Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis

Abstract

Background: Successful 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.

Results: Tools 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.

Conclusions: The 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.

Keywords: Cyanobacterial cell factories; Cyanobacterial chassis; Cyanobacterial genetic engineering; Cyanobacterial host; Synechococcus; Synechococcus 7002; Synechococcus sp. PCC 7002.

Fig. 1

Transformation efficiency of Synechococcus sp. PCC 7002 as a function of incubation time with varying lengths of homology arms (250–1250 bp) for genome integration at desB

Fig. 2

Fluorescence emission spectra of Synechococcus sp. PCC 7002 (wild type) and engineered strains expressing hGFP, Ypet, and mOrange with excitation at 488 nm

Fig. 3

Fluorescence microscopy images of wild type Synechococcus sp. PCC 7002 (a, b) and engineered strains expressing hGFP (c, d), Ypet (e, f), and mOrange (g, h). Chlorophyll-a fluorescence (Ex 484/Em >600) is shown in a, c, e, and g while the recombinant fluorescent protein fluorescence (Ex 484/Em 520—GFP, Ex 500/Em 520—Ypet, and Ex 534/Em 597.6—mOrange) is shown in b, d, f, and h. Scale bar 10 µm

Fig. 4

Average normalized expression levels of native Synechococcus sp. PCC 7002 promoters, as measured by Ypet fluorescence (Ex 485 nm, Em 528 nm) under continuous and diurnal light conditions. Ypet fluorescence was measured in each engineered strain across a 3 week period of growth and normalized to the wild type fluorescence. The normalized fluorescence values are averaged across the growth period, and at least three biological replicates are included. Normalized Ypet fluorescence values having a change that is statistically significant (two-tail p value <0.05) compared to the wild type are indicated with an asterisk. Continuous light conditions are approximately 60 µmol photons m⁻² s⁻¹, and diurnal light conditions consist of 12:12 light dark cycles using the same light intensity