Daniel C Ducat
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Researcher at Michigan State University, East Lansing, Michigan, USA
Microbial Biotechnology, 2020-02-16
In contrast to the current paradigm of using microbial monocultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to program into individual strains. Additionally, the division of labor between specialist species found in natural consortia can lead to increased catalytic efficiency and stability relative to a monoculture or a community composed of generalists. In this work, we have designed a synthetic co-culture for phototrophic degradation of xenobiotics, composed of a cyanobacterium, (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). 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 as the only carbon source and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). The synthetic consortium is able to degrade 2,4-DNT with only light and CO2 as inputs for the system, and it was stable over time through repeated backdilutions. Furthermore, cycling this consortium through low nitrogen medium promoted the accumulation of polyhydroxyalkanoate (PHA)-an added-value biopolymer-in P. putida, thus highlighting the versatility of this production platform. Altogether, the synthetic consortium allows for simultaneous bioproduction of PHA and remediation of the industrial pollutant 2,4-DNT, using light and CO2 as inputs. Importance: In this study, we have created an artificial consortium composed of two bacterial species that enables the degradation of the industrially-produced environmental pollutant 2,4-DNT while simultaneously producing PHA bioplastic. In these co-cultures, the photosynthetic cyanobacteria fuel an engineered P. putida strain programmed both to use sucrose as a carbon source and to perform the biotransformation of 2,4-DNT. The division of labor in this synthetic co-culture is reminiscent of that commonly observed in microbial communities and represents a proof-of-principle example of how artificial consortia can be employed for bioremediation purposes. Furthermore, this co-culture system enabled the utilization of freshwater sources that could not be utilized in classical agriculture settings, reducing the potential competition of this alternative method of bioproduction with current agricultural practices, as well as remediation of contaminated water streams.
Journal of Biological Engineering, 2017-01-23
BACKGROUND: Microbial consortia composed of autotrophic and heterotrophic species abound in nature, yet examples of synthetic communities with mixed metabolism are limited in the laboratory. We previously engineered a model cyanobacterium, Synechococcus elongatus PCC 7942, to secrete the bulk of the carbon it fixes as sucrose, a carbohydrate that can be utilized by many other microbes. Here, we tested the capability of sucrose-secreting cyanobacteria to act as a flexible platform for the construction of synthetic, light-driven consortia by pairing them with three disparate heterotrophs: Bacillus subtilis, Escherichia coli, or Saccharomyces cerevisiae. The comparison of these different co-culture dyads reveals general design principles for the construction of robust autotroph/heterotroph consortia. MAIN FINDINGS: We observed heterotrophic growth dependent upon cyanobacterial photosynthate in each co-culture pair. Furthermore, these synthetic consortia could be stabilized over the long-term (weeks to months) and both species could persist when challenged with specific perturbations. Stability and productivity of autotroph/heterotroph co-cultures was dependent on heterotroph sucrose utilization, as well as other species-independent interactions that we observed across all dyads. One interaction we observed to destabilize consortia was that non-sucrose byproducts of photosynthesis negatively impacted heterotroph growth. Conversely, inoculation of each heterotrophic species enhanced cyanobacterial growth in comparison to axenic cultures Finally, these consortia can be flexibly programmed for photoproduction of target compounds and proteins; by changing the heterotroph in co-culture to specialized strains of B. subtilis or E. coli we demonstrate production of alpha-amylase and polyhydroxybutyrate, respectively. CONCLUSIONS: Enabled by the unprecedented flexibility of this consortia design, we uncover species-independent design principles that influence cyanobacteria/heterotroph consortia robustness. The modular nature of these communities and their unusual robustness exhibits promise as a platform for highly-versatile photoproduction strategies that capitalize on multi-species interactions and could be utilized as a tool for the study of nascent symbioses. Further consortia improvements via engineered interventions beyond those we show here (i.e. increased efficiency growing on sucrose) could improve these communities as production platforms.
Carboxysomes are protein-based bacterial organelles that encapsulate a key enzyme of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we show that a small protein, termed McdB, localizes to carboxysomes through interactions with carboxysome shell proteins to drive emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly interacts to stimulate McdA ATPase activity, and indicate that carboxysome-dependent McdA depletion zone formation on the nucleoid is required for directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a new class of self-organizing proteins that follow a Brownian-ratchet mechanism, challenging the cytoskeletal model of organelle transport, for equidistant positioning of carboxysomes in cyanobacteria. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria more broadly.