LAUSR

Highly prevalent in nature, cytochrome P450 enzymes are powerful biocatalysts for selective C–H functionalization [1]. These heme-containing enzymes activate inert C–H bonds within complex molecules for a plethora of transformations with exquisite regio-, chemoand stereoselectivity. Many plant P450s are of industrial relevance in the production of pharmaceuticals, fragrances, pesticides and vitamins, yet few have been employed commercially [2]. Key examples include the P450 CYP71AV1 used for large-scale production of the antimalarial drug artemisinin, and CYP75 enzymes exploited for their differential hydroxylation abilities to manipulate color patterns in top-selling flowers [3]. These cases illustrate a minuscule portion of the synthetic potential of P450s. Unveiling the molecular basis for selective C–H oxidation in plantderived P450s can facilitate their engineering towards increased commercial applications. However, despite their widespread occurrence [1,2], little is known about the basis for their late-stage selectivity given the limited number of crystal structures available [4]. Contrary to bacterial P450s, which are generally soluble and expressed in high levels [1,3], most plant P450s are found in low yields in native tissues complicating their isolation. Moreover, they are typically membranebound through the N-terminus in the endoplasmic reticulum (Fig. 1A), and insoluble when produced in bacterial expression systems such as Escherichia coli [2]. Other challenges include the need for co-expression of native reductase partners, proper incorporation of the heme cofactor, differences in codon preference and genetic instabilities when large or multiple plasmids are employed [5]. Eukaryotic-based heterologous hosts exist, including Saccharomyces cerevisiae and Pichia pastoris, yet similar challenges remain. In their recent ACS Catalysis article [6], authors Chun Li and coworkers highlight an interesting avenue towards the rational engineering of plant P450s that are difficult to express in vitro. This work deepens our understanding of the selectivity and iterative mechanism involved in the biosynthesis of medicinally relevant molecules. Elucidating the role P450s play in functionalizing plant natural products is fundamental for guiding engineering efforts towards improving the performance of these enzymes. This may include redirecting the selectivity of the biocatalyst, designing self-sufficient P450-redox partner chimeric fusions, allowing the use of light-driven cofactor regeneration processes, or enabling new-to-nature chemical reactions [7]. The work of Chun Li et al. [6] is focused on decoding and finetuning the molecular factors controlling selectivity and iterative oxidation in the CYP72A63 fromMedicago truncatula towards the synthesis of high-value, bioactive licorice triterpenoids. When efforts towards expressing CYP72A63 in E. coli failed, Chun Li et al. engineered a yeast strain to produce 11-oxo-β-amyrin in vivo by introducing the genes responsible for its biosynthesis along with CYP72A63, which was previously determined to synthesize glycyrrhetinic acid from 11-oxo-βamyrin. Although efficient, the P450 does not catalyze formation of glycyrrhetinic acid selectively as other intermediates and by products, including glycyrrhetol, glycyrrhetaldehyde and 29-OH-11-oxo-βamyrin, are also generated. Based on this catalytic promiscuity, Chun Li et al. postulated that key residues in the P450 active site may govern selectivity. Although recombinantly producing plant P450s is challenging, there is an increasing number of strategies to do so (Fig. 1B). Truncating the membrane-anchoring segment can increase the solubility of a P450 as exemplified by Nagano et al. in their work to crystallize the plant CYP90B1 [4]. If this deletion approach adversely impacts the ability of the P450 to couple with CYP reductase for electron transfer, modifying the transmembrane helix is an alternative option [5]. In some cases, the need of P450s for NADPH, which is provided by the reductase, can stress an organism leading to metabolic imbalances. Although often low yielding, this can be resolved by co-overexpressing the reductase or creating the corresponding P450 fusion protein. Co-expressing chaperones to facilitate protein folding can also promote protein expression. Additional approaches include media supplementation and optimization of cultivation parameters such as pH, temperature and