Simple Purification of Nicotiana benthamiana-Produced Recombinant Colicins: High-Yield Recovery of Purified Proteins with Minimum Alkaloid Content Supports the Suitability of the Host for Manufacturing Food Additives

Abstract

Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA) and U. S. Department of Agriculture (USDA) as food-processing antibacterials through the GRAS (generally recognized as safe) regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins—including biopharmaceuticals, vaccines, and biomaterials—but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins.

Techno‐economic analysis of a plant‐based platform for manufacturing antimicrobial proteins for food safety

“…For example, recent literature highlights the challenge of the "viable but nonculturable" (VBNC) state of microorganisms in these foodsanitizing treatments. 11 In fact, various preparations of bacteriophages, such as the Salmonella-specific bacteriophage cocktail SalmoFresh™, 12 endolysins, 13,14 and bacteriocins, such as colicins 15,16 and nisins, 17 have already been granted Generally Recognized as Safe (GRAS) status as food antimicrobials by the US Food and Drug Administration (FDA). 10 Biotic approaches to food sanitization have high potential as supplementary treatments to de-risk the supply chain by employing efficacious and orthogonal protection against high-risk pathogens.…”

“…11,14,15,[22][23][24] In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. Plant-based platforms have the potential for producing market-relevant volumes of AMPs at competitive costs, because they do not require expensive bioreactors and culture media.…”

“…Based on this reduction, the maximum daily intake of nicotine from the use of colicin as a food safety AMP would be much lower than is encountered in everyday consumption of Solanaceae plants such as peppers, tomatoes, or potatoes 15. (b) Top individual factors and the respective cost category, contributing to the AOC of the base case scenario.…”

“…Therefore, there is a strong and intuitive case for acceptance of certain bacteriophage-and bacteriocin-derived antimicrobial treatments for food safety applications. 11 In fact, various preparations of bacteriophages, such as the Salmonella-specific bacteriophage cocktail SalmoFresh™, 12 endolysins, 13,14 and bacteriocins, such as colicins 15,16 and nisins, 17 have already been granted Generally Recognized as Safe (GRAS) status as food antimicrobials by the US Food and Drug Administration (FDA). It is anticipated that similar antimicrobial preparations will be granted GRAS status by FDA in the future, as the popularity of these technologies grows and additional regulatory notices are filed.…”

“…We have previously reported expression levels as high as 3 g/kg plant fresh weight (FW). 11,14,15,[22][23][24] In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. We used laboratoryscale results and working process knowledge from pilot and commercial processes to develop a process simulation model using SuperPro Designer ® to assess the commercial viability of the production platform and to identify economic "hotspots" to help guide future research and development.…”