turnover of miltiradiene to ferruginol,and CYP76AH3 oxidized ferruginol to form 11-hydorxy ferruginol.In this study,the functions of CYP76AH1 and CYP76AH3 were expanded on the basis of the previous work by our group.（1）Miltiradiene,ferruginol,11-hydroxy ferruginol,sugiol and 11-hydroxy sugiol were used as substrates for in vitro enzyme assay.The results showed that the in vitro catalytic function of CYP76AH1 and CYP76AH3 were the same.Both of them could catalyze miltiradiene,ferruginol,11-hydroxy ferruginol and sugiol.Among them,ferruginol,11-hydroxy ferruginol and sugiol were both substrates and products.However,the catalytic activities of them for different substrates were different.（2）CYP76AH1 with cytochrome P450 reductase（SmCPR1）and CYP76AH3 with SmCPR1 were constructed into vectors,separately,and transformed into yeast strain YJ14,which could produce miltiradiene.Comparing the product of engineered yeast strains to identify the function of CYP76AH1 and CYP76AH3 in vivo.The in vivo results showed that the engineered yeast strains with CYP76AH1 or CYP76AH3 could produce ferruginol,11-hydroxy ferruginol,sugiol and 11-hydroxy sugiol,in addition,two compounds with molecular weight of 316.1961 and 300.2035 were produced.However,the contents of these compounds were different among the two engineered yeast strains.In vivo results showed that CYP76AH1 and CYP76AH3 had the same catalyzing function with different catalytic efficiency.Both in vitro enzyme assay and in vivo experiments have proved that CYP76AH1 and CYP76AH3 are multifunctional enzymes with same functions.Both of them can catalyze miltiradiene to form ferruginol,and further oxidize ferruginol at two different carbon centers to form 11-hydroxy ferruginol,sugiol and 11-hydroxy sugiol.However,their catalytic efficiencies are different.The main product of CYP76AH1 is ferruginol,and the main products of CYP76AH3 are 11-hydroxy ferruginol and 11-hydroxy sugiol.2.Function of CYP76AK1Based on formal research of structural modification roles of P450s,we predict that P450s will still play key roles in biosynthesis of tanshinones.For further screening the genes involved in tanshinones biosynthesis,the transcriptome database of salvia miltiorrhiza hairy root was analyzed.4 new genes co-expressed with CYP76AH1 and CYP76AH3 were chosen as candidate genes.Total of 9 P450s containing 5 genes choosed previously were obtained as candidate genes for further functional study.To identify the function of these candidate genes rapidly,CYP76AH3 and candidate gene were combined to construct into a yeast expression vector pESC-His and expressed in WAT11U.The microsome with recombinant protein were followed by in vitro enzyme assay using ferruginol as substrate.The result revealed that the recombinant protein of CYP76AK1 and CYP76AH3 could catalyze ferruginol to form new products.The function of CYP76AK1 was further studied.（1）Miltiradiene,ferruginol,11-hydroxy ferruginol,sugiol and 11-hydroxy sugiol were used as substrates for in vitro enzyme assay.The result showed that CYP76AK1 could catalyze 11-hydroxy ferruginol and 11-hydroxy sugiol.Two new compounds with molecular weight of 318 and 316 were produced with CYP76AK1 and 11-hydroxy ferruginol as substrate.CYP76AK1 catalyzed 11-hydroxy sugiol to form a compound with molecular weight of 332,which was identified as 11,20-dihydroxy sugiol by MS and NMR.（2）We further constructed the inducible engineering yeast strain YJ61 and constitutive engineering yeast strain YJ62 to investigate the in vivo function of CYP76AK1 in yeast and identify the compounds with molecular weight of 318 and 316.Both YJ61 and YJ62 could produce 11-hydroxy ferruginol,sugiol,the compounds with molecular weight of 318 and 316,11,20-dihydroxy sugiol and 11-hydroxy sugiol,and the two new compounds with molecular weight of 318 and 316 were the main products.The two new compounds were gained by extracting and separating the fermentation products of YJ62 and identified as 11,20-dihydroxy ferruginol and 10-hydroxymethyl tetrahydromiltirone by MS and NMR.（3）The relative expression level of CYP76AK1 in root,stem and leaf of annual S.miltiorrhiza was analyzed using real-time PCR.The relative expression analysis revealed that CYP76AKl1,CYP76AH1 and CYP76AH3 were more abundant in the root,consistent with the accumulation site of tanshinones.Both the substrates and products of CYP76AK1 were detected in Danshen.These results further identified the key function of CYP76AK1 in the biosynthesis of secondary metabolites in Danshen.¹³C labeled 11,20-dihydroxy ferruginol and 10-hydroxymethyl tetrahydromiltirone were produced by YJ62 with ¹³C labeled glucose.¹³C labeled 11,20-dihydroxy ferruginol was converted to miltirone with protein of salvia miltiorrhiza hairy root,which provided direct evidence for the function of CYP76AK1 in tanshinones biosynthesis.Resules of in vitro enzyme assay and in vivo test showed that CYP76AK1 was involved in the biosynthesis of tanshinones.CYP76AK1 catalyzed 11-hydroxy ferruginol and 11-hydroxy sugiol to form 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol,respectively.11,20-dihydroxy ferruginol was unstable under ambient conditions and underwent spontaneous oxidization to 10-hydroxymethyl tetrahydromiltirone.Here,we studied the functions of three P450s involved in biosynthesis of tanshinones.We expanded the functions of CYP76AH1 and CYP76AH3,and found a new functional P450——CYP76AK1.The complex structural modification of these P450s increased our knowledge of multifunctional P450s.This study laid the foundation for researching the downstream biosynthesis of tanshinones.The engineering yeast strain constructed in this study could provide substrates and chassis bacteria.The experimental methods used in this study could be a represtentative example for the study of tanshinones biosynthesis,and could also be used in research of P450s in other plants with remarkable medicinal value.