BackgroundAs autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions.ResultsFirst, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain.ConclusionsHigh expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0514-7) contains supplementary material, which is available to authorized users.

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Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth–associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of Synechococcus 7942. We also determined that the fast-growth–associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD+ kinase with significantly improved kinetics. The rpaA SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.

At the genome level, Synechococcus elongatus UTEX 2973 (Synechococcus 2973) is nearly identical to the model cyanobacterium Synechococcus elongatus PCC 7942 (Synechococcus 7942) with only 55 single nucleotide differences separating the two strains. Despite the high similarity between the two strains, Synechococcus 2973 grows three times faster, accumulates significantly more glycogen, is tolerant to extremely high light intensities, and displays higher photosynthetic rates. The high homology between the two strains provides a unique opportunity to examine the factors that lead to increased photosynthetic rates. We compared the photophysiology of the two strains and determined the differences in Synechococcus 2973 that lead to increased photosynthetic rates and the concomitant increase in biomass production. In this study, we identified inefficiencies in the electron transport chain of Synechococcus 7942 that have been alleviated in Synechococcus 2973. Photosystem II (PSII) capacity is the same in both strains. However, Synechococcus 2973 exhibits a 1.6-fold increase in PSI content, a 1.5-fold increase in cytochrome b6f content, and a 2.4-fold increase in plastocyanin content on a per cell basis. The increased content of electron carriers allows a higher flux of electrons through the photosynthetic electron transport chain, while the increased PSI content provides more oxidizing power to maintain upstream carriers ready to accept electrons. These changes serve to increase the photosynthetic efficiency of Synechococcus 2973, the fastest growing cyanobacterium known.

cyanobacteria are attractive microbial hosts for production of chemicals using light and co 2. However, their low productivity of chemicals is a major challenge for commercial applications. this is mostly due to their relatively slow growth rate and carbon partitioning toward biomass rather than products. Many cyanobacterial strains synthesize sucrose as an osmoprotectant to cope with salt stress environments. in this study, we harnessed the photosynthetic machinery of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce sucrose under salt stress conditions and investigated if the high efficiency of photosynthesis can enhance the productivity of sucrose. By expressing the sucrose transporter cscB, Synechococcus 2973 produced 8 g L −1 of sucrose with a highest productivity of 1.9 g L −1 day −1 under salt stress conditions. the salt stress activated the sucrose biosynthetic pathway mostly via upregulating the sps gene, which encodes the rate-limiting sucrose-phosphate synthase enzyme. to alleviate the demand on high concentrations of salt for sucrose production, we further overexpressed the sucrose synthesis genes in Synechococcus 2973. The engineered strain produced sucrose with a productivity of 1.1 g L −1 day −1 without the need of salt induction. the engineered Synechococcus 2973 in this study demonstrated the highest productivity of sucrose in cyanobacteria. Microbial production of fuels and commodity chemicals provides alternative solutions to reduce the reliance on fossil fuel. However, the requirement of sugar feedstock is one of the challenges for sustainable bioproduction. Cyanobacteria are photosynthetic prokaryotes that use light, CO 2 , and trace amounts of minerals for growth. Compared to terrestrial plants, cyanobacteria have higher efficiencies to utilize solar energy 1. In recent years, many synthetic biology tools have been developed for cyanobacteria 2. These tools have enabled metabolic engineering of cyanobacteria to produce various chemicals, including fuels 3 , petrochemicals 4 , sugars 5 , fragrances 6 , and biopolymers 7. Although cyanobacteria demonstrate the potential of converting CO 2 into desired products, most of the reported titers and productivities are still too low for commercial applications 8,9. A more efficient photosynthetic chassis is needed to improve CO 2 utilization and carbon partitioning toward products. Sucrose is an important feedstock in food industry and bioethanol production. Cyanobacteria synthesize sucrose as a compatible solute to tolerate high salt environments. By synthesizing sucrose, the osmotic pressure can be maintained to avoid desiccation in salt stress conditions. Studies of various cyanobacterial strains showed that more than 60 strains accumulate sucrose under high salt conditions 10. In cyanobacterial cells, sucrose is synthesized from uridine diphosphate glucose (UDP-Glu) and fructose 6-phosphate (F6P) by sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP) (Fig. 1A). CscB is a sucrose/H + symporter which b...