Unlabelled: Ergosterol is widely used in skin care products and drug preparation. Lanosterol 14α-demethylase (Erg11p, 14DM, CYP51) is the rate-limiting enzyme for the biosynthesis of various steroid compounds in Saccharomyces cerevisiae. Herein, Erg11p was engineered to extend the in vivo catalytic half-life and increase the turnover rate. Single mutations resulting in lower folding energy were selected, and mutant P201H had an ergosterol yield of 576.9 mg·L^-1. Through consensus design, single mutations resulting in higher sequence identity to homologs were tested and mutant K352L had an ergosterol yield of 677.9 mg·L^-1. The key residues for substrate binding were confirmed via alanine scanning mutagenesis and mutant F384A had an ergosterol yield of 657.8 mg·L^-1. Molecular dynamics (MD) simulation was conducted to investigate the contributions of pocket residues and eight residues were found to engage in weak interactions with lanosterol. Saturation mutagenesis was applied to these residues to enhance binding to lanosterol, and mutant F384E had an ergosterol yield of 733.8 mg·L^-1. Meanwhile, MD simulations were conducted to assess the impact of mutant F384E on enzyme activity. The results consistently showed that single point mutation F384E had the greatest effect, outperforming the combination mutations. Batch fermentation increased the ergosterol yield of mutant F384E to 3067.5 mg·L^-1, the highest reported to date. The successful engineering of Erg11p may pave the way for industrial-scale production of ergosterol and other steroids.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-024-04136-x.

• PMID: 39554987
• DOI full text
• PMC full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Liang R, Xu K, Wang X, Wei W, Chen Q, Qin Z, Zeng W, Zhou J

Primary Lit For

Additional Lit For

Review For