Cell-cell interaction in a synthetic co-culture: PHA production from sunlight and CO2 co-cultivating Synechococcus elongatus and Pseudomonas putida

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Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications, as the division of labor allows them to perform tasks otherwise not possible. However, the application of such a consortium in a bioprocess requires tight and individual controllability of the involved microbes. A prerequisite towards the full control is the thorough understanding of the individual events taking place in each strain and their interaction. The artificial synthetic consortium under examination in this project is the association of a cyanobacterium providing the feedstock for the heterotrophic bacterium, Pseudomonas putida. The combination of the cyanobacterial carbohydrates as feedstock together with the biosynthetic potential of P. putida, an emerging workhorse in Synthetic Biology, opens a wide range of applications. In more detail, the synthetic consortium consisting of Synechococcus elongatus PCC7942 cscB and P. putida produces polyhydroxyalkanoates (PHA) from light and CO2.The cyanobacterium S. elongatus cscB employed in this co-cultivation was engineered to excrete sucrose, when exposed to elevated salt concentrations. This sugar then in turn serves as carbon source for P. putida and is converted to PHA. The starting point of the last funding period was this one-pot process, where S. elongatus cscB secreted sufficient sucrose to support the growth and PHA accumulation of P. putida with a maximal titer of ~150 mg PHA L-1. In the first funding period, this process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually and defined conditions could be set. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg L-1 and a PHA production rate of 42.1 mg L -1 day -1.In this proposal for the renewal of the project, we pursue to explore the potential of the artificial synthetic consortium on different levels. On the process level, we plan to employ high cell density cultivations and the better suited S. elongatus UTEX2973 to improve PHA yield. On the cellular level, we will analyse the regulatory events identified by omics technologies, investigate the influence of the bacterial surface structures on the cell-cell interaction, assess the stability of the co-culture by targeted contaminations, and set up a population model. In a collaboration with the Kohlheyer group, we will follow the co-cultivation on single cell level. We aim to to develop and analyze stoichiometric models for both populations that will result in a Inter species core model, giving a first picture of the metabolic fluxes and providing a basis for further process improvements.