Pyrrolysine is the 22nd amino acid that is encoded by the natural genetic code. In the archaebacterial family Methanosarcinaceae, its incorporation into three proteins (MtmB, MtbB, and MttB) involved in the methylamine catabolic pathway is specified by the amber stop codon UAG. The unusual amino acid was discovered in 2002 by crystallography and mass spectrometry of MtmB, MtbB, and MttB. Asophisticated bioinformatics strategy has shown a set of five genes (pylBCDST) to be both necessary and sufficient for the biosynthesis and utilization of Pyrrolysin. More specifically, pylT and pylS code for a pyrrolysine tRNA and its cognate amino acyl tRNA synthetase, whereas three enzymes specified by pylB, pylC, and pylD catalyze pyrrolysine biosynthesis. Incorporation experiments using lysine and recombinant Escherichia coli strains engineered for expression of mtmB and pylBCDST genes of Methanosarcina barkeri, showed that all the carbon and nitrogen atoms of Pyrrolysine are derived from lysine.