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 CO 2 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 CO 2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrosedependent 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 CO 2 as the primary inputs.

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.

16In contrast to the current paradigm of using microbial monocultures in most 17 biotechnological applications, increasing efforts are being directed towards engineering 18 mixed-species consortia to perform functions that are difficult to program into individual 19 strains. Additionally, the division of labor between specialist species found in natural 20 consortia can lead to increased catalytic efficiency and stability relative to a monoculture or 21 a community composed of generalists. In this work, we have designed a synthetic co-22 sucrose as a carbon source and to perform the biotransformation of 2,4-DNT. The division 41 of labor in this synthetic co-culture is reminiscent of that commonly observed in microbial 42 communities and represents a proof-of-principle example of how artificial consortia can be 43 employed for bioremediation purposes. Furthermore, this co-culture system enabled the 44 utilization of freshwater sources that could not be utilized in classical agriculture settings, 45 reducing the potential competition of this alternative method of bioproduction with 46 current agricultural practices, as well as remediation of contaminated water streams. 47 48 49 consortia; bioproduction 50 polyhydroxyalkanoate (PHA) (Figure 1). This was accomplished through the pairing of an 131 engineered strain of P. putida, containing the genes needed to both metabolize sucrose and 132 to degrade 2,4-DNT with the sucrose-exporting S. elongatus CscB encapsulated in alginate 133 hydrogel beads. Approaching the bioremediation of this compound via the synthetic 134 consortium method allows for the bioprocess to be photosynthetically powered while 135 avoiding the complexities of introducing a new enzymatic pathway into photosynthetic 136 cyanobacteria. We demonstrated that these co-cultures can successfully execute the 137 biotransformation of 2,4-DNT via the engineered pathway and are also able to accumulate 138Eng 11:1-36. 595

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