Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

0534894 - MBÚ 2021 RIV DE eng J - Journal Article
Borčinová, M. - Raschmanová, H. - Zamora, I. - Looser, V. - Marešová, Helena - Hirsch, S. - Kyslík, Pavel - Kovar, K.
Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris.
Applied Microbiology and Biotechnology. Roč. 104, č. 13 (2020), s. 5787-5800. ISSN 0175-7598. E-ISSN 1432-0614
Institutional support: RVO:61388971
Keywords : fed-batch * protein-production * cultivation strategies * providencia-rettgeri * current state * growth-rate * expression * perspectives * Pichia pastoris * Penicillin G acylase
OECD category: Microbiology
Impact factor: 4.813, year: 2020
Method of publishing: Open access
https://link.springer.com/article/10.1007/s00253-020-10669-x

To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate q(p)), includes productivity of secreted (q(p,extra)) plus intracellularly accumulated (q(p,intra)) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (q(p,extra)) and product retention (q(p,intra)) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (mu). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and q(p,extra), which depended on mu in a bell-shaped manner, (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing, (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, q(p,extra) was linearly related to mu and was up to three times higher than in initial phase (I), while q(p,intra) decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.
Permanent Link: http://hdl.handle.net/11104/0313037