Screening And Functional Analysis Of The Genes Involved In Diosgenin Biosynthesis In Dioscorea Zingiberensis

Diosgenin serves as the precursor of more than 200 steroid hormone drugs in the world,and it shows diversely medicinal effects,such as anti-cancer and prevention of nervous diseases.The annual demand for diosgenin in the world-wide market is above 2,000 tons,which at present are predominantly supplied by China and Mexico.Although diosgenin is synthesized in several monocot and eudicot species,the monocotyledonous plant Dioscorea zingiberensis is the only source for the industrial production of diosgenin in China.In the late 1960 s,it was found that diosgenin is derived from cholesterol,and the conversion from cholesterol to diosgenin involves successive oxidation at its carbon 22,16 and 26 positions,respectively.In 2019,the P450 enzymes responsible for diosgenin biosynthesis in the dicotyledonous plant fenugreek have been identified by the scientists of the United States.However,before this study,the genes involved in diosgenin biosynthesis in the Dioscorea zingiberensis have not been identified yet,and it is unclear whether the biosynthetic pathways leading to diosgenin are different between the monocot and eudicot species.In order to address the scientific questions mentioned above,the Dioscorea zingiberensis was investigated in this study for the identification and functional characterization of the P450-encoding genes that participate in diosgenin biosynthesis in it through biochemical and structural biology techniques.The findings of this study are summarized as follows:1.Identification of the P450 s responsible for diosgenin biosynthesis in the Dioscorea zingiberensisWith a focus on the 19 P450 candidates that were positively correlated with diosgenin synthesis in the Dioscorea zingiberensis,which was previously discovered by our laboratory,this study embarked on the functional analysis of these P450 candidates under the cholesterol-producing yeast strain RH6829 background.Finally,we have found that among the 19 candidates,four P450 s,namely CYP90B71,CYP90G6,CYP94N8 or CYP94D143,are involved in the conversion of cholesterol to diosgenin in the Dioscorea zingiberensis.Starting with cholesterol as the substrate,CYP90B71 and CYP90G6 successively catalyze the 22R-and 16S-hydroxylating reactions,respectively,and CYP94N8 or CYP94D143 is responsible for the C26-oxidation during the biosynthesis of diosgenin.2.Comparing of the biosynthetic steps in the conversion from cholesterol to diosgenin between the monocot and eudicot plantsThrough analyzing the biochemical functions of the P450 enzymes that participate in the biosynthesis of diosgenin in the Dioscorea zingiberensis,we have found that 22R-hydroxycholesterol and 16 S,22R-dihydroxycholesterol are the intermediates of diosgenin in the monocotyledonous plants.However,this finding is inconsistent with the previous report with the dicotyledonous plant fenugreek,in which the authors believed that 22S-hydroxycholesterol and16 S,22S-dihydroxycholesterol are the precursors of diosgenin.In order to explore whether there are any differences in the biosynthetic steps toward diosgenin biosynthesis between the monocot and the eudicot,the diosgenin-producing P450 genes isolated from both the monocotyledonous plant Dioscorea zingiberensis and the dicotyledonous species fenugreek were expressed in the same yeast strain WAT11,and their biochemical functions were carefully investigated using the yeast microsome-based in vitro assays.It was found that the diosgenin biosynthetic steps in the both species are essentially the same,using 22R-hydroxycholesterol and16 S,22R-dihydroxycholesterol as the intermediates of diosgenin.Surprisingly,our data suggested that 16 S,22S-dihydroxycholesterol,which was previously proposed to be an intermediate of diosgenin by others,is highly likely a by-product of the diosgenin synthesis pathway.3.Structural basis explaining the functional evolution of diosgenin-producing CYP90sThe P450 enzymes responsible for the conversion of cholesterol to diosgenin belong to the CYP90 family.As a member of the CYP90 family in the brassinosteroid pathway,the CYP90B1 from Arabidopsis thaliana has been successfully investigated for its crystal structure in 2019.In this study,we have discovered that CYP90B1 shows the activity of catalyzing both 22R-and22S-hydroxylating reactions on the substrate cholesterol.Using the crystal structure of CYP90B1 as a template,the protein structures of CYP90B71(22R-hydroxylase)and CYP90G6(16S-hydroxylase)were simulated,and the key residues lining the substrate-binding sites in their catalytic cavities were predicted.Through the directional mutagenesis of the substrate binding sites,we have found that the specific 22R-and 16S-hydroxylation activities required for diosgenin biosynthesis are controlled by these substrate binding sites,and these activities can be acquired by rational mutations on the CYP90 s that occur in other metabolic pathways.The data that we have obtained from the mutagenesis experiment may echo the evolutional trajectory of the diosgenin-producing CYP90 s in nature.For example,we speculate that CYP90B71 is likely to evolve from CYP90s(such as CYP90B1)in the brassinosteroid synthesis pathway,and during the evolutionary process its22S-hydroxylation activity was lost,whereas its 22R-hydroxylation activity was retained,which led to the formation of a specific cholesterol 22R-hydroxylase.