Linjiang Zhu

The plasticity of the enzyme active site is widespread in naturally evolved homologous enzymes, but has been less explored in engineered enzymes. In this study, the plasticity of the active site in sucrose phosphorylases (SPs) for 3‐O‐α‐glucopyranosyl‐resveratrol synthesis was obtained. The mutant R135L/Y344Q of Bl SP from Bifidobacterium lactis showed the same catalytic activity with the previous mutant Q345F, but displayed different microenvironment of active site and enzymatic properties. The new mutant could almost completely convert up to 15 g/L resveratrol into resveratrol glycoside without cosolvent, meanwhile, generate a new impurity 3‐O‐α‐diglucoside‐resveratrol, which was identified as 3‐O‐α‐maltosyl‐resveratrol.

Enzyme engineering drives green biocatalysis but lacks diagnostic tools to distinguish transport-limited from binding-limited catalytic bottlenecks, causing unnecessary trial and error. We developed the combined pocket-tunnel diagnosis (CPTD) framework to identify the rate-limiting step. It integrates static structural analysis with molecular dynamics to diagnose geometric and energetic barriers in the substrate transport and binding. Applying this to glycosyltransferase BsYjiC for the synthesis of bulky sterol glycosides revealed a steric tunnel bottleneck. A compact 44 mutant library achieved 5.5-fold, 9.4-fold, and 4.3-fold activity enhancements for DHEA, pregnenolone, and diosgenin, respectively. Subsequent cavity engineering boosted diosgenin activity 9.29-fold and expanded the substrate scope to cholesterol. Comparative validation confirmed diagnostic accuracy, as a pocket-first strategy failed for both DHEA and pregnenolone and yielded only marginal gains for diosgenin. In contrast, the same pocket mutations produced substantially greater enhancements after the tunnel bottleneck was removed.

Ergothioneine (EGT) is a unique natural chiral compound endowed with potent antioxidative, anti-inflammatory, and cytoprotective properties. Currently, EGT is primarily produced via bioextraction from mushrooms and chemical synthesis; however, the low efficiency and high costs associated with these methods hinder their ability to meet the growing market demand. Consequently, heterologous EGT production in non-native host strains (e.g., Escherichia coli and Corynebacterium glutamicum) has garnered increasing attention. With the rapid advancement of synthetic biology and metabolomics, remarkable progress has been achieved in EGT production in recent years, with the high titers have reached 7.2 g/L in E. coli and 9.3 g/L in Yarrowia lipolytica. Meanwhile, the development of a "chemoenzymatic catalytic cascade″ route has achieved the highest titer: 47.3 g/L. This review focuses on the latest advances in the discovery and identification of key enzymes involved in EGT biosynthetic and catabolic pathways and metabolic engineering strategies for EGT production. Additionally, the multifunctional roles and practical applications of EGT in the food, cosmetics, and pharmaceutical industries are summarized.

Paenibacillus, a plant-growth-promoting rhizobacterium, exhibits broad-spectrum biocontrol activity through the production of diverse antibacterial metabolites, competitive niche colonization, induction of systemic resistance, and enhancement of nutrient uptake. The review summarizes recent advances in elucidating the synergistic interactions among its biocontrol mechanisms and their responses to environmental factors. Subsequently, it outlines gene editing and regulatory technologies applicable to Paenibacillus. Next, key synthetic biology strategies employed to enhance biosynthetic capabilities are examined. Finally, future prospects and challenges associated with advancing Paenibacillus toward precision engineering and high-efficiency applications are discussed. Notably, its role in industrial biotechnology—particularly in the scalable production of industrial enzymes and high-value chemicals—is increasingly recognized as a focal point of growing scientific and commercial interest.

This study presents an approach combining molecular dynamics (MD) simulations and near‐attack conformation (NAC) analysis to mine and engineer UDP‐glucosyltransferases. Using the MD‐NAC strategy, we identified that BsYjiC from Bacillus subtilis possesses menthol glycosylating activity for the first time, and it exhibited a 28% higher k_cat/K_M value compared to the known l‐menthol glycosylating enzyme, BlYjiC from Bacillus licheniformis. The strategy was further employed to modify BsYjiC by preventing nonproductive substrate motion and stabilizing NACs through engineering key residues identified by MD. Based on this strategy, 14 positive mutants were identified out of 16 candidates, and the double mutant A76D/V108L (M2) demonstrated a 10.64‐fold increase in k_cat/K_M value compared to the starting point. Whole‐cell cascade reactions with sucrose synthase yield 160 mM glucoside after catalyzing for 96 h, demonstrating its application merit. This work underscores MD‐driven enzyme engineering as a promising strategy to overcome limitations of static structure‐based approaches.

Sucrose phosphorylase (SPase) is widely used for the glycosylation of polyhydroxy compounds, while low regioselectivity will limit its application. In this study, the regioselectivity of wild‐type SPase from Limosilactobacillus reuteri for the synthesis of 2‐O‐α‐d‐glucosylglycerol (2‐αGG) (84.7% ± 0.5%) was enhanced by combinational semi‐rational strategies. A key residue R137 was identified by sequence alignment analysis of non‐conserved residues in the catalytic pocket, molecular docking analysis, and alanine scanning at the key flexible loops. The regioselectivity of R137M increased to 95.4% ± 0.4%. Iteration mutation, loop engineering, and triple code saturation mutagenesis were used to further increase the 2‐OH selectivity (>97%) and double mutant R137M/L337T (98.0% ± 0.5%) was the best one. Whole‐cell transformation with R137M/L337T produced a higher yield of 2‐αGG with the increased final 2‐OH selectivity (>99%); besides, the amount of byproduct glucose greatly declined, meaning a decreased hydrolysis activity. Hence, the combinational semi‐rational mutagenesis methods are effective for improving the regioselectivity of SPase.