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. 2020 Jul;104(13):5787-5800.
doi: 10.1007/s00253-020-10669-x. Epub 2020 May 18.

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

Martina Borčinová  1   2 , Hana Raschmanová  3   4 , Iwo Zamora  3   5 , Verena Looser  3   4 , Helena Marešová  6 , Sven Hirsch  7 , Pavel Kyslík  6 , Karin Kovar  3   8
Affiliations

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

Martina Borčinová et al. Appl Microbiol Biotechnol. 2020 Jul.

Abstract

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.

Keywords: Fedbatch bioreactor cultivation; Penicillin G acylase; Pichia pastoris; Process optimisation; Secretion of a heterologous protein; Specific rate of product formation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic representation of the typical time course of specific rates of product formation. The shift in qp was determined at the time point tk when qp equalled 0.5 Δ qp. Both qp,total and qp,intra followed the visualised trend, and qp,extra was subsequently subtracted
Fig. 2
Fig. 2
Quantification of intra- and extracellular recombinant protein. Recombinant protein (dark grey circles) folding and maturation takes place in the endoplasmic reticulum. Mature protein is then translocated into the Golgi apparatus and subsequently encapsulated in vesicles and secreted to the extracellular environment. Concentrations of the enzyme inside the cells cp,intra (U l−1) and in the supernatant cp,extra (U l−1) are quantified as enzyme activity per litre of culture volume. Specific rate of intracellular retention qp,intra (U (gcdw)−1 h−1) represents the activity of product per gram cdw per hour that is retained within the maturation and secretory machinery inside the cell. The specific rate qp,extra (U (gcdw)−1 h−1) corresponds to the activity of secreted product (extracellular enzyme) per gram of cdw per hour
Fig. 3
Fig. 3
Time course of PGA production with distinguished phases with respect to qp. The displayed data were acquired in the cultivation ENS-C and represent the typically observed trend in PGA formation in all cultivations enlisted within this work. a Amount of PGA (in kU) during the production phase of cultivation. The symbols represent measured enzyme: crossed squares, total amount; full squares, intracellular amount; empty squares, extracellular amount. The solid lines represent the calculated theoretical values for the respective measured enzyme (kU). b The coloured lines marked by respective square symbols represent calculated qp(t) values: green (crossed squares)—qp,total, blue (full squares)—qp,intra, red (empty squares)—qp,extra (U (gcdw)−1 h−1). The black bold line represents the time development of intracellular PGA activity per gram cdw (U (gcdw)−1), which indicates the saturation of the cell with product. The time course of the specific production rate of PGA qp(t) was divided into three phases as indicated by the vertical dotted lines: initial, transition and saturation phase. Production time 0 indicates the time, from which methanol was fed
Fig. 4
Fig. 4
Relationship between specific growth rate of biomass and the specific rates of product secretion and intracellular retention in different phases of the bioprocess a Specific rate of protein secretion (qp,extra, U (gcdw)−1 h−1) in initial phase (I) (full symbols) and saturation phase (III) (empty symbols) plotted against specific growth rate (μset). b Specific rate of intracellular protein retention (qp,intra, U (gcdw)−1 h−1) in initial phase (full symbols) and saturation phase (empty symbols) plotted against specific growth rate (μset). Error bars corresponds to calculated standard error. The grey lines approximate the underlying relationship. Not shown are data for PGA secretion from ENS-D cultivation (from the 50th hour onwards) since they were biased by the post-harvesting treatment
Fig. 5
Fig. 5
Relationship between saturation of the intracellular environment with PGA and biomass specific growth rate (ENS B-E) during transition phase (II). Full symbols represent the maximum (interpolated) value of the intracellular concentration of active PGA-enzyme per gram of biomass (U (gcdw)−1). Open symbols represent the time point (hours) corresponding to the maximum intracellular saturation relative to the start of induction by methanol, which was set to 0 h. Lines in grey, with 95% confidence bands (in black), were computed by linear regression
Fig. 6
Fig. 6
Comparison of time courses of specific production rates (with respect to the extracellular product) for different lipases produced in both Mut+ and MutSP. pastoris strains, all being under the control of the pAOX1 promoter, with the exception of one under the pFLD1 promoter. Squares represent the time courses of qp,extra (U (gcdw)−1 h−1) as published by respective authors or calculated from their experimental data. The solid curves represent the data interpolation using equation 5. a Arnau et al. (2010): Mut+ phenotype, pAOX1 promoter, Rhizopus oryzae lipase (ROL) production; b Barrigón et al. (2013): Mut+ phenotype, pAOX1 promoter, ROL production; c Resina et al. (2005) A: Mut+ phenotype, pFLD1 promoter, ROL production; d Resina et al. (2005) B: Muts, phenotype, pAOX1 promoter, ROL production; e Sha et al. (2013) A: Muts, phenotype, pAOX1 promoter, Rhizopus chinensis lipase (r27RCL) production; f Sha et al. (2013) B: Mut phenotype, pAOX1 promoter, r27RCL production, co-expressed with secretion factors ERO1 and PDI
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