Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (HPO, Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (HPO, Pi). We then identified and disrupted the two indigenous Pi transporters, pst (Synpcc7942_2441 to 2445) and pit (Synpcc7942_0184). The resultant strain failed to grow on any media containing various types of P compounds other than Pt. The strain did not yield any escape mutants for at least 28 days with a detection limit of 3.6 × 10 per colony forming unit, and rapidly lost viability in the absence of Pt. Moreover, growth competition of the Pt-dependent strain with wild-type cyanobacteria revealed that the Pt-dependent strain could dominate in cultures containing Pt as the sole P source. Because Pt is rarely available in aquatic environments this strategy can contribute to both biosafety and contamination management of genetically engineered cyanobacteria.

Contamination by the predatory zooplankton Poterioochromonas malhamensis is one of the major threats that causes catastrophic damage to commercial-scale microalgal cultivation. However, knowledge of how to manage predator contamination is limited. Previously, we established a phosphite (Pt)-based culture system by engineering Synechococcus elongatus, which exerted a competitive growth advantage against microbial contaminants that compete with phosphate source. Here, we examined whether Pt is effective in suppressing predator-type contamination. Co-culture experiment of Synechococcus with isolated P. malhamensis revealed that, although an addition of Pt at low concentrations up to 2.0 mM was not effective, increased dosage of Pt (~20 mM) resulted in the reduced grazing impact of P. malhamensis. By using unsterilized raw environmental water collected from rivers or ponds, we found that the suppression effect of Pt was dependent on the type of environmental water used. Eukaryotic microbial community analysis of the cultures using environmental water samples revealed that Paraphysomonas, a colorless Chrysophyceae, emerged and dominated under high-Pt conditions, suggesting that Paraphysomonas is insensitive to Pt compared to P. malhamensis. These findings may provide a clue for developing a strategy to reduce the impact of grazer contamination in commercial-scale microalgal cultivation.

Metrics & MoreArticle RecommendationsI n the original manuscript, we have incorrectly described the order of the fifth author's name as Abdel-Hady Gamal Nasser. We correct the name as Gamal (first name) Nasser (middle name) Abdel-Hady (family name).