METHOD FOR PRODUCING PHOSPHOLIPASE А2 KOMAGATAELLA PHAFFII (PICHIA PASTORIS) YIB ΔLEU2_PLA2S PRODUCTION STRAIN Russian patent published in 2021 - IPC C12N15/81 

FIELD: biotechnology.

SUBSTANCE: invention relates to industrial biotechnology and genetic engineering and concerns the production of an auxotrophic recipient strain for genetic engineering, plasmid vectors for gene expression and a production strain of phospholipase A2 which can be used in the food industry in the production of lysophospholipids and surfactants with emulsifying properties. Claimed is a method for producing a phospholipase А2 Komagataella phaffii (Pichia pastoris) YIB Δleu2_PLA2Sv production strain which involves: the creation of an integrative plasmid vector pMAY-LEU2 used for producing an auxotrophic strain of leucine Komagataella phaffii (Pichia pastoris) YIB Δleu2 which contains an AOX1 inducible promoter, ZeoR, a zeocin resistant gene, LEU2, sites for integration into the gene responsible for the synthesis of leucine, FLP, a FLP-recombinant gene and a constructed plasmid pMAY-LEU2 has a size of 9509 bp, the creation of an integrative plasmid vector pSEA-LEU-PLA2Sv for expression of genes in yeast containing a selective marker for selection of transformers in E.coli cells, a replication site, an integration site and an expression cassette comprising a promoter, transcription terminator and selective marker, as a selective marker the DNA sequence encoding the Leu2 gene is used, the creation of the yeast-recipient strain Komagataella phaffii (Pichia pastoris) YIB Δleu2, based on the wild strain Y-3489 deposited in the VKPM, and this strain auxotrophic on leucine is obtained by transforming the vector pMAY-LEU2 on the sites of integration into the LEU2 gene with subsequently extracting a sequence containing antibiotic resistance genes, the creation of a Komagataella phaffii (Pichia pastoris) YIB Δleu2_PLA2Sv strain of yeast on the basis thereof obtained by the transformation of the strain Komagataella phaffii (Pichia pastoris) YIB Δleu2 by the plasmid vector pSEA-LEU-PLA2Sv, with subsequent selection on the YNB environment, with the strain providing the synthesis of recombinant phospholipase A2 and containing no antibiotic resistance genes being deposited in the VKPM under the number Y-4513.

EFFECT: invention can be used in the food industry in the production of lysophospholipids and surfactants with emulsifying properties.

1 cl, 4 dwg