Incze_2023_Life.(Basel)_13_1885

Industrial enzyme production with the Pichia pastoris expression system requires a well-characterized production strain and a competitively priced fermentation medium to meet the expectations of the industry. The present work shows a workflow that allows the rapid and reliable screening of transformants of single copy insertion of the target production cassette. A constitutive expression system with the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) with homology arms for the glycerol kinase 1 (GUT1) was constructed for the targeted integration of the expression plasmid in a KU70 deficient Pichia pastoris and the production of a bacterial fumonisin esterase enzyme (CFE). A robust colony qPCR method was developed for the copy number estimation of the expression cassette. Optimization of the protein production medium and the scale-up ability was aided by design of experiments (DOE) approach resulting in optimized production conditions at a semi-industrial scale. A novel fermentation medium containing 3% inactivated yeast and 2% dextrose in an ammonium-citrate buffer (IYD) was shown to be a promising alternative to YPD media (containing yeast extract, peptone, and dextrose), as similar protein titers could be obtained, while the cost of the medium was reduced 20-fold. In a demonstration-scale 48 h long fed-batch fermentation, the IYD media outperformed the small-scale YPD cultivation by 471.5 +/- 22.6%.