Corynebacterium glutamicum is a cornerstone microbial chassis for industrial amino acid production, yet metabolic engineering in this organism still relies on static gene expression strategies that lack temporal flexibility and impose metabolic burden. Optogenetics offers a non-invasive and reversible alternative for dynamically coordinating cellular processes, but its application in C. glutamicum remains limited. Here, we establish an EL222-based, one-component optogenetic system enabling bidirectional, dynamic transcriptional control in C. glutamicum. By mitigating EL222-associated phototoxicity and optimizing illumination parameters, we developed blue light-induced and blue light-repressed expression modules with tunable responses. Applying these tools to L-lysine biosynthesis allowed light-responsive regulation of key pathway genes. In shake-flask batch fermentation, strain CG62 produced 2.96 ± 0.11 g L–1 L-lysine under blue light after 60 h, compared with 1.47 ± 0.12 g L–1 in the dark, representing a 101.36% increase in L-lysine titer. We establish a comparatively simple one-component EL222-based framework that supports both blue light-induced and blue light-repressed transcriptional regulation in C. glutamicum and demonstrate its application to dynamic control of L-lysine biosynthesis. This work expands the optogenetic toolkit for C. glutamicum and highlights light-driven dynamic regulation as a versatile strategy for optimizing industrial microbial cell factories.
Qing-Wei Jiang, Cheng Wang, Juan Shen et al.· ACS Sustainable Chemistry &a...· 0 citations
Deoxynivalenol (DON), a trichothecene mycotoxin commonly found in cereal grains and their derived products, poses significant risks to human and animal health. In previous work, a fusion enzyme composed of the dehydrogenase DADH and the aldo-keto reductase AKR13B3 was engineered to convert DON into the non-toxic 3-epi-DON in a single step. However, the poor thermal stability of this fusion enzyme limited its industrial application. In this study, EVcouplings and the GRAPE-WEB platform were utilized to identify key amino acid residues governing the thermal stability of the fusion enzyme AKR13B3–DADH. Through single-point mutation screening and the combination of beneficial mutation sites, a triple mutant M361L/T508Y/Y603F (M1) was obtained. The half-life of M1 at 50 °C reached about 500 min, representing a 16.4-fold increase compared with the wild type, while its catalytic activity increased by 2.7-fold. The apparent melting temperature increased by approximately 4 °C. Molecular dynamics simulations verified that the improved thermostability results from reduced conformational flexibility in key regions, enhanced structural packing, and a strengthened hydrogen bond network. These results demonstrate the successful development of a thermostable DON-detoxifying fusion enzyme and provide a practical basis for its industrial application.
Yi-Ting Pan, Hao Zhu, Qing-Wei Jiang et al.· International Journal of Mol...· 0 citations
Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins, widely contaminating agricultural products and posing a serious threat to food safety and human health. Enzymatic degradation is considered a promising detoxification strategy due to its high efficiency, strong specificity, and lack of secondary pollution. AKR13B3, a member of the aldo-keto reductase family, possesses intrinsic catalytic activity for AFB1 degradation; however, its low natural activity severely limits practical application. In this study, the binding mode of the AKR13B3-NADPH complex with AFB1 was first determined using AlphaFold 3.0 and AutoDock Vina. Through interaction analysis, Trp102 and Asp41 were identified as key targets for enhancing catalytic activity. Following site-directed mutagenesis screening, two mutants, D41H and D41T, with significantly improved catalytic activity were obtained, exhibiting 52.32% and 46.44% higher activity than the wild-type enzyme, respectively. Three-dimensional structural simulation revealed that D41H and D41T form stable interactions with the carbonyl group on the lactone ring of AFB1, thereby polarizing the carbonyl group and reducing the activation energy of the reaction, ultimately enhancing catalytic activity. Substrate channel analysis demonstrated that, compared with the wild-type, the D41H and D41T mutants significantly increased the bottleneck radius of the substrate channel (by 25% and 22%, respectively) and shortened the channel length (by 23% and 33%, respectively), thereby partially relieving steric hindrance and diffusion limitations and improving catalytic efficiency. In summary, this study elucidates the molecular basis by which D41H and D41T enhance the catalytic activity of AKR13B3 toward AFB1 through the dual mechanisms of external/hydrogen bond catalysis and channel remodeling, providing an important theoretical foundation for the rational design and directed engineering of AFB1-degrading enzymes.
Qing-Wei Jiang, Juan Shen, Zhang-Hu Chen et al.· International Journal of Mol...· 0 citations
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