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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.


PROVIDER: S-EPMC5674047 | BioStudies | 2017-01-01T00:00:00Z

REPOSITORIES: biostudies

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