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.

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.

Continuous reports of foodborne illnesses worldwide and the prevalence of antibiotic-resistant bacteria mandate novel interventions to assure the safety of our food. Treatment of a variety of foods with bacteriophage-derived lysins and bacteriocin-class antimicrobial proteins has been shown to protect against high-risk pathogens at multiple intervention points along the food supply chain. The most significant barrier to the adoption of antimicrobial proteins as a food safety intervention by the food industry is the high production cost using current fermentation-based approaches. Recently, plants have been shown to produce antimicrobial proteins with accumulation as high as 3 g/kg fresh weight and with demonstrated activity against major foodborne pathogens. To investigate potential economic advantages and scalability of this novel platform, we evaluated a highly efficient transgenic plant-based production process. A detailed process simulation model was developed to help identify economic "hot spots" for research and development focus including process operating parameters, unit operations, consumables, and/or raw materials that have the most significant impact on production costs. Our analyses indicate that the unit production cost of antimicrobial proteins in plants at commercial scale for three scenarios is $3.00-6.88/g, which can support a competitive selling price to traditional food safety treatments.

Colicins and Salmocins - New Classes of Plant-Made Non-antibiotic Food Antibacterials.

Recently, several plant-made recombinant proteins received favorable regulatory review as food antibacterials in the United States through the Generally Recognized As Safe (GRAS) regulatory procedure, and applications for others are pending. These food antimicrobials, along with approved biopharmaceuticals and vaccines, represent new classes of products manufactured in green plants as production hosts. We present results of new research and development and summarize regulatory, economic and business aspects of the antibacterial proteins colicins and salmocins as new food processing aids.

Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins.

Gram-negative bacteria belonging to the genus Klebsiella are important nosocomial pathogens, readily acquiring resistance to all known antibiotics. Bacteriocins, non-antibiotic antibacterial proteins, have been earlier proposed as potential therapeutic agents for control of other Gram-negative species such as Escherichia, Pseudomonas and Salmonella. This study is the first report describing pore-forming and peptidoglycan-degrading bacteriocins klebicins from Klebsiella. We have identified, cloned, expressed in plants and characterized nine pore-forming and peptidoglycan-degrading bacteriocins from different Klebsiella species. We demonstrate that klebicins can be used for broad and efficient control of 101 of the 107 clinical isolates representing five Klebsiella species, including multi-drug resistant pathovars and pathovars resistant to carbapenem antibiotics.

Plant-made Salmonella bacteriocins salmocins for control of Salmonella pathovars.

Salmonella enterica causes an estimated 1 million illnesses in the United States each year, resulting in 19,000 hospitalizations and 380 deaths, and is one of the four major global causes of diarrhoeal diseases. No effective treatments are available to the food industry. Much attention has been given to colicins, natural non-antibiotic proteins of the bacteriocin class, to control the related pathogen Escherichia coli. We searched Salmonella genomic databases for colicin analogues and cloned and expressed in plants five such proteins, which we call salmocins. Among those, SalE1a and SalE1b were found to possess broad antimicrobial activity against all 99 major Salmonella pathovars. Each of the two salmocins also showed remarkably high potency (>106 AU/µg recombinant protein, or >103 higher than colicins) against major pathogenic target strains. Treatment of poultry meat matrices contaminated with seven key pathogenic serovars confirmed salmocin efficacy as a food safety intervention against Salmonella.