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Schizosaccharomyces pombeis a popular model organism in molecular biology and cell physiology. With its ease of genetic manipulation and growth, supported by in-depth functional annotation in the PomBase database and genome-wide metabolic models,S. pombeis an attractive option for synthetic biology applications. However,S. pombecurrently lacks modular tools for generating genetic circuits with more than one transcriptional unit. We have developed a toolkit to address this issue. Adapted from the MoClo- YTK plasmid kit forSaccharomyces cerevisiaeand using the same Golden Gate grammar, our POMBOX toolkit is designed to facilitate the fast, efficient and modular construction of genetic circuits inS. pombe. It allows for interoperability when working with DNA sequences that are functional in bothS. cerevisiaeandS. pombe(e.g. protein tag, antibiotic resistance cassette, coding sequences). Moreover, POMBOX enables the modular assembly of multi-gene pathways and increases possible pathway length from 6 to 12 transcriptional units. We also adapted the stable integration vector homology arms to Golden Gate assembly and tested the genomic integration success rate depending on different sequence sizes, from four to twenty-four kilobases. We included fourteenS. pombepromoters that we characterized for two fluorescent proteins, in both minimal defined media (EMM2) and complex media (YES). Then we tested sixS. cerevisiaeand six synthetic terminators inS. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes inS. pombeto produce specialized metabolite precursors, namely methylxanthine, amorpha-4,11-diene and cinnamic acid from the purine, mevalonate and amino acid pathways. |
