POMBOX: a fission yeast toolkit for molecular and synthetic biology

Schizosaccharomyces pombe is 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. pombe is an attractive option for synthetic biology applications. However, S. pombe currently 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 for Saccharomyces cerevisiae and using the same Golden Gate grammar, our POMBOX toolkit is designed to facilitate the fast, efficient and modular construction of genetic circuits in S. pombe. It allows for interoperability when working with DNA sequences that are functional in both S. cerevisiae and S. 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 fourteen S. pombe promoters that we characterized for two fluorescent proteins, in both minimal defined media (EMM2) and complex media (YES). Then we tested six S. cerevisiae and six synthetic terminators in S. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes in S. pombe to produce specialized metabolite precursors, namely methylxanthine, amorpha-4,11-diene and cinnamic acid from the purine, mevalonate and amino acid pathways.