Engineered gene clusters for the production of the antimicrobial peptide microcin J25

Si Jia Pan, Wai Ling Cheung, A. James Link

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

Microcin J25 (MccJ25) is an antimicrobial peptide produced by isolates of Escherichia coli with activity against closely related species. Production and export of mature MccJ25 in E. coli requires four genes that are organized on a plasmid-borne cluster in natural producer strains. In these strains, MccJ25 production does not commence until the cells reach stationary phase, and, according to previous literature, the highest titers of MccJ25 are obtained from cells grown in nutrient-poor medium. We sought to design an engineered MccJ25 gene cluster that alleviated the growth phase and media limitations of the natural cluster. In contrast to previous reports, we observe here that production of MccJ25 from its natural cluster is efficient in rich media, such as Luria-Bertani (LB). The engineered gene cluster functions in several E. coli strains and produces titers of MccJ25 that are moderately increased (1.5- to 2-fold) relative to the natural cluster. RT-PCR experiments and translational GFP fusion experiments confirm that the engineered cluster produces MccJ25 throughout exponential phase. Furthermore, we provide evidence that control of the natural MccJ25 gene cluster is at the transcriptional level. The observations herein provide design parameters for large-scale production of MccJ25 for biotechnological applications.

Original languageEnglish (US)
Pages (from-to)200-206
Number of pages7
JournalProtein Expression and Purification
Volume71
Issue number2
DOIs
StatePublished - Jun 1 2010

All Science Journal Classification (ASJC) codes

  • Biotechnology

Keywords

  • Antimicrobial peptide
  • Gene cluster
  • Microcin
  • Microcin J25
  • Posttranslational modification

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