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.

• Keywords
• Antibiotic contamination, Fosmid library, Industrial production, Metagenome, Microbial consortia, Penicillin G acylase,
• MeSH
• Anti-Bacterial Agents biosynthesis   MeSH
• Bacteria classification   genetics   isolation & purification   metabolism   MeSH
• Biodiversity MeSH
• DNA, Bacterial genetics   MeSH
• Escherichia coli genetics   MeSH
• Phylogeny MeSH
• Soil Pollutants MeSH
• Metagenome MeSH
• Metagenomics MeSH
• Microbiota * genetics   MeSH
• Industrial Microbiology MeSH
• Soil MeSH
• Soil Microbiology * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• Soil Pollutants MeSH
• Soil MeSH
• RNA, Ribosomal, 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.

• Keywords
• Fedbatch bioreactor cultivation, Penicillin G acylase, Pichia pastoris, Process optimisation, Secretion of a heterologous protein, Specific rate of product formation,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biomass MeSH
• Bioreactors MeSH
• Extracellular Space metabolism   MeSH
• Intracellular Space metabolism   MeSH
• Kinetics MeSH
• Penicillin Amidase genetics   metabolism   MeSH
• Promoter Regions, Genetic MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Saccharomycetales genetics   growth & development   metabolism   MeSH
• Batch Cell Culture Techniques MeSH
• Models, Theoretical MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Penicillin Amidase MeSH
• Recombinant Proteins MeSH

Strain Pantoea agglomerans JM1 was isolated from the soil of a microbiome that had been exposed to polluting pharmaceuticals. The bacterium exhibited enzymatic activities useful for the biotransformation of beta-lactams. The genome of the strain was assembled and described, and the gene encoding valacyclovir-like hydrolase was identified.

• Publication type
• Journal Article MeSH

This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.

• MeSH
• Achromobacter genetics   metabolism   MeSH
• Bioreactors microbiology   MeSH
• Gene Expression MeSH
• Genetic Vectors MeSH
• Gene Dosage MeSH
• Cloning, Molecular MeSH
• Codon genetics   MeSH
• Penicillin Amidase genetics   metabolism   MeSH
• Pichia genetics   metabolism   MeSH
• Promoter Regions, Genetic MeSH
• Industrial Microbiology methods   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Transformation, Genetic MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Codon MeSH
• Penicillin Amidase MeSH
• Recombinant Proteins MeSH