Uridine diphosphate glycosyltransferases (UGTs) are among the key rate-limiting enzymes in the biosynthesis of salidroside. Plant-derived UGTs often exhibit poor solubility and low catalytic activity, whereas microbial UGTs typically show insufficient regioselectivity for salidroside production. In this study, we performed stepwise engineering of the UGT from Paenibacillus durus (PdUGT) to generate a highly regioselective biocatalyst for salidroside production. Through stepwise reshaping of the active site and the access tunnel, we progressively enhanced the regioselectivity for tyrosol glycosylation from 81.2 to 99.9%, reaching a level comparable to that of natural plant enzymes. Mutations prioritized by SaProt and ΔΔG calculations further increased the melting temperature by 11.2 °C. The final variant, M4, exhibited a 20-fold increase in catalytic efficiency, with a specific activity of 58.5 U·mg−1. In a UDP-glucose recycling cascade, M4 enabled the production of 193.1 mM salidroside with 99.0% conversion and a space-time yield of 5.7 g·L−1·h−1. Molecular dynamics simulations and substrate docking suggested that the improved performance of M4 is associated with tighter binding of tyrosol, suppression of unproductive tyrosol reorientation, and enhanced protein compactness under thermal stress. Substrate profiling confirmed that PdUGT exhibits broad substrate promiscuity, while M4 displays high specificity toward tyrosol and related aromatic alcohols. This work establishes PdUGT as a valuable microbial UGT scaffold and provides experimentally supported design principles for engineering glycosyltransferases. Moreover, this study lays a solid foundation for the industrial bioproduction of salidroside and other glycosides.
Mu-Yang Li, Yi-Wei Meng, Ji-Shan Li et al.· ACS Catalysis· 0 citations
The direct asymmetric synthesis of chiral amides remains a focal point in green manufacturing, particularly for (S)-2-aminobutyramide whose traditional production is often hampered by low atom economy and arduous chiral resolution steps. Here, we report the creation of a non-native bio-amidation activity in Escherichia coli asparagine synthetase A (AsnA) for the proof-of-concept one-step biosynthesis of (S)-2-aminobutyramide, a critical intermediate for anti-epileptic drugs. To overcome the bottleneck of high-throughput screening, we engineered the AmiC–AmiR transcription factor into a highly sensitive biosensor (RC-4-Y83H) by remodeling its ligand-binding pocket, achieving a 23-fold induction response specifically toward (S)-2-AB. Utilizing this biosensor to screen saturation mutagenesis libraries, we identified an evolved variant (K77L/E120L/R255W) that showed a reversed substrate preference between the two substrates tested, converting (S)-2-aminobutyric acid to (S)-2-AB with retention of configuration. Molecular dynamics simulations and free-energy landscape analysis reveal that the mutations fundamentally remodel the active site, inducing a 180° flip of the substrate binding mode. This structural reorientation positions the non-native carboxyl group in a catalytically productive geometry relative to ATP, a conformation inaccessible in the wild-type enzyme. As a proof of concept, this work does not aim to deliver an industrially competitive biocatalyst; instead, it shows that the specificity of a native amide-forming enzyme can be reprogrammed toward a non-native alpha-amino acid, and it provides a generalizable, biosensor-guided framework for such reprogramming.
Qianpeng Lai, Yiwei Meng, Zhongmei Liu et al.· Chemical Science· 0 citations
Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.
