Development of a Producer of Recombinant Maral Chimosine based on the Kluyveromyces lactis Yeast

Abstract

Abstract-A pSVB-Cer plasmid vector has been developed that allows integrating an expression cassette containing the maral prochymosin gene into the genome of the commercial strain GG799 of the Kluyveromyces lactis yeast. Controlled expression of the maral prochymosin gene in the recombinant producer was achieved using a hybrid auto-inducible P350 promoter, which is activated in the absence of glucose in the culture medium. This promoter included a 3ʹ-fragment of the core sequence of the promoter of glyceraldehyde-3-phosphate dehydrogenase (GAP1) and a 5ʹ-fragment of the regulatory sequence of the promoter of isocitrate lyase (ICL1). As a selective marker, the gene of acetamidase (amdS) controlled by the promoter of the gene of alcohol dehydrogenase (ADH1) was introduced into the pSVB-Cer vector. Producer clones, which ensure the accumulation of recombinant maral prochymosin in the culture medium, were obtained. The influence of the initial glucose concentration, temperature, cultivation duration, as well as amounts of phosphoric acid salts and B group vitamins on the level of the target pro-enzyme accumulation in the culture fluid was established. The maximum yield of recombinant maral chymosin with an activity of 152 U/ ml was obtained by cultivating the producer strain for 114 hours at 22 oC in a medium containing 3% glucose, 0.75 g/l potassium hydrophosphate and 4.5 mg/l calcium pantothenate. Key words: recombinant maral chymosin, milk-clotting activity, producer, hybrid auto-inducible promoter, Kluyveromyces lactis The work was performed within the framework of the state task of the Ministry of Science and Higher Education of the Russian Federation (topic number FZMW-2020-0002, "Development of Recombinant Enzyme Producers for Cheese Making") and by financial support of Russian Foundation for Basic Research and the government of NSO according to the research project №19-44-543018