Zhenbo Yuan

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.

C ₁₉ H ₁₆ BrF ₂ NO ₂ , monoclinic, C 2/ c (no. 15), a = 29.4837(7) Å, b = 7.9890(2) Å, c = 20.0516(5) Å, β = 130.448(1)°, V = 3594.23(16) Å ³ , Z = 8, R _gt ( F ) = 0.0558, wR _ref ( F ² ) = 0.1626, T = 296(2) K.

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.

A bioinspired one-pot one-step photoenzymatic cascade integrating a metal-free photocatalyst and an engineered bifunctional arylformidase directly convert l -Trp and its esters into l -Kyn under mild conditions with excellent green metrics evaluation.

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.

Dearomatization offers a powerful route towards three-dimensional spirocyclic architectures, which are pivotal in bioactive molecules and pharmaceuticals, particularly for enantioselective construction of congested quaternary spirocenters. However, biocatalytic dearomatization towards these attractive spirocyclic frameworks under mild and green conditions remain underexplored. Here, we report a photoenzymatic platform synergizing visible-light activation with engineered nicotinamide-dependent ketoreductase (KRED) catalysis to achieve highly enantioselective dearomative spirocyclization of N-hydroxyphthalimide esters. Semi-rational engineering of KRED P2-D12 enables the synthesis of diverse chiral spirocyclohexadienones, also including the challenging ortho-cyclohexadienones, with exceptional functional group tolerance and stereocontrol (up to >99% ee). The utility of this methodology is underscored significantly by the enantioselective total synthesis of the cytotoxic natural product (+)-denobilone A (93% ee). Overall, this work establishes a chemobiocatalytic platform for the synthesis of complex and chiral spiroarchitectures with high enantioselectivity, expanding the biocatalytic toolbox in sustainable synthetic chemistry.

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