ABSTRACT
Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.
Subject(s)
Ginsenosides , Inteins , Inteins/genetics , Protein Splicing , Proteins/metabolismABSTRACT
Bakanae disease is a destructive rice disease in South Korea caused by Fusarium fujikuroi infection. Chemical fungicides have been used to manage the disease, but the emergence of fungicide-resistant strains has gradually increased. Two chelating agents, chitosan oligosaccharides (COS) and ethylenediaminetetraacetatic acid (EDTA), are well known as biosafe and biocompatible antimicrobial agents. In this study, we compared the actions of COS and EDTA to gain a better understanding of the underlying antimicrobial activities and to evaluate them as eco-friendly fungicides against F. fujikuroi. While COS exhibited a rapid fungicidal effect on hyphal growing cells within 5 min, EDTA had a fungistatic effect on reversible growth inhibition. Scanning electron microscopy revealed that COS treatment resulted in pore-formation and cellular leakage along the growing hyphae, whereas EDTA caused no significant morphological changes. COS activity was greatly suppressed by the addition of Ca(2+) to the medium, and EDTA action was largely suppressed by Mn(2+) and slightly by Ca(2+), respectively. Taken together, these results indicated that two chelating agents, COS and EDTA, have different modes of antimicrobial action on F. fujikuroi. Thus, the combination of chelating agents having different modes of action might be an effective disease management strategy to prevent or delay the development of fungicide-resistant strains.
