Kundral Simran, Giang Peter D, Grundon Leah R, Supper Jenna M, Khare Sunil K, Bernhardt Paul V, Evans Paul, Bell Stephen G, De Voss James J
The University of Queensland, Australia - Indian Institute of Technology Delhi Research Academy (UQIDRA), Delhi, India.
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072, Australia.
Appl Microbiol Biotechnol. 2025 May 31;109(1):133. doi: 10.1007/s00253-025-13521-2.
Cytochrome P450 enzymes (P450s) have gained significant attention due to their remarkable ability to oxidise unactivated C-H bonds with high regio- and stereoselectivity. Their industrial utility is often limited by challenges such as low stability, poor expression, and dependence on elusive redox partners. These issues have driven the search for more robust P450s, especially those that are inherently stable under extreme conditions typical of industrial processes. "Self-sufficient P450s" in which the P450 heme domain is naturally fused to redox domains in a single polypeptide chain eliminates the need to identify and separately express required redox partners. Furthermore, P450s from thermophilic organisms are more temperature tolerant with fewer stability issues. This study presents a self-sufficient P450, CYP116B305, identified from metagenomically assembled genomes from Innot Hot Springs (71 °C), located in North Queensland, Australia. CYP116B305 was heterologously expressed in Escherichia coli and purified using standard protocols. Investigation of the thermal stability of CYP116B305 revealed a robust heme domain with a T value of 56.9 ± 0.1 °C, while the reductase domain exhibited slightly lower stability, with a T value of 52.5 ± 0.5 °C. Further characterisation revealed that CYP116B305 efficiently bound to and hydroxylated 2-hydroxyphenylacetic acid (2-HPA) at the C-5 position, yielding homogentisic acid. The catalytic parameters, including the coupling efficiency and rate of electron transfer from the NADPH cofactor to the P450 heme, were shown to improve at an elevated temperature (45 °C) compared to 25 °C. The combination of the self-sufficiency and improved stability makes CYP116B305 a promising platform for biotechnological applications and biocatalyst engineering. KEY POINTS: • Hot spring metagenomics reveals thermostable P450s of biocatalytic value. • CYP116B305 shows enhanced catalytic activity at elevated temperature (45 °C). • CYP116B305 is a promising platform enzyme for diverse biotechnological use.
细胞色素P450酶（P450s）因其能够以高区域选择性和立体选择性氧化未活化的C-H键的卓越能力而备受关注。它们在工业上的应用常常受到诸如稳定性低、表达不佳以及依赖难以捉摸的氧化还原伙伴等挑战的限制。这些问题促使人们寻找更强大的P450s，特别是那些在工业过程典型的极端条件下具有固有稳定性的P450s。“自给自足的P450s”，即P450血红素结构域在单条多肽链中与氧化还原结构域自然融合，消除了识别和分别表达所需氧化还原伙伴的必要性。此外，来自嗜热生物的P450s具有更高的温度耐受性，稳定性问题更少。本研究展示了一种自给自足的P450，CYP****116B305，它是从位于澳大利亚北昆士兰的伊诺特温泉（71°C）的宏基因组组装基因组中鉴定出来的。CYP116B305在大肠杆菌中进行了异源表达，并使用标准方案进行了纯化。对CYP116B305热稳定性的研究表明，其血红素结构域具有很强的稳定性，T值为56.9±0.1°C，而还原酶结构域的稳定性略低，T值为52.5±0.5°C。进一步的表征显示，CYP116B305能有效地结合2-羟基苯乙酸（2-HPA）并在其C-5位进行羟基化反应，生成尿黑酸。与25°C相比，包括偶联效率和电子从NADPH辅因子转移到P450血红素的速率在内的催化参数在升高的温度（4°C）下有所改善。自给自足和稳定性提高的结合使CYP116B305成为生物技术应用和生物催化剂工程的一个有前景的平台。要点：•温泉宏基因组学揭示了具有生物催化价值的耐热P450s。•CYP116B305在升高的温度（45°C）下显示出增强的催化活性。•CYP116B305是一种有前景的用于多种生物技术用途的平台酶。
