In vivo production, purification, characterization ofdeglycolysed colicine M and K forms in plant and usage asnatural food additives

Today, increasing resistance of bacterial pathogens common in food poses a critical threat to global public health and food security. In addition, the increase in consumers' demand for natural food without chemical additives has led scientists to find alternative food preservatives. Colicins are antimicrobial proteins produced by certain strains of Escherichia coli (E. coli), which kill non-host E. coli strains and are released into the environment to reduce competition with other bacterial strains. Colicin-M is one member of this group that acts on other closely related bacterial strains by destroying their outer cell membrane and is recognized as safe (GRAS) by the FDA, therefore, it has great potential as a potent antimicrobial agent. Colicin M has been recombinantly expressed in plants using the transient expression system and its antimicrobial activity was demonstrated. Colicin M as bacterial protein is not glycosylated in native hosts, and therefore, aberrantly glycosylated in any eukaryotic system including plants. A robust technology to produce proteins in eukaryotic system including plants in non-glycosylated, in their native-like form by co-expression target proteins with bacterial Endo H deglycosylation enzyme was developed. This technology has been successfully applied to produce vaccines against, malaria, Covid-19, rabies, and some therapeutics. The goal of this study is to produce recombinant Colicin M in Nicotiana benthamiana (N.benthamiana) in non-N-glycosylated, native-like form using Endo H deglycosylation technology. In this study, using enzymatic Endo H deglycosylation technology, we produced recombinant colicin M in the N. benthamiana plant in both glycosylated (gCM) and deglycosylated (dCM) forms, and their antimicrobial activity and stability were comparatively studied. Our collective findings demonstrated superipor stability and activity of dCM compare with that of glycosylated form. For comparison, Colicin K (CK) was also produced in N. benthamiana with or without Endo H by co-expression. Although, CK has several potential N-glycosylated sites, but our results confirmed that CK is not glycosylated when expresses in N. benthamiana plant. Both (with or without co-expression by Endo H) recombinant plant produced proteins were partially purified using Ni-column affinity chromatography and partial purified CK protein we characterized for stability and activity. No significant differences were observed between CKs, which were produced with or without co expression with Endo H. In conclusion, our results demonstrate that the deglycosylated Endo H technology can be used to produce other colicins in non-glycosylated, in their native-like form, which would be important to increase the commercialization potential of these colicins as a food additive.