Dataset Information

High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin.

ABSTRACT: Drug resistance is a major problem in antibacterial chemotherapy. Apidaecins, which refer to a series of small, proline-rich antimicrobial peptides, are predominantly active against many drug-resistant bacteria. The apidaecins have special antibacterial mechanisms, and are non-toxic for human cells, a prerequisite for using them as novel antibiotic drugs. However, no efficient non-tagged apidaecin expression system has been reported, which is the limiting factor for their application. Here we successfully generated a Pichia pastoris transformant expressing and secreting apidaecin. However, expression was unstable and poor. Analysis of this revealed that the integration plasmid was frequently lost and that apidaecin expression resulted in cell death. Using N-methyl-N-nitro-N-nitroso-guanidine mutagenesis and selection, a mutant strain Apmu4 was derived, in which the rate of loss of the integration plasmid was much lower after induction, and which produced improved titres of apidaecin. Additionally, we discovered that using glucose as the sole carbon source to pre-culture the strain before induction could greatly enhance apidaecin production. A pilot-scale 10?L fermentation yielded 418?mg/L of recombinant apidaecin, which represents the highest reported yield of apidaecin. Consequently, this study reports the first super heterologous expression and secretion of apidaecin in yeast.

SUBMITTER: Chen X

PROVIDER: S-EPMC5674047 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Publications

High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin.

Chen Ximing X Li Juan J Sun Haili H Li Shiweng S Chen Tuo T Liu Guangxiu G Dyson Paul P

Scientific reports 20171106 1

Drug resistance is a major problem in antibacterial chemotherapy. Apidaecins, which refer to a series of small, proline-rich antimicrobial peptides, are predominantly active against many drug-resistant bacteria. The apidaecins have special antibacterial mechanisms, and are non-toxic for human cells, a prerequisite for using them as novel antibiotic drugs. However, no efficient non-tagged apidaecin expression system has been reported, which is the limiting factor for their application. Here we su ...[more]

PMID: 29109421

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Project description: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 (?). 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 ? 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 ? 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.

Dataset Information

High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin.

Publications

High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin.

Similar Datasets

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