Systematic identification of a neutral site on chromosome of Synechococcus sp. PCC7002, a promising photosynthetic chassis strain

•A paradigm for mining new neutral sites on cyanobacteria chromosomes was provided.•51 promising neutral sites were extracted from chromosome of PCC7002.•An intergenic space between two neutral sites was identified as a more ideal site.•An ethanol synthesis pathway was integrated in PCC7002 chromosome on the new site. Cyanobacteria based photosynthetic biomanufacturing is supposed to be one of the alternative routes for sustainable production of biofuels and biochemicals. Synechococcus sp. PCC 7002 is a promising cyanobacterial chassis strain possessing desired properties for scaled cultivation in future. Development of cyanobacterial cell factories requires modifications of PCC7002 chromosome to expand the photosynthesis-based metabolism networks. Therefore, mining genomic neutral sites for stable integration of heterologous genes and pathways would be of great significance for expanding the toolbox for PCC7002 genome engineering. Here we demonstrate a paradigm for identification of potential neutral sites from chromosome of PCC7002 based on genomic and transcriptomics data. By refining the massive omics information from database, 51 putative sites with no significant physiological or metabolism effects were extracted as candidates. Combining genomic context analysis, a locus termed as NS0027 between two neighboring putative neutral genes was selected and evaluated as a neutral site for genetic integration. In addition, an ethanol synthesis pathway was introduced into the NS0027 site to assess the functionality of this site. The sites we identified and the strategy we adopted in this work would benefit the development of effective genetic toolbox and efficient photosynthetic cell factories based on PCC7002.