Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell's physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia's potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. KEY POINTS: • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.

• Klíčová slova
• Chaperone, Co-expression strategy, Folding and secretion, Pichia pastoris, Productivity of recombinant protein production, Unfolded protein response (UPR),
• MeSH
• fungální proteiny * genetika   metabolismus   MeSH
• Pichia * genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomycetales MeSH
• signální dráha UPR MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fungální proteiny * MeSH
• rekombinantní proteiny MeSH

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 qp), includes productivity of secreted (qp,extra) plus intracellularly accumulated (qp,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 (qp,extra) and product retention (qp,intra) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (μ). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and qp,extra, which depended on μ 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, qp,extra was linearly related to μ and was up to three times higher than in initial phase (I), while qp,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.Key Points• A novel concept for industrial bioprocess development.• A Relationship between biomass growth and product formation in P. pastoris.• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter.• A Proof of concept in production of industrially relevant penicillin G acylase.

• Klíčová slova
• Fedbatch bioreactor cultivation, Penicillin G acylase, Pichia pastoris, Process optimisation, Secretion of a heterologous protein, Specific rate of product formation,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biomasa MeSH
• bioreaktory MeSH
• extracelulární prostor metabolismus   MeSH
• intracelulární prostor metabolismus   MeSH
• kinetika MeSH
• penicilinamidasa genetika   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomycetales genetika   růst a vývoj   metabolismus   MeSH
• techniky vsádkové kultivace MeSH
• teoretické modely MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• penicilinamidasa MeSH
• rekombinantní proteiny MeSH