Latent Functions and Applications of Cytochrome P450 Monooxygenases from Thamnidium elegans: A Novel Biocatalyst for 14α-Hydroxylation of Testosterone

Abstract

Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with high availability and diversity in nature. Fungi provide a diverse and complex array of P450s, and these enzymes play essential roles in various secondary metabolic processes. Besides the physiological impacts of P450s on fungal life, their versatile functions are attractive for use in advanced applications of the biotechnology sector. Herein, we report gene identification and functional characterization of P450s from the zygomycetous fungus Thamnidium elegans (TeCYPs). We identified 48 TeCYP genes, including two putative pseudogenes, from the whole-genome sequence of T. elegans . Furthermore, we constructed a functional library of TeCYPs and heterologously expressed 46 TeCYPs in Saccharomyces cerevisiae . Recombinants of S. cerevisiae were then used as whole-cell biocatalysts for bioconversion of various compounds. Catalytic potentials of various TeCYPs were demonstrated through a functionomic survey to convert a series of compounds, including steroidal substrates. Notably, CYP5312A4 was found to be highly active against testosterone. Based on nuclear magnetic resonance analysis, enzymatic conversion of testosterone to 14α-hydroxytestosterone by CYP5312A4 was demonstrated. This is the first report to identify a novel fungal P450 that catalyzes the 14α-hydroxylation of testosterone. In addition, we explored the latent potentials of TeCYPs using various substrates. This study provides a platform to further study the potential use of TeCYPs as catalysts in pharmaceutical and agricultural industries and biotechnology.

Biocatalyst collection and heterologous expression of sesquiterpene synthases from basidiomycetous fungi: Discovery of a novel sesquiterpene hydrocarbon

“…Polymorphic differences might be common in basidiomycetes (Bezmenova et al, 2020; Ide et al, 2012). Several candidates were amplified as immature cDNAs whose open reading frame was shifted by intronic sequences, which were incorrectly spliced; filamentous fungi may sometimes allow a deleterious splicing event (Ide et al, 2012; Nazir et al, 2010; Permana et al, 2022). In addition, 31 candidate genes were not amplified by RT‐PCR.…”

A Modular Chemoenzymatic Approach to C14-Functionalized Steroids

“…With the best mutants, each substrate's the catalytic specificity and conversion can reach 83-94% and 62.1-78.5% respectively by a 6-12 hrs biotransformation. Taken together, these engineered P450 hydroxylases were far better than the previously reported biocatalysts [21][22][23] in regiospecificity, substrate promiscuity and catalytic reactivity, which enabled a highly efficient and practical biocatalytic way to the synthesis of C14α-hydroxylated steroids with a C17 substitution.…”

“…A few filamentous fungi and two P450 hydroxylases have been identified with steroidal C14-H α-hydroxylation activity. [16][17][18][19][20][21][22][23] These biocatalysts, especially the P450 enzymes overexpressed in Saccharomyces cerevisiae are very attractive in the preparation of C14α-OH steroids due to the easy operation, low cost and high step-economy features. However, their poor regiospecificity and low catalytic reactivity for the steroids with a C17-side chain, [21][22][23] constitute a significant obstacle to their broad utility.…”

“…[16][17][18][19][20][21][22][23] These biocatalysts, especially the P450 enzymes overexpressed in Saccharomyces cerevisiae are very attractive in the preparation of C14α-OH steroids due to the easy operation, low cost and high step-economy features. However, their poor regiospecificity and low catalytic reactivity for the steroids with a C17-side chain, [21][22][23] constitute a significant obstacle to their broad utility. Thus, the aforementioned limitations of the state-of-the-art methods severely impede their applications in preparing C14-functionalized steroids and the downstream biological and pharmaceutical studies.…”

“…For instance, although the C17 substitution is pivotal for most of the bioactive steroids and related medicines, currently there are lack of efficient C14α-hydroxylases for these C17substituted steroidal substrates. [16][17][18][19][20][21][22][23] In addition, at the ∆14 olefin hydrofunctionalization stage, a complicated chemoselectivity issue may arise from the reactive functional groups of the A/B/C rings and side chain. 35 Herein, we describe a modular chemoenzymatic approach to access C14-functionalized steroids.…”