Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida.

In contrast to the current paradigm of using microbial mono-cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co-culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). By encapsulating S. elongatus within a barium-alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4-DNT, enhancing the performance of the co-culture. The synthetic consortium was able to convert 2,4-DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose-dependent accumulation of polyhydroxyalkanoate, an added-value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4-DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs.

Cyanobacterial Surface Display System Mediates Engineered Interspecies and Abiotic Binding.

Cyanobacteria are uniquely suited for the development of sustainable bioproduction platforms but are currently underutilized in scaled applications in part due to a lack of genetic tools. Here, we develop a surface display system in the cyanobacterial model Synechococcus elongatus PCC7942 via expression of modified versions of the outer membrane porin SomA. Importantly, we demonstrate accessibility of heterologous functional groups on the recombinant porin to the external environment in living cells. We show that this requires the removal of occluding factors that include lipopolysaccharides and a putative surface layer protein. Displayed epitopes on SomA can be utilized to mediate physical adhesion between living cyanobacteria and abiotic surfaces or an engineered Saccharomyces cerevisiae partner strain. We show that >80% of cyanobacterial cells attach to functionalized magnetic beads, allowing for magnet-assisted recovery. This work showcases the development of a functional surface display system in cyanobacteria with wide-ranging applications.