A comparison of different constitutive expression systems for the production of bovine chymosin in Komagataella phaffii.

Bovine chymosin is a protease of commercial interest in the food industry for milk clotting during cheese production. Today, chymosin is mainly produced by recombinant DNA technology in fungi. The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) represents one of the best microbial platforms for heterologous protein production in the context of industrial biotechnology. Regulated gene expression in K. phaffii is typically based on the use of the inducible alcohol oxidase I promoter (PAOX1), which relies on the presence of methanol, a toxic and flammable compound. In this work, we aimed to compare the use of different constitutive methanol-free expression systems to produce bovine chymosin in K. phaffii. For this purpose, the expression of a synthetic prochymosin B gene (CHYMB) was assessed from vectors containing 3 different constitutive glycolytic promoters (PPGK, PGAP, and PTEF). Best production was achieved when CHYMB was placed under control of PTEF and cells were grown at 28°C. Secreted chymosin was purified in a single molecular exclusion chromatography step, and the recombinant enzyme presented the same characteristics as commercial enzymes in terms of substrate specificity and thermostability. Together, our results showed that the yield of recombinant chymosin produced under the control of constitutive promoters is better than that previously reported for PAOX1. We propose that constitutive expression of bovine chymosin in K. phaffii should be considered as a viable alternative for industrial production.