Yan Zhang

An ATP-free, single-strain, one-pot whole-cell cascade couples PPi-driven phosphorylation with H ₂ O ₂ -managed oxidation, delivering 69.8 g L ⁻¹ PLP at 4.36 g L ⁻¹ h ⁻¹ while simplifying PLP bioconversion.

Mogroside VI (Mog VI) is a rare triterpene glycoside from Siraitia grosvenorii with promising bioactivities. However, its biosynthesis is limited by a single rate-limiting glycosylation step converting mogroside V, catalyzed by the inherently low-activity plant glycosyltransferase UGT73–327–2. In this study, we applied a structure-guided engineering strategy to overcome this catalytic bottleneck. By combining substrate-channel expansion with catalytic pocket remodeling, the double mutant W192F/K206E was generated, showing a 22.2-fold increase in catalytic activity. Molecular dynamics simulations and kinetic analyses indicated that the enhanced performance results from an enlarged substrate-access channel, improved substrate-binding stability, and a more favorable active-site geometry that reduces key catalytic distances. Furthermore, coupling the engineered UGT with Arabidopsis thaliana sucrose synthase enabled an in situ UDP-glucose regeneration system, achieving a Mog VI titer of 5.6 g·L–1 with a 76.8% molar conversion. This work establishes an efficient biocatalytic route for Mog VI production and highlights the potential of structure-based glycosyltransferase engineering for the synthesis of rare natural glycosides.

Enhancing the thermostability of UDP-glycosyltransferases is essential for their industrial application in natural product biosynthesis. Here, we developed a structure-guided, computation-assisted strategy to improve the stability and efficiency of UGT94B1_M0, which catalyzes the conversion of rebaudioside A to the high-value sweetener rebaudioside D. By integrating multiple computational tools and energy-based analyses, a focused mutation library was constructed and experimentally screened. The optimal variant, UGT94B1_M3, displayed an 8.40 °C higher melting temperature, a 20.65-fold longer half-life, and a 1.45-fold enhancement in catalytic efficiency relative to M0. Molecular dynamics simulations revealed that these improvements were associated with increased structural rigidity and favorable electrostatic interactions. When coupled with Arabidopsis thaliana sucrose synthase for UDP-glucose regeneration, M3-AtSuSy produced 33.87 mM Reb D within 1 h at a molar conversion rate of 84.67%, 2.81-fold higher than M0-AtSuSy. This work establishes a generalizable strategy for thermostability engineering of UDP-glycosyltransferases toward efficient and sustainable glycoside biosynthesis.

Consecutive two-step oxidations catalyzed by a single cytochrome P450 are often reported in the biosynthesis of plant natural products (PNPs). However, due to unbalanced catalytic activity, intermediates and byproducts are always substantially accumulated, creating a major bottleneck in the efficient heterologous synthesis of many important PNPs. Despite this, efforts to balance these consecutive two-step oxidations to enhance the production of final PNPs are often overlooked. Here, guided by rational design, we engineered RoCYP01 by modulating substrate/product channels, enzyme-substrate interactions, the electron transfer chain, and O₂/H₂O exchange tunnels, effectively rebalancing the consecutive two-step oxidations from R-CH₃ to R-CH₂OH to R-COOH. Consequently, the product, betulinic acid, was greatly improved in an engineered yeast strain, substantially reducing the accumulation of intermediates. Thus, this work establishes a mechanism-driven strategy to rebalance consecutive two-step oxidations, providing a potential strategy for the efficient biosynthesis of other PNPs that involve P450-catalyzed consecutive two-step oxidations.

Sustainable and scalable production of plant natural products remains a major challenge at the interface of synthetic biology, metabolic engineering, and drug discovery, often constrained by plant-membrane-bound cytochrome P450s. To overcome the bottleneck in P450-catalyzed methylenedioxy-bridge (MDB) formation, we designed a concise, modular Escherichia coli pathway to the alkaloid berberine that bypasses multiple native steps and enables high-level accumulation of the P450-dependent intermediate. We then implemented integrated engineering of CjCYP719A1, including expression optimization, structure-guided design, consensus mutagenesis, and machine learning, to obtain a variant with 8.9-fold higher activity and improved thermostability. This enabled a berberine titer of 353 mg/L in scaled-up preparations, the highest reported to date, demonstrating industrial potential. Furthermore, mechanistic studies reveal their improved catalytic activity and thermostability. Together, this work provides a practical framework for functional expression and systematic optimization of plant-derived P450s to enable efficient production of plant natural products in E. coli.

Precise three-dimensional arrangements of atoms and functional groups govern molecular reactivity and biological function, making stereocontrol a central goal in modern organic and medicinal chemistry. Yet reconciling broad substrate generality with high enantioselectivity remains a core limitation of enantioselective catalysis, motivating the pursuit of privileged chiral catalysts. Here we design a new-to-nature photoenzyme that unlocks enzymatic deracemization of structurally diverse allenes, an entropically disfavored process inaccessible to natural biocatalysts. Structure-guided design created substrate-specific interaction networks, delivering high enantioenrichment for axially chiral allenic carboxylic acids, esters, and amides via triplet energy transfer under aerobic conditions. X-ray crystal structures of enzyme–substrate complexes reveal tailored chiral pockets that accommodate distinct substrates and direct enantiocontrol, rationalizing the breadth and selectivity observed. This work reconciles generality with high selectivity on an enzymatic platform, establishes deracemization as an evolvable route to axially chiral molecules, and broadens activation strategies for stereochemical editing.

Efficient biosynthesis of Reb M from substrates by designing a concise biosynthetic pathway.

Spirocyclic architectures, which feature two rings sharing a single atom, are common in natural products and exhibit beneficial biological and material properties. Due to the significance of these architectures, biocatalytic dearomative spirocyclization has recently emerged as a powerful approach for constructing three-dimensional spirocyclic frameworks under mild, sustainable conditions and with exquisite stereocontrol. This review surveys the latest advances in biocatalyzed spirocyclization of all-carbon arenes (phenols and benzenes), aza-aromatics (indoles and pyrroles), and oxa-aromatics (furans). We highlight cytochrome P450s, flavin-dependent monooxygenases, multicopper oxidases, and novel metalloenzyme platforms that effect regio- and stereoselective oxidative coupling, epoxidation/semi-pinacol rearrangement, and radical-mediated cyclization to produce diverse spirocycles. Mechanistic insights gleaned from structural, computational, and isotope-labeling studies are discussed where necessary to help the readers further understand the reported reactions. Collectively, these examples demonstrate the transformative potential of biocatalysis to streamline access to spirocyclic scaffolds that are challenging to prepare through traditional methods, underscoring biocatalysis as a transformative tool for synthesizing pharmaceutically relevant spiroscaffolds while adhering to green chemistry paradigms to ultimately contribute to a cleaner and more sustainable future.

The creation of enzymes with abiological abilities offers exciting opportunities to access new-to-nature biocatalysis beyond that found in nature. Here, we repurpose a novel protein scaffold, CTB10, as an artificial photoenzyme through genetic code expansion. It enables catalytic deracemization of cyclopropane, a process that remains inaccessible to traditional biocatalysis due to its thermodynamically unfavorable nature. Following structural optimization through directed evolution, a broad substrate scope with high enantioselectivities is achieved. Furthermore, the crystal structure of the CTB10-based photoenzyme-substrate complex well demonstrates how the catalytic chiral cavity is sculpted to promote efficient and selective light-enabled deracemization. Therefore, this study unlocks the potential for achieving challenging deracemization through biocatalysis.