[Analysis of the properties of Komagataella phaffii CF-st401, a genetically modified producer of the sweet protein brazzein by using in silico methods].

Abstract

Currently, in order to reduce the consumption of mono- and disaccharides in diets, sweeteners are widely used. At the same time, none of the sweeteners approved for food industry usage matches the organoleptic properties of natural sugars. This circumstance was the basis for the development of technology for producing a new type of food ingredient with a sweet taste - brazzein using the producer strain Komagataella phaffii CF-st401. The purpose of the study was the application of in silico methods to assess the safety of K. phaffii CF-st401 genetically modified (GM) microorganism, which is the producer of the "Sweet protein Brazzein". Material and methods. The research object was the map of K. phaffii CF-st401 plasmid obtained by Biryuch LLC as a result of sequencing the plasmid of GM strain K. phaffii CF-st401, including: a synthetic nucleotide sequence similar to the sequence encoding the brazzein protein in the plant (Pentadiplandra brazzeana) and optimized for expression in the DNA of the recipient strain; the nucleotide sequence of plasmid M4794 from Saccharomyces cerevisiae and a linear fragment of the flanking DNA regions from K. phaffii used for integration into the genome of the recipient strain K. phaffii YIB Δleu2 VKPM Y-476, as well as amino acid sequence data of recombinant brazzein. By using bioinformatics methods (in silico), we investigated the DNA structure of the vector sequence of K. phaffii CF-st401, including the presence of operons responsible for toxin production, antibiotic resistance, and allergenicity. Results. As a result of the studies of K. phaffii CF-st401 vector plasmid, introduced into K. phaffii YIB Δleu2 VKPM Y-4761, it was shown that its regions responsible for the structure of the "Sweet protein Brazzein" coincide by more than 70% with elements of the brazzein P56552 reference protein from the plant P. brazzeana. The absence of selective markers and allergenicity in the vector plasmid was confirmed. Conclusion. The analysis of the structure of the DNA vector sequence of the K. phaffii CF-st401 GM strain confirmed the feasibility of using bioinformatics methods to predict the properties of technological microorganisms when assessing their safety for consumers.