Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources

Abstract

Cyanobacteria, such as Synechococcus sp. PCC 7002 (Syn7002), are promising chassis strains for "green" biotechnological applications as they can be grown in seawater using oxygenic photosynthesis to fix carbon dioxide into biomass. Their other major nutritional requirements for efficient growth are sources of nitrogen (N) and phosphorus (P). As these organisms are more economically cultivated in outdoor open systems, there is a need to develop cost-effective approaches to prevent the growth of contaminating organisms, especially as the use of antibiotic selection markers is neither economically feasible nor ecologically desirable due to the risk of horizontal gene transfer. Here we have introduced a synthetic melamine degradation pathway into Syn7002 and evolved the resulting strain to efficiently use the nitrogen-rich xenobiotic compound melamine as the sole N source. We also show that expression of phosphite dehydrogenase in the absence of its cognate phosphite transporter permits growth of Syn7002 on phosphite and can be used as a selectable marker in Syn7002. We combined these two strategies to generate a strain that can grow on melamine and phosphite as sole N and P sources, respectively. This strain is able to resist deliberate contamination in large excess and should be a useful chassis for metabolic engineering and biotechnological applications using cyanobacteria.

Metabolic engineering of phosphite metabolism in Synechococcus elongatus PCC 7942 as an effective measure to control biological contaminants in outdoor raceway ponds

“…PC6803, S. elongatus PCC 7942, Synechococcus sp. PCC 7002 [21,22,26]. Furthermore, the system was also re ned to develop biocontainment strategies through more complex molecular strategies [21,22,23].…”

“…However, no Phi transporters have been experimentally identi ed to date in many other microalgae or cyanobacteria, including S. elongatus PCC 7942 and Synechococcus sp. PCC 7002, and efforts to identify them using genomic information available for have been also unsuccessful [21,22,38]. Although the physicochemical properties of Phi and Pi are distinct, it has been proposed that due to their structural similarities, Phi enters plant and microalgae cells using the same protein that transport Pi into the cell [35,39,40].…”

“…Previous works demonstrated that the PtxD/Phi system can be used as an effective alternative selectable marker in Synechococcus sp. PCC 7002 [22]. To test whether the system can be also implemented in S. elongatus PCC 7942, an aliquot of cells (8.7 x10 8 cells/mL) were naturally transformed with the psyn6_PtxDopt vector and spread directly onto agar plates with 1.8 mM Phi as sole P source, that resulted a very effective Phi concentration to suppress cell growth ( Supplementary Figure 1, Additional le).…”

“…This system allows the establishment of a highly selective environment by using Phi as a growth control agent and P source at the same time. More recently, the Phi metabolism was also established as a biocontainment strategy, that allows the control of proliferation of genetically modi ed S. elongatus in case an accidental release to the environment, as well as a selectable marker for microalgae, cyanobacteria, and several plant species [21,22,23,24,25]. Biotechnological applications of the selective nutrition based on Phi and the ptxD gene for microalgae/cyanobacteria cultivation have been successful as a proof of concept using small volume closed or semi-closed systems [20,21,23,26].…”

Progress and challenges in engineering cyanobacteria as chassis for light‐driven biotechnology

“…Furthermore, the metabolic load of multiple resistances can negatively affect cell performance, and antibiotic use is economically and ecologically undesirable in scaled‐up ‘real world’ applications. Strategies utilizing alternative phosphorus (phosphite) and nitrogen (melamine) sources for selection circumvent the antibiotic issue (Polyviou et al , ; Selão et al , ), but the problem of marker recycling remains. Marker‐less editing is therefore desirable and two‐stage selection/counter‐selection (Lea‐Smith et al , ) and site‐specific recombinase (Tan et al , ) systems can be used in cyanobacteria.…”