N(epsilon)-acetyl-b-lysine is an archaeal compatible solute whose synthesis is mediated by the sequential reactions of the lysine-2,3-aminomutase AblA and the acetyltransferase AblB. alpha-Lysine serves as the precursor and is converted by AblA to beta-lysine, and AblB then acetylates this intermediate to N(epsilon)-acetyl-beta-lysine. The biochemical and biophysical properties of N(epsilon)-acetyl-beta-lysine have so far not been studied intensively due to restrictions in the supply of this compound. A search for ablAB-like genes in the genomes of members of the family Bacillaceae revealed the yodP-kamA genes that encode a AblA-related lysine-2,3-aminomutase and AblB-related putative acetyltransferase. In Bacillus subtilis, the yodP-kamA genes are part of a transcriptional unit (yodT-yodS- yodR-yodQ-yodP-kamA) whose expression is upregulated during sporulation and controlled by the mother-cellspecific transcription factor SigE. N(epsilon)-acetyl-beta-lysine was not detectable in vegetatively growing or osmotically stressed B. subtilis cells, and the deletion of the yodT-yodS-yodRyodQ- yodP-kamA region had no noticeable effects on growth in rich or minimal media or osmotic stress resistance. However, when we expressed the yodP-kamA genes outside their natural genetic context from an isopropyl beta-D-1-thiogalactopyranoside- inducible promoter on a plasmid in B. subtilis, the recombinant strain synthesized considerable amounts (0.28 mu mol/mg protein) of N(epsilon)-acetyl-beta-lysine. The data reported here thus open the bottleneck for the large-scale production of N(epsilon)-acetyl-beta-lysine to investigate its properties as a compatible solute.