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.
Highlights
Antimicrobial peptides are evolutionarily ancient weapons, which appear to be ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms[1,2]
N-terminal mutant forms of apidaecin have stronger activity to inhibit gram-negative bacteria compared to the wild type apidaecin[22]
Drug resistance is a major problem in antibacterial chemotherapy
Summary
Antimicrobial peptides are evolutionarily ancient weapons, which appear to be ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms[1,2]. As expression systems based on Pichia pastoris have been used successfully for the production of various recombinant heterologous proteins[14] including some bacteriocin peptides EntL50A and EntL50B15,16 and plectasin-derived peptides[17,18], this offers a potential improved system for synthesis of recombinant apidaecin. This expression system has many advantages such as ease of genetic manipulation, inexpensive culture to high cell densities, posttranslational modifications of proteins, no toxicity from intracellularly accumulated material, and easy purification with very low secretion of endogenous proteins[19,20,21]. Inclusion of peptone and yeast extract in the growth medium can effectively protect against proteolysis of apidaecin following induction
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