Ecosystems worldwide are exposed to pollutants connected to the industrial production of pharmaceuticals. The objective of this study was to study the composition and characteristics of the soil microbial communities that had been exposed to long-term selection pressure caused by the industrial production of penicillin G. Soil samples from four sites among the penicillin G production plant were analysed using 16S rRNA profiling via Illumina MiSeq platform and were compared with the control samples from four sites outside the plant. Total metagenomic DNA from the impacted soil was also used for the preparation of E. coli T1R-based fosmid library which was consequently qualitatively tested for the presence of penicillin G acylase (PGA)-encoding genes using the method of sequence homology. Analyses of alpha diversity revealed that the long-term antibiotic presence in the soil significantly increased the microbial diversity and richness in terms of Shannon diversity index (p = 0.002) and Chao estimates (p = 0.004). Principal component analysis showed that the two types of communities (on-site and control) could be separated at the phylum, class and genus level. The on-site soil was enriched in Betaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetia, while a significant decrease in Actinobacteria was observed. Metagenomic fosmid library revealed high hit rates in identifying PGAs (14 different genes identified) and confirmed the biotechnological potential of soils impacted by anthropogenic activity. This study offers new insights into the changes in microbial communities of soils exposed to anthropogenic activity as well as indicates that those soils may represent a hotspot for biotechnologically interesting targets.

• Klíčová slova
• Antibiotic contamination, Fosmid library, Industrial production, Metagenome, Microbial consortia, Penicillin G acylase,
• MeSH
• antibakteriální látky biosyntéza   MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• biodiverzita MeSH
• DNA bakterií genetika   MeSH
• Escherichia coli genetika   MeSH
• fylogeneze MeSH
• látky znečišťující půdu MeSH
• metagenom MeSH
• metagenomika MeSH
• mikrobiota * genetika   MeSH
• průmyslová mikrobiologie MeSH
• půda MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• DNA bakterií MeSH
• látky znečišťující půdu MeSH
• půda MeSH
• RNA ribozomální 16S 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