l-Glutamate exporters/importers responsible for diazotrophic l-glutamate production in engineered Klebsiellapasteurii.

Diazotrophic L-glutamate (L-Glu) fermentation, independent of the Haber-Bosch process, is a sustainable strategy. Following our successful production of L-Glu from aerial nitrogen by an engineered Klebsiella pasteurii NG13 overproducing citrate synthase and citrate transporters (+CC strain), we herein examined L-Glu transporters in this bacterium. Extracellular L-Glu accumulation was abolished in a K. pasteurii strain in which a gene homologous to Escherichia coli ybjL was deleted, suggesting that the YbjL homologous protein (herein designated as GluE) was the main exporter of the +CC strain for diazotrophic L-Glu production. However, the overexpression of gluE did not increase diazotrophic L-Glu production, suggesting that the export of L-Glu was not a rate-limiting step in diazotrophic L-Glu production in the +CC strain. To sustain and increase diazotrophic L-Glu production, we constructed a triple knockout K. pasteurii +CC strain lacking the putative gltS, gltP, and gltIJKL orthologs, all the counterparts of which encode L-Glu importers in E. coli. In glucose-fed cultures, while L-Glu produced extracellularly by the +CC strain decreased during a long-term cultivation, the triple knockout strain sustained L-Glu production over the culture period and finally reached 2.67 g L-1. These results suggest the potential of the practical application of our system to the production of nitrogen-containing compounds from aerial nitrogen.