Characterization and structure‐guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

Abstract

Summary One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non‐activated C‐H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP 109Q5 from Chondromyces apiculatus DSM 436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β‐ionone in non‐allylic positions. The crystal structure of CYP 109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure‐guided approach to engineering the enzyme. Some variants of CYP 109Q5 displayed a switch in selectivity towards the non‐allylic positions of β‐ionone, allowing the simultaneous production of 2‐ and 3‐hydroxy‐β‐ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole‐cell system finally enabled the production of up to 0.5 g l −1 hydroxylated products of β‐ionone; this system can be applied to product identification in further biotransformations. Overall, CYP 109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP 109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.

A Career in Catalysis: Bernhard Hauer

“…The group also constructed a highly active whole-cell catalyst from E. coli that expresses CYP1232A24 and forms up to 1.77 g/L of 3,4-dihydroxyphenylacetic acid. In another study, five P450s were isolated from the myxobacterium Chondromyces apiculatus , two of which were further characterized . Both enzymes are capable of accepting a wide range of substrates and can even functionalize the β-ionone scaffold in non-allylic positions.…”

“…In another study, five P450s were isolated from the myxobacterium Chondromyces apiculatus, two of which were further characterized. 133 Both enzymes are capable of accepting a wide range of substrates and can even functionalize the β-ionone scaffold in non-allylic positions. A targeted mutant library of CYP109Q5 further shifted the selectivity to non-allylic positions and allowed for the first time the simultaneous synthesis of products 2-and 3hydroxy-β-ionone, which are important for carotenoid synthesis.…”

Cytochrome P450 Monooxygenases Catalyse Steroid Nucleus Hydroxylation with Regio‐ and Stereo‐Selectivity

“…1 and 3 were converted at moderate conversion. The crystal structure of CYP109Q5 (6GMF) revealed flexibility within the active site pocket, permitting the accommodation of bulky substrates [73] …”

Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

“…These enzymes are broadly distributed in all life kingdoms having the ability to catalyse a vast number of chemically challenging reactions, making them powerful biocatalytic tools for biotechnology and synthetic biology (Bernhardt and Urlacher, 2014; Urlacher and Girhard, 2019). Cytochromes P450 act on a wide range of structurally different substrates, such as fatty acids, alkanes, steroids, vitamins, antibiotics, and diverse xenobiotics and drugs, playing key roles in biosynthesis and detoxification of a huge array of compounds (Van Bogaert et al ., 2011; Peschke et al ., 2016; Bhattacharya and Yadav, 2018; Ichinose and Kitaoka, 2018; Felpeto‐Santero et al ., 2019; Klenk et al ., 2019). Electron equivalents required in cytochrome P450 reactions are usually derived from NAD(P)H through an electron transfer chain represented by two main class of redox partners (Munro et al ., 2007; McLean et al ., 2015).…”