Promoting nitrogen fixation by threonine synthesis in co-culture of Azotobacter vinelandii and Escherichia coli.

Amino acid production directly from N2 is a big challenge in green biomanufacturing. Merging biological nitrogen fixation (BNF) with target amino acid biosynthesis in co-culture system offers a promising solution. Here, we constructed a Nitrogen Fixation-Utilization Coupling (NFUC) system that enabled threonine production directly from N2 and ethanol, by co-culturing engineered Azotobacter vinelandii for BNF and Escherichia coli for threonine biosynthesis. Systematic metabolic engineering increased threonine titer 3.4-fold over the initial co-culture through optimizing NH4+ release, threonine pathway and energy supply. The nitrogen fixation activity of Azotobacter vinelandii was enhanced in co-culture by the demand for threonine synthesis, resulting in an improvement in total fixed nitrogen compared to its monoculture. The observed downregulation of glutamine synthetase activity in co-cultured Azotobacter vinelandii revealed its prioritization of N2 fixation over endogenous amino acid synthesis, enhancing NH4+ availability for Escherichia coli with an increase in extracellular NH4+ level. This NFUC system establishes a platform for sustainable bioproduction of amino acids directly from N2.