Difucosyllactose (DFL), a fucosylated human milk oligosaccharide, exhibits significant application potential in the food and pharmaceutical industries. However, the microbial production of DFL faces challenges such as the accumulation of the intermediate 2'-fucosyllactose (2'-FL) and the high costs associated with the use of antibiotics and inducers. In this study, we constructed a plasmid- and inducer-free Escherichia coli MG1655 strain to enhance the biosynthesis of DFL while minimizing 2'-FL accumulation. Initially, the de novo DFL biosynthetic pathway was established by introducing different α-1,3-fucosyltransferases (α-1,3-FucT) into a 2'-FL-producing strain. Combinatorial metabolic engineering strategies were then employed to improve DFL accumulation. Subsequently, guided by the in silico multienzyme assembling by reshaping space (iMARS) framework, a fusion enzyme, FucTaY218K-L60-FutC, was rationally designed to effectively alleviate 2'-FL accumulation. Furthermore, modulation of the hydrophobic microenvironment in the substrate-binding pocket of FucTaY218K yielded the beneficial mutant FucTaY218K/W31R. Finally, modifications to the lactose operon significantly improved strain growth while concurrently enhancing DFL production. The best-performing strain achieved a DFL titer of 74 g/L in a 5-L bioreactor, corresponding to a productivity of 0.96 g/L/h, representing the highest productivity reported to date. This study establishes a robust and environmentally friendly platform for the industrial-scale production of DFL and provides a strategy for the microbial synthesis of other high-value human milk oligosaccharides.
Zhiqiang Liu, Binglin Li, Jieying Deng et al.· ACS Synthetic Biology· 0 citations
Trehalose is a nonreducing disaccharide widely used for its biomolecule-protective properties. However, multienzyme cascade production remains limited by low enzyme expression and suboptimal catalytic performance. To address this, thermostable maltooligosyltrehalose synthase (TreY) and trehalohydrolase (TreZ) from Arthrobacter ramosus were individually expressed intracellularly in Bacillus subtilis, and the crude lysates were combined for trehalose biosynthesis, achieving 281.4 g/L trehalose and a yield of 0.7 g trehalose/g maltodextrin. Integrated computational screening identified MalQ-3 from Cyanobacterium stanieri as a suitable 4-α-glucanotransferase for soluble expression. Subsequent semirational engineering generated MalQ-3-M2 (S54P/V472F), with enhanced activity and stability associated with improved substrate-pocket dynamics, thereby facilitating glucan-chain rearrangement and short-chain reutilization. MalQ-3-M2 was separately expressed in B. subtilis and incorporated into the crude-lysate cascade, increasing the trehalose titer to 338 g/L and the yield to 0.85 g trehalose/g maltodextrin. Overall, this work establishes a scalable B. subtilis platform for efficient trehalose production.
Haidong Huang, Yangyang Li, Jin-Song Song et al.· Journal of Agricultural and...· 0 citations
Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene into diverse triterpenoids, yet their intrinsically low catalytic efficiency restricts biosynthetic productivity. Here, we establish a mechanism-guided synergistic engineering strategy that extends beyond conventional active-site engineering by integrating distal substrate access regulation with catalytic microenvironment optimization to enhance the catalytic performance of CrAS from Catharanthus roseus. Structural modeling and mechanistic analyses revealed a conserved catalytic framework involving carbocation-mediated polycyclization and identified a surface-exposed constriction region that regulates substrate access. Guided by these insights, distal surface engineering of the constriction region was synergistically combined with active pocket optimization. The resulting combinatorial mutant, M3 (L323A/T327K/N565I), exhibited a 95.2% increase in catalytic efficiency and enhanced α-amyrin and β-amyrin by 53.2% and 49.7%, reaching 158 mg/L and 63 mg/L, respectively. Multi-scale analyses combining molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the enhanced catalytic performance is attributable to increased flexibility of the substrate access pathway, reinforced electrostatic and cation-π interactions, and reduced reaction energy barriers. Notably, distal mutation T327K improved substrate ingress through dynamic modulation of the protein surface, while N565I optimized the catalytic microenvironment by enhancing hydrophobic packing and stabilizing key intermediates. Overall, our findings establish a generalizable framework for engineering complex cyclases and provide a foundation for the sustainable microbial production of high-value triterpenoids.
Yangyang Li, K. Jin, Jiangong Lu et al.· Biotechnology and Bioenginee...· 0 citations