Function Verification of Genes Involved in 22(R)-hydroxycholesterol Biosynthesis in Veratrum nigrum and Their Heterologous Synthesis
[Objective]22(R)-hydroxycholesterol is an important precursor of veratrol alkaloid biosynthesis.The function of cholesterol C-22 hydroxylase in Veratrum nigrum was verified by heteroexpression,and the yeast chassis was constructed to produce 22(R)-hydroxycholesterol,which lays a foundation for deciphering the biosynthetic pathway of veratrol alkaloid.[Method]Based on the transcriptome data of V.nigrum,three full-length CYP90B subfamily gene sequences were cloned from the root of V.nigrum,the yeast Y33 expression vector was constructed and transferred into the cholesterol yeast chassis for functional verification.The yeast fermentaed products in shake flask were detected by high performance liquid chromatography and liquid chromatography-mass spectrometry.The enzyme VnCYP90B27-1 with cholesterol C-22 hydroxylase catalytic function was screened.VnCYP90B27-1 was integrated into the yeast chromosome by multi-fragment assembly,homologous recombination and lithium acetate conversion to construct a yeast chassis for 22(R)-hydroxycholesterol.[Result]RT-qPCR showed that the expressions of the three candidate genes in the roots and leaves was consistent with the trend of transcriptome expression.The expression of VnCYP90B27-1 in the roots was significantly higher than that in the leaves.The results of phylogenetic tree showed that VnCYP90B27-1 had high homology with VcCYP90B27 of Veratrum californicum and VnCYP90B27 of V.nigrum,and belonged to CYP90B subfamily.In addition,the results of heterologous expression in the yeast showed that VnCYP90B27-1 had the function of cholesterol C-22 hydroxylase,and successfully achieved the heterologous synthesis of 22(R)-hydroxycholesterol in S.cerevisiae,with the yield of(5.37±0.37)mg/L in shake flask.[Conclusion]The function of 22(R)-hydroxycholesterol synthesis gene VnCYP90B27-1 from V.nigrum is successfully cloned and verified.The 22(R)-hydroxycholesterol yeast chassis is constructed.It is proved that the catalytic activity of VnCYP90B27-I in S.cerevisiae is higher than that of VcCYP90B27 from V.californicum,which provides genetic resources for heterologous synthesis of steroid alkaloids.
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