Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii

The diamine oxidase from Glutamicibacter halophytocola (DAO-GH) was recombinantly produced in K. phaffii using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter for methanol-free production. Firstly, K. phaffii clones were generated for intracellular and secretory DAO-GH production that still possessed antibiotic resistance due to the cloning procedure. For intracellular production, a maximum intracellular DAO activity of 15,404 nkat/L_culture was achieved in fed-batch bioreactor cultivations, while for secretory production, the highest extracellular DAO activity of 6,078 nkat/L_culture was achieved using the αMF signal peptide without its EAEA sequence. The intracellularly produced DAO-GH was partially purified in several purification steps with a yield of 80%, a purification factor of about 10 and specific DAO activity of 16.7 nkat/mg_protein. The secretory DAO-GH production resulted in a specific DAO activity of 15.4 nkat/mg_protein already in the cell-free culture supernatant at the end of cultivation without further purification steps. The food industry aims to avoid the use of antimicrobial resistance in enzyme production, therefore, a new cassette plasmid with self-excisable antibiotic resistance markers was constructed for secretory DAO-GH production. The antibiotic-resistance-free K. phaffii clone generated with this plasmid achieved a maximum extracellular DAO activity of 4,770 nkat/L_culture in a fed-batch bioreactor cultivation. The DAO-GH obtained in this cultivation was spray-dried, resulting in a storable powder with 23 nkat/g_powder DAO activity and a water activity value of 0.12. This study demonstrated the secretion of recombinant DAO in a microbial host such as K. phaffii for the first time and provides a strategy for generating antibiotic-resistance-free K. phaffii clones.