Zhengshan Luo

Mogrosides are intensely sweet cucurbitane-type triterpenoid glycosides produced by Siraitia grosvenorii, but their heterologous biosynthesis is constrained by inefficient squalene epoxidation and utilization of epoxidized intermediates. Here, enzyme engineering, metabolic engineering, and fermentation optimization were integrated to enhance 2,3;22,23-diepoxysqualene (SDO) production and achieve the first quantitative biosynthesis of 24,25-epoxycucurbitadienol (24,25-ECD). Screening plant-derived squalene epoxidases (SQEs) and engineering the substrate-binding pocket of SgSQE1 yielded variants P353A and L410A, which supported detectable SDO production at 5.83 and 5.14 mg/L, respectively. To enhance the conversion of epoxidized substrates toward 24,25-ECD, substrate-pocket engineering of cucurbitadienol synthase (SgCBS) yielded the F553G variant, increasing 24,25-ECD accumulation by 42.86% relative to the wild type. At the pathway level, BTS1 deletion, ERG9-SgERG1 fusion, and COQ1 promoter replacement further increased SDO production to 42.12 mg/L. Finally, optimization of dissolved oxygen during fed-batch fermentation enabled 24,25-ECD production to reach 522.27 mg/L in a 5-L bioreactor. This study integrates precursor supply, substrate utilization, and process optimization to improve 24,25-ECD biosynthesis, providing a foundation for microbial production of mogrol and mogrosides.

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.

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.

Steviol glycosides (SGs) are highly valued for their sweetness, safety, and zero calories, but their bitter taste and low solubility limit their application. Modifying glycosyl units is a promising strategy to improve sensory qualities. In this study, we identified the enzyme UGT94E13 through phylogenetic analysis and enzyme screening, which catalyzes the glycosylation of rebaudioside M2 (Reb M2) at the C-13 position, producing the novel β-1,6-O-glycosylated product rebaudioside M9 (Reb M9). Subsequently, the catalytic activity of UGT94E13 toward Reb M2 was enhanced 12.0-fold through a structure-guided enzyme engineering strategy, and the mechanism behind this enhancement was analyzed. Finally, an enzymatic cascade system comprising the optimal mutant UGT94E13-F169A/I185A and sucrose synthase AtSuSy was constructed and optimized, achieving efficient synthesis of Reb M9 with a yield of 98.3% and a titer of 42.8 g·L^-1. Overall, this study provides an effective method for enhancing glycosylation of SGs and a reference for the glycosylation modification of natural products.