POMBOX: A Fission Yeast Cloning Toolkit for Molecular and Synthetic Biology.

The fission yeast 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 annotations 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 1 transcriptional unit. We developed a toolkit to address this gap. Adapted from the MoClo-YTK plasmid kit for Saccharomyces cerevisiae and using the same modular cloning grammar, our POMBOX toolkit is designed to facilitate fast, efficient, and modular construction of genetic circuits inS. pombe. It allows for interoperability when working with DNA sequences that are functional in bothS. cerevisiae and S. pombe (e.g., protein tags, antibiotic resistance cassettes, and coding sequences). Moreover, POMBOX enables the modular assembly of multigene pathways and increases the 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 rates depending on different sequence sizes, from 4 to 24 kb. We included 14 S. pombe promoters that we characterized using two fluorescent proteins, in both minimally defined (EMM2─Edinburgh minimal media) and complex (YES─yeast extract with supplements) media. Then, we examined the efficacy of 6 S. cerevisiae and 6 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 aromatic amino acid pathways.

Natural Variation in OASC Gene for Mitochondrial O-Acetylserine Thiollyase Affects Sulfate Levels in Arabidopsis.

Sulfur plays a vital role in the primary and secondary metabolism of plants, and carries an important function in a large number of different compounds. Despite this importance, compared to other mineral nutrients, relatively little is known about sulfur sensing and signalling, as well as about the mechanisms controlling sulfur metabolism and homeostasis. Sulfur contents in plants vary largely not only among different species, but also among accessions of the same species. We previously used associative transcriptomics to identify several genes potentially controlling variation in sulfate content in the leaves of Brassica napus, including an OASC gene for mitochondrial O-acetylserine thiollyase (OAS-TL), an enzyme involved in cysteine synthesis. Here, we show that loss of OASC in Arabidopsis thaliana lowers not only sulfate, but also glutathione levels in the leaves. The reduced accumulation is caused by lower sulfate uptake and translocation to the shoots; however, the flux through the pathway is not affected. In addition, we identified a single nucleotide polymorphism in the OASC gene among A. thaliana accessions that is linked to variation in sulfate content. Both genetic and transgenic complementation confirmed that the exchange of arginine at position 81 for lysine in numerous accessions resulted in a less active OASC and a lower sulfate content in the leaves. The mitochondrial isoform of OAS-TL is, thus, after the ATPS1 isoform of sulfurylase and the APR2 form of APS reductase 2, the next metabolic enzyme with a role in regulation of sulfate content in Arabidopsis.