Comparative expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide

Abstract

Currently, the Russian market of phospholipase A2 enzyme preparations is represented by commercial preparations of foreign manufacturers: Nagase (Japan) and Maxapal (the Netherlands). However, the growing demand and the need to reduce the cost of production of phospholipase A2 require the development of new super-producers of phospholipase A2. In this connection, the aim of the work is to compare the expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide. The object of the study is the recipient yeast strain Komagataella phaffii X-33. The studies were conducted in accordance with generally accepted norms and approaches. Phospholipase A2 genes from Streptomyces violaceoruber were used for this worK. The target sequences were synthesized in the company "Eurogen" (Russia) and cloned as part of the TE vector pUC57. In the course of the work, the genetic constructs pPICZaA-Pla2 and PPICZmf4iA-Pla2 containing the Streptomyces violaceoruber phospholipase A2 gene were assembled under the native signal a-MF and its modification mf4i. The transformation of the yeast Komagataella phaffii X-33 with the obtained genetic constructs was also carried out. As a result of the conducted studies, it was shown that on average, there were no significant differences in the level of expression and specific activity of recombinant phospholipase A2 in methylotrophic yeast K. Phaffii X-33 when using the native a-MF secretion signal and its modified version mf4i. However, the use of the secretion factor mf4i allows for higher production of phospholipase A2 in individual clones. The obtained data indicate the prospects of using the secretion factor mf4i to create super-producers of enzymes based on yeast K. Phaffii X-33.