Aug 2026· Journal of Agricultural and Food Chemistry· Vol 74 34, pp.
27168-27178
· 0 citations· 41 references
Medicine
TL;DR
This study achieves simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.
Abstract
Rhamnosyltransferases are remarkable biocatalysts for the synthesis of rhamnosylated natural products with valuable physicochemical properties and bioactivities. However, their application is hindered by poor stability and low catalytic efficiency. Here, we achieved simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy. The variant M9 exhibited a 589.43-fold extension in half-life, a 2.5 °C increase in Tm, a 13.6 °C increase in T50, and an 8- to 763-fold increase in activity toward diverse flavonoids compared with the wild type. Molecular dynamics simulations provided insights into enhanced thermostability and catalytic efficiency. To demonstrate its synthetic utility, a whole-cell biocatalytic system was constructed in E. coli by coexpressing M9 and UDP-rhamnose synthase, enabling a neohesperidin titer of 1.14 g L-1 without exogenous sugar donor supplementation. This study presents a practical enzyme engineering strategy for simultaneous activity-stability enhancement in glycosyltransferases and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.
The rational engineering of an α-L-rhamnosidase (DthRha) to address limitations and enhance its performance for flavonoid production highlights the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.
Haoyu Jia, Luran Wang, Tong Yan et al.· International Journal of Bio...· 1 citation
Daphnetin is a clinically established coumarin, but its native biosynthetic pathway remains elusive. In this study, a 2-oxoglutarate-dependent dioxygenase (2OGD), AtS8H, was identified that hydroxylates umbelliferone (UMB) to daphnetin , enabling the design of an artificial biosynthetic pathway. However, the pathway efficiency was constrained by the poor solubility of AtS8H and the low catalytic activity of the upstream 2OGD enzyme IbC2'H. To enhance AtS8H solubility, we developed an integrated strategy combining ProteinMPNN-guided sequence redesign with surface charge engineering. The obtained quintuple mutant S8H 2-6 exhibited significantly improved solubility and a 7.2-fold increase in catalytic efficiency. For IbC2'H, we developed a fluorescence-based high-throughput screening method, and a quadruple mutant C6 was obtained by directed evolution, which displayed a 2.7-fold higher kcat and a 3.2 °C improvement in thermal stability. Implementing both engineered enzymes into an optimized Escherichia coli strain enabled the de novo production of daphnetin at a titer of 46 mg/L. This work reports, to the best of our knowledge, the first microbial de novo production of daphnetin from a simple carbon source and demonstrates an integrated enzyme engineering approach that synergistically refines biosynthetic pathways for efficient microbial production.
Shunmin Ji, Chong Xie, Yanyan Wang et al.· ACS Synthetic Biology· 0 citations
Isobutene is an important platform chemical that is still predominantly produced from petroleum-derived feedstocks. The ferulic acid decarboxylase (Fdc) catalyzed decarboxylation of 3-methylcrotonic acid provides a green and sustainable route to bio-based isobutene. However, the poor activity of native Fdc toward this non-natural substrate severely limits its practical application. Here, we developed an integrated enzyme-engineering strategy that combines sequence-cluster mining with distal-site engineering to improve catalytic performance. The engineered triple-site mutant V132C/Y298F/S484A (3MUT) exhibited 5.3-fold the isobutene production of WT. Coupling this mutant with reaction-process optimization and cofactor engineering increased the isobutene yield to 67.9%, representing the highest reported yield to date. Mechanistic studies revealed that distal-site mutations in 3MUT reshaped the active site by disrupting the M293-mediated hydrogen bond that blocks the substrate to active site, then further promoting favorable hydrogen-bond interactions with the substrate via R183 and E292, respectively. These findings establish an efficient route for sustainable bio-based isobutene production and demonstrate the potential of distal-site engineering for improving UbiD-family decarboxylases.
Ting Feng, Xuanyu Cao, Liran Yang et al.· Bioresource Technology· 0 citations
A bidirectional loop-engineering strategy is employed to decipher the function of a conserved loop (XDXXP) in GH46 chitosanases and reveals that the loop modulates the geometry, electrostatics, and hydrophilicity of the substrate-binding cleft, thereby influencing substrate recognition and product specificity.
Mao-Yue Li, Minjuan Gui, Yingying Wei et al.· Journal of Agricultural and...· 0 citations
α-L-Rhamnosidase is a glycoside hydrolase (GH) that cleaves α-L-rhamnosidic bonds in diverse natural substrates, including oligosaccharides, glycoproteins, flavonoids, and glycolipids that contain terminal L-rhamnose. Owing to its broad substrate specificity, it has attracted growing interest in biotechnology, food processing, pharmaceuticals, and natural product modification. Fungal and bacterial α-L-rhamnosidases show diversity in molecular architecture, catalytic efficiency, thermostability, and pH tolerance, while sharing a conserved inverting mechanism mediated by acidic residues within the GH78 and GH106 families. Structural studies reveal catalytic cores, substrate recognition motifs, and aromatic residues involved in rhamnose binding. These enzymes enable the production of bioactive compounds such as prunin, isoquercitrin, quercetin, hesperetin, icariin, and myricetin. Recent advances in computational design, protein engineering, and directed evolution have improved enzyme performance. This review summarizes the current knowledge of microbial α-L-rhamnosidases, emphasizing their sources, biochemical characteristics, substrate specificity, structural organization, catalytic mechanisms, therapeutic relevance, and recent advances in protein engineering.
Kunwar Vishal, Vinita Yadav, Pramod K. Yadav· Journal of Agricultural and...· 0 citations
Simultaneously enhancing the thermostability and catalytic activity of acetylxylan esterases (AxEs) remains a significant challenge due to the inherent stability-activity trade-off. Here, a previously uncharacterized CE7 family acetylxylan esterase (TsAxE) from Thermoclostridium stercorarium was identified and engineered through a parallel multidimensional engineering strategy integrating consensus motif engineering, molecular docking, and interface engineering. The best-performing mutant BCF (D218L/D245P/G183Y) exhibited a 44.10 ± 0.44% increase in activity toward p-nitrophenyl acetate (pNPA). Notably, the thermostability of BCF was substantially improved, with the half-life (t1/2) at 60 °C extended from 0.63 ± 0.04 h to 43.82 ± 3.90 h. In addition, BCF showed improved catalytic efficiency toward p-nitrophenyl butyrate (pNPB). Molecular docking and molecular dynamics analyses suggested that these mutations may reshape substrate-binding pocket and improve structural stability. This study demonstrates the effectiveness of integrating complementary engineering strategies for the rational improvement of AxEs activity and thermostability.
Yujiao Tao, Xinrui Tang, Mei Zhao et al.· Journal of Agricultural and...· 0 citations
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