Potential of Pichia pastoris for the production of industrial penicillin G acylase

Marešová, Helena; Palyzová, Andrea; Plačková, Martina; Grulich, Michal; Rajasekar, Vyasa Williams; Štěpánek, Václav; Kyslíková, Eva; Kyslı́k, Pavel

doi:10.1007/s12223-017-0512-0

Cited by 10 publications

(10 citation statements)

References 41 publications

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“…In this work, the up-regulation was most significant in the case of Ec PGA (60% of the Ec PGA-producing cells exhibited up-regulated UPR), which strongly accumulated in the cells (up to 50–70% of the total active Ec PGA). As already discussed in the work of Marešová et al (2017), the reason for poor secretion of Ec PGA might be its incorrect maturation in P. pastoris cells, which results in ER stress. A co-expression of E. coli chaperone genes might improve the secretion of proteins of bacterial origin (Summpunn et al, 2018).…”

Section: Discussionmentioning

confidence: 89%

“…The plasmid P KAR2(FL) -sfGFP was integrated into the P. pastoris strain producing Ec PGA to monitor the UPR during a bioreactor cultivation. Because the Ec PGA was previously observed to intracellularly accumulate in P. pastoris (Borcinova et al, in preparation; Marešová et al, 2017), we assumed that UPR would be up-regulated, and sfGFP would be strongly produced. The up-regulation of UPR upon production of Ec PGA was proved both by flow cytometric analysis of sfGFP fluorescence (Supplementary Figures 4, 5) and qPCR analysis of KAR2 expression (Figure 3), when comparing the strain producing Ec PGA with a control strain producing no recombinant protein, both having the P KAR2(FL) -sfGFP cassette integrated into their genome.…”

Section: Resultsmentioning

confidence: 99%

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Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

et al. 2019

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Pichia pastoris ( Komagataella sp .) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli ( Ec PGA), lipase B from Candida antarctica ( Ca LB) and xylanase A from Thermomyces lanuginosus ( Tl XynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing Ec PGA, which accumulated in the cells (50–70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing Ca LB (20%) and Tl XynA (27%). The proportion of cells with up-regulated UPR in Ca LB-producing (35%) and Tl XynA-producing (30%) strains was lower in comparison to the Ec PGA-producing strain, and a smaller proportion of Ca LB and Tl XynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.

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Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

et al. 2019

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“…was used in this study. Construction of the strain is described by Maresova et al (2017). Briefly, plasmid pENS2, based on the vector pPICZαA (Invitrogen, CA, USA), contained a codonoptimised pga gene fused to the α-mating factor leader signal sequence from S. cerevisiae under the control of the pAOX1 promoter.…”

Section: Strainmentioning

confidence: 99%

“…Previous studies with P. pastoris strain X33 producing penicillin G acylase from Achromobacter sp. (ENS strain) showed that production of this enzyme in yeast hosts was significantly hindered and that the titres obtained were not satisfactory from an economic point of view (Ljubijankic et al 2002;Maresova et al 2010;Maresova et al 2017;Senerovic et al 2006). The majority of the enzyme produced remained inside the cells, while only a small proportion was secreted.…”

Section: Introductionmentioning

confidence: 99%

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

Borčinová

Raschmanová

Zamora

et al. 2020

Appl Microbiol Biotechnol

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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.

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“…The aim of the study was to identify new enzymes useful for the biotransformation of beta-lactams, with activities analogous to alpha-amino acid ester hydrolases (AEHs) ( 13 ) or penicillin G acylases (PGAs) ( 14 , 15 ). The enzymes were detected by the hydrolysis of a chromogenic substrate, 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB) ( 16 , 17 ). The strain JM1 showed a weak-positive phenotype for NIPAB hydrolysis.…”

Section: Genome Announcementmentioning

confidence: 99%

Draft Genome Sequence of Pantoea agglomerans JM1, a Strain Isolated from Soil Polluted by Industrial Production of Beta-Lactam Antibiotics That Exhibits Valacyclovir-Like Hydrolase Activity

et al. 2017

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Potential of Pichia pastoris for the production of industrial penicillin G acylase

Cited by 10 publications

References 41 publications

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

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

Draft Genome Sequence of Pantoea agglomerans JM1, a Strain Isolated from Soil Polluted by Industrial Production of Beta-Lactam Antibiotics That Exhibits Valacyclovir-Like Hydrolase Activity

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