Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton.

The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual two-step mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

Synthetic Biology-based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities.

The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.

Biosynthesis of Natural and Unnatural Perylenequinones for Drug Development.

A wide range of perylenequinones (PQs) with diverse structures and versatile bioactivities have long been isolated, positioning them as highly promising agents for photodynamic therapy (PDT). However, the lack of an efficient and cost-effective method to obtain these compounds and to introduce structural diversity and complexity currently hinders their further research and application. In this concept, we present a comprehensive overview of the advancements in the biosynthetic pathways of natural PQs based on their structural classification, and also summarize recent progress in the biosynthesis of natural PQs and derivatives. These pioneering efforts may pave the way for structure modification and large-scale bioproduction of natural and unnatural PQs through synthetic biology strategies to promote their drug development.

Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids.

Plant-derived alkaloids are an important class of pharmaceuticals. However, they still rely on phytoextraction to meet their diverse market demands. Since multistep biocatalytic cascades have begun to revolutionize the manufacture of natural or unnatural products, to address the synthetic challenges of alkaloids, herein we establish an artificially concise four-enzyme biocatalytic cascade with avoiding plant-derived P450 modification for synthesizing phenethylisoquinoline alkaloids (PEIAs) after enzyme discovery and enzyme engineering. Efficient biosynthesis of diverse natural and unnatural PEIAs is realized from readily available substrates. Most importantly, the scale-up preparation of the colchicine precursor (S)-autumnaline with a high titer is achieved after replacing the rate-limiting O-methylation by the plug-and-play strategy. This study not only streamlines future engineering endeavors for colchicine biosynthesis, but also provides a paradigm for constructing more artificial biocatalytic cascades for the manufacture of diverse alkaloids through synthetic biology.

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13.

Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.