To clone the full-length cDNA of CYP6620B1 gene, which is a key gene in the downstream pathway of triterpenoid synthesis in S. baumii and perform bioinformatics analysis and eukaryotic expression analysis of the gene, with a view to providing a theoretical basis for the elucidation of the mechanism of triterpenoid biosynthesis pathway of S. baumii and the enhancement of triterpenoid production.

The transcript level of CYP6620B1 gene was detected by qRT-PCR at different developmental stages of the mycelium, protocorm and zygote of S. baumii. The full-length cDNA sequence of the target gene was obtained by PCR amplification, and the physicochemical properties and spatial structure of the protein were analyzed by bioinformatics online software. The recombinant Saccharomyces cerevisiae strain INVSc/pYES2-CYP6620B1 was obtained by LiAC/PEG method, and the total triterpene content of it was detected by microplate reader.

The transcript level of CYP6620B1 gene in different developmental stages of S. baumii showed a trend of increasing and then decreasing, which coincided with the reported trend of total triterpenoids in S. baumii, suggesting that it might be involved in the regulation of triterpenoid synthesis, and the full-length cDNA of CYP6620B1 was cloned and obtained as 1 629 bp, encoding 542 amino acids, without signal peptide and transmembrane structure, and the sub The results of phylogenetic tree showed that the CYP6620B1 protein of Taiwanofungus camphoratus had the highest homology with that of S. baumii, and the molecular docking results showed that the CYP6620B1 protein had the lowest binding energy and good docking conformation with lanosterol; the CYP6620B1 gene was successfully expressed in the Saccharomyces cerevisiae strain INVSc, and the total triterpene content of recombinant Saccharomyces cerevisiae strain INVSc/pYES2-CYP6620B1 was significantly higher than that of the control strain.

In this study, the CYP6620B1 gene, which may be involved in the triterpene synthesis pathway of S. baumii, was successfully cloned and analysed, which lays the foundation for an in-depth investigation of the gene's function in the triterpene biosynthesis pathway of S. baumii.

This study aimed to clone and explore the differential expression of the C-24 sterol methyltransferase gene (ERG6), which is the key gene involved in the ergosterol biosynthesis pathway of S. baumii, to characterize the role of ERG6 in ergosterol biosynthesis pathway and provide a theoretical basis for obtaining the ergosterol high-yielding strains by biotechnology.

The full-length cDNA of ERG6 was cloned by PCR amplification and subsequently sequenced, and the prokaryotic expression of ERG6 was detected by SDS-PAGE. The sequence of the ERG6 promoter was cloned and analyzed by bioinformatics software. qRT-PCR was used to analyze the transcriptional level of ERG6 in S. baumii at different developmental stages. The content of ergosterol was determined by the spectrophotometer.

The full-length cDNA of ERG6 was successfully cloned. SDS-PAGE showed that the target ERG6 protein could be successfully expressed in the prokaryotic system, which expression was significantly higher than other proteins. At the same time, it was found that the protein expression increased gradually with the increase of induction time. Bioinformatics analyzed and found the gene sequence of ERG6 was 996 bp, encoding 331 amino acids, which had 42 potential phosphorylation sites and no signal peptides as well as transmembrane structures. The results of promoter sequence analysis showed that the ERG6 promoter had a core promoter region and 4 transcription factors that may be involved in its expression regulation. In addition to the typical promoter action elements, it also had a variety of response elements such as methyl jasmonate, gibberellin and abscisic acid. The results of fluorescence quantification and ergosterol content determination showed that the expression of ERG6 was positively correlated with the changing trend of the ergosterol content at different development stages.

ERG6 plays a positive regulatory role in ergosterol biosynthesis, which lays a foundation for the cultivation of ergosterol high-yielding strains in S. baumii.