Abstract
BackgroundSuccessful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. The cyanobacterium Synechococcus sp. PCC 7002 embodies key attributes for an industrial host, including a fast growth rate and high salt, light, and temperature tolerances. This study addresses key limitations in the advancement of Synechococcus sp. PCC 7002 as an industrial chassis.ResultsTools for genome integration were developed and characterized, including several putative neutral sites for genome integration. The minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 was determined to be approximately 250 bp. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) were characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002. Of these three proteins, the yellow fluorescent protein (Ypet) had the best optical properties for minimal interference with the native photosynthetic pigments and for detection using standard microscopy and flow cytometry optics. Twenty-five native promoters were characterized as tools for recombinant gene expression in Synechococcus sp. PCC 7002 based on previous RNA-seq results. This characterization included comparisons of protein and mRNA levels as well as expression under both continuous and diurnal light conditions. Promoters A2520 and A2579 were found to have strong expression in Synechococcus sp. PCC 7002 while promoters A1930, A1961, A2531, and A2813 had moderate expression. Promoters A2520 and A2813 showed more than twofold increases in gene expression under light conditions compared to dark, suggesting these promoters may be useful tools for engineering diurnal regulation.ConclusionsThe genome integration, fluorescent protein, and promoter tools developed in this study will help to advance Synechococcus sp. PCC 7002 as a cyanobacterial chassis. The long minimum homology arm length for Synechococcus sp. PCC 7002 genome integration indicates native exonuclease activity or a low efficiency of homologous recombination. Low correlation between transcript and protein levels in Synechococcus sp. PCC 7002 suggests that transcriptomic data are poor selection criteria for promoter tool development. Lastly, the conventional strategy of using promoters from photosynthetic operons as strong promoter tools is debunked, as promoters from hypothetical proteins (A2520 and A2579) were found to have much higher expression levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0584-6) contains supplementary material, which is available to authorized users.
Highlights
Successful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis
Genetic tools developed for one cyanobacterial host are often directly used in another [13, 17], and cellular processes studied in one cyanobacterium are frequently assumed to be similar, if not identical, in another cyanobacterium
In order to achieve advancements similar to that achieved in E. coli, we recommend that metabolic engineers focus on a single cyanobacterial host or chassis
Summary
Successful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. Strain development efforts for cyanobacteria are often limited by the availability of Ruffing et al Microb Cell Fact (2016) 15:190 characterized genetic tools and our insufficient understanding of cyanobacterial metabolism and regulation. Complicating these efforts is the fact that multiple, diverse cyanobacterial hosts are used. While some tools and cellular processes may be universal to cyanobacteria or eubacteria, this generalization among cyanobacterial species may limit their advancement as industrial hosts. In order to achieve advancements similar to that achieved in E. coli, we recommend that metabolic engineers focus on a single cyanobacterial host or chassis
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