Co-cultures are attractive microcosms for studying metabolic interactions in a controlled system. Co-cultures involving diazotrophs, or organisms capable of reducing atmospheric nitrogen into ammonia, are of particular interest sustainable alternatives to the Haber-Bosch process a source of nitrogen fertilizer. While diazotrophs like Azotobacter vinelandii have successfully been engineered to produce ammonium aerobically, these strains also exhibit a high substrate demand. Indeed, we have previously shown that the majority (88-90%) of the carbon consumed by A. vinelandii is spent on respiration, ultimately to protect its nitrogenase from oxygen, rather than ATP production for fueling nitrogen fixation and other cellular processes. Thus, a major challenge in engineering stable co-cultures with nitrogen-fixers is to identify partners that can meet the high substrate demands of these diazotrophs while receiving limited nitrogen in return. We investigated the performance of four, fully nitrogen self-sufficient co-cultures involving an ammonium-secreting strain of A. vinelandii paired with both wild-type and various engineered E. coli strains. 13C-metabolic flux analysis of these co-cultures unexpectedly revealed that A. vinelandii shares the majority of its fixed nitrogen. For example, even in commensalist pairings, E. coli constituted more than 60-75% of an exponentially growing co-culture population. Furthermore, using 13C-MFA, we identified additional cross-fed nutrients that are not otherwise secreted by either organism when grown in isolation. Finally, in evolution studies, we were able to isolate a clone of one of our engineered E. coli strains that had gained the ability to continue producing glucose for A. vinelandii even under complete nitrogen starvation.

Engineering obligate mutualism in co-cultures of ammonium-secreting Azotobacter vinelandii and glucose-secreting Escherichia coli

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.