Development of a P450 Fusion Enzyme for Biaryl Coupling in Yeast

Abstract

Despite the diverse and potent bioactivities displayed by axially chiral biaryl natural products, their application in drug discovery is limited by restricted access to these complex molecular scaffolds. In particular, fundamental challenges remain in controlling the site- and atroposelectivity in biaryl coupling reactions. In contrast, Nature has a wealth of biosynthetic enzymes that catalyze biaryl coupling reactions with catalyst-controlled selectivity. In particular, a growing subset of fungal P450s have been identified to catalyze site- and atroposelective biaryl couplings. Herein, we optimize a whole-cell biocatalytic platform in Pichia pastoris to synthesize biaryl molecules through the recombinant production of the fungal P450 KtnC. Moreover, engineering redox self-sufficient fusion enzymes further improves the efficiency of the system. Altogether, this work provides a platform for biaryl coupling reactions in yeast that can be applied to engineering a currently underexplored pool of fungal P450s into selective biocatalysts for the synthesis of complex biaryl compounds.

Fusion Enzymes Involved in Biosynthetic Tailoring Reactions in Fungi

“…[71] Enzyme fusion has been widely used as one of the most promising approaches for enzyme engineering and optimization in academia and industry. For example, the fusion of cytochrome P450s with their redox partners [72][73][74] and Baeyer-Villiger monooxygenases (BVMOs) with alcohol dehydrogenases [75][76] have been extensively used for the efficient synthesis of valuable chemicals. However, the artificial fusion of different enzymes together remains challenging.…”

Recent advances in oxidative phenol coupling for the total synthesis of natural products

“…Recently, the fungal P450 KtnC was also expressed in yeast to accomplish the in vivo biosynthesis of P -orlandin ( 8i ) by Narayan and co-workers. 67…”

Radical-Mediated Regiodivergent C(sp3)–H Functionalization of N-Substituted Indolines via Enzymatic Carbene Transfer

“…[13][14][15][16][17][18][19][20][21] In the area of C-H functionalization, various classes of oxidizing enzymes, including cytochromes P450, flavin-dependent halogenases and non-heme iron-dependent dioxygenases and halogenases, have proven useful for the selective functionalization of aromatic and aliphatic C-H bonds in small molecules. [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] Furthermore, through protein engineering, regiodivergent selectivity has been achieved, for example, for the halogenation of aromatic compounds using evolved flavin-dependent halogenases 22,[29][30] or for late-stage C(sp 3 )-H hydroxylation of complex natural products using engineered P450 enzymes and halogenases (Figure 1B), [31][32][33][34][35][36][37][38] thus creating new opportunities for the diversification and/or chemoenzymatic synthesis of these molecules. More recently, important progress has been made also in the development of biological catalysts for C-H functionalization via 'non-native' chemistry such as carbene transfer catalysis.…”