Permana Dani, Niesel Ksenia, Ford Mark James, Ichinose Hirofumi
Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Research Center for Environmental and Clean Technology, The National Research and Innovation Agency of the Republic of Indonesia (BRIN), Bandung Advanced Science and Creative Engineering Space (BASICS), Jl. Cisitu, Bandung 40135, Indonesia.
ACS Omega. 2022 Apr 18;7(16):13932-13941. doi: 10.1021/acsomega.2c00430. eCollection 2022 Apr 26.
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 (TeCYPs). We identified 48 TeCYP genes, including two putative pseudogenes, from the whole-genome sequence of . Furthermore, we constructed a functional library of TeCYPs and heterologously expressed 46 TeCYPs in . Recombinants of 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.
细胞色素P450单加氧酶（P450s）是自然界中普遍存在且具有高丰度和多样性的酶。真菌提供了种类繁多且复杂的P450s，这些酶在各种次级代谢过程中发挥着重要作用。除了P450s对真菌生命的生理影响外，它们的多种功能在生物技术领域的先进应用中具有吸引力。在此，我们报告了接合菌纲真菌（TeCYPs）中P450s的基因鉴定和功能表征。我们从[具体真菌名称]的全基因组序列中鉴定出48个TeCYP基因，包括两个推定的假基因。此外，我们构建了TeCYPs的功能文库，并在[具体宿主名称]中异源表达了46个TeCYPs。然后将[具体宿主名称]的重组体用作全细胞生物催化剂，用于各种化合物的生物转化。通过功能组学研究，对一系列化合物（包括甾体底物）的转化证明了各种TeCYPs的催化潜力。值得注意的是，发现CYP5312A4对睾酮具有高活性。基于核磁共振分析，证明了CYP5312A4将睾酮酶促转化为14α-羟基睾酮。这是首次报道鉴定出一种新型真菌P450，它催化睾酮的14α-羟基化反应。此外，我们使用各种底物探索了TeCYPs的潜在潜力。本研究提供了一个平台，以进一步研究TeCYPs在制药、农业和生物技术产业中作为催化剂的潜在用途。
