Nε-acetyl-β-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. α-Lysine serves as the precursor and is converted by AblA to β-lysine, and AblB then acetylates this intermediate to N(ε)-acetyl-β-lysine. The biochemical and biophysical properties of N(ε)-acetyl-β-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-cell-specific transcription factor SigE. N(ε)-acetyl-β-lysine was not detectable in vegetatively growing or osmotically stressed B. subtilis cells, and the deletion of the yodT-yodS-yodR-yodQ-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 β-D-1-thiogalactopyranoside-inducible promoter on a plasmid in B. subtilis, the recombinant strain synthesized considerable amounts (0.28 μmol/mg protein) of N(ε)-acetyl-β-lysine. The data reported here thus open the bottleneck for the large-scale production of N(ε)-acetyl-β-lysine to investigate its properties as a compatible solute.