AbstractBiotin is an essential cofactor required for carboxylation and decarboxylation reactions in all domains of life. While biotin biosynthesis in most Bacteria and Eukarya is well studied, the complete pathway for this vitamer in Archaea is still not known. Detailed genome searches indicated the presence of possible bio gene clusters only in Methanococcales and Thaumarchaeota. Therefore, we analysed the functionality of the predicted genes bioA, bioB, bioD and bioF in the Thaumarchaeon Nitrososphaera gargensis Ga2.9 which are essential for the later steps of biotin synthesis. In complementation tests, the gene cluster-encoded N. gargensis bioABD genes except bioF restored growth of corresponding E. coli Rosetta-gami 2 (DE3) deletion mutants. To find out how biotin biosynthesis is initiated, we searched the genome for a possible bioH analogue encoding a pimeloyl-ACP-methylester carboxylesterase. The respective amino acid sequence of the ORF estN1 showed weak conserved domain similarity to this class of enzymes (e-value 3.70e⁻⁴²). Remarkably, EstN1 is a promiscuous carboxylesterase that complements E. coli ΔbioH and Mesorhizobium loti ΔbioZ mutants for growth on biotin-free minimal medium. Additional 3D-structural models support the hypothesis that EstN1 is a BioH analogue. Thus, this is the first report providing experimental evidence that Archaea carry functional bio genes.