In addition, genetic engineering relying on integration at neutral sites is time-consuming when performed sequentially, because all chromosomes must be segregated to stably retain the genetically modified constructs in polyploid cyanobacteria. Due to their polyploidy, chromosomal expression systems in these cyanobacteria are expected to be more effective than those in monoploid organisms, although genetic manipulation is limited to a few cyanobacteria that have natural competence and chromosomal recombination abilities. 7002 ( Wang et al., 2019), integration of genetic constructs into chromosomal neutral sites has been used for exogenous gene expression. 7120), which performs fixation of both nitrogen and carbon, is particularly suitable for the production of nitrogenous substances, and has recently been studied for ammonia production ( Higo et al., 2016). The multicellular filamentous cyanobacterium, Anabaena sp.
7002) have been used in synthetic biology studies for the biosynthesis of multiple products including biofuels ( Lan and Liao, 2011 Liu et al., 2011), antioxidants ( Shimada et al., 2020), flavors/fragrances ( Formighieri and Melis, 2015), and pharmaceuticals ( Choi et al., 2016). 6803), Synechococcus elongatus PCC 7942 ( S. Recently, three cyanobacterial model strains Synechocystis sp. Cyanobacteria have oxygen-producing photosynthetic capabilities, meaning that they can produce biomass using solar energy and CO 2, and have recently gained attention for their potential as green cell factories for CO 2-neutral biosynthesis of various products ( Knoot et al., 2018 Farrokh et al., 2019). The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.Ĭyanobacteria are the predominant phototrophs in ocean and freshwater ecosystems, and are among the most widespread phylogenetic clades. Furthermore, pYS could be used together with the conventional vector pEX, which was constructed from an endogenous plasmid in S. 7942, GFP expression in the pYS-based system was tightly regulated by IPTG, achieving 10-fold higher levels than in the chromosome-based system. A reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in not only S. PCC 6803 revealed that a certain region encoding a Rep-related protein (here named Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. Our comprehensive screening using a genomic library of Synechocystis sp. In this study, we characterized a Rep protein, exhibiting replication activity in multiple cyanobacteria and established an expression vector using this protein. However, compared to other bacteria, tools for genetic engineering, especially expression vector systems, are limited. Owing to their photosynthetic capabilities, cyanobacteria are regarded as ecologically friendly hosts for production of biomaterials. 2Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan.1Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.Yutaka Sakamaki 1, Kaisei Maeda 1,2, Kaori Nimura-Matsune 1, Taku Chibazakura 1 and Satoru Watanabe 1
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