AbstractFormamide is rarely used as nitrogen source 
by microorganisms. Therefore, formamide and formamidase have been used as protection system to allow for growth under non-sterile conditions and for non-sterile production of acetoin, a product lacking nitrogen. Here, we equippedCorynebacterium glutamicum, a renowned workhorse for industrial amino acid production for 60 years, with formamidase fromHelicobacter pylori26695, enabling growth with formamide as sole nitrogen source. Thereupon, the formamide/formamidase system was exploited for efficient formamide-based production of the nitrogenous compounds L-glutamate, L-lysine,N-methylphenylalanine, and dipicolinic acid by transfer of the formamide/formamidase system to established producer strains. Stable isotope labeling verified the incorporation of nitrogen from formamide into biomass and the representative product L-lysine. Moreover, we showed ammonium leakage during formamidase-based access of formamide to be exploitable to support growth of formamidase-deficientC. glutamicumin co-cultivation and demonstrated that efficient utilization of formamide as sole nitrogen source benefitted from overexpression of formate dehydrogenase.Key points• C. glutamicum was engineered to access formamide.• Formamide-based production of nitrogenous compounds was established.• Nitrogen cross-feeding supported growth of a formamidase-negative strain.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-023-12592-3.