Lasso peptide biosynthesis requires at least three genes, referred to as the A, B, and C proteins. The A gene encodes the precursor peptide, which is modified by the B and C proteins into the mature natural product. The B protein is an adenosine triphosphate-dependent cysteine protease that cleaves the leader region from the precursor peptide. The C protein displays homology to asparagine synthetase and is thought to activate the carboxylic acid side chain of a glutamate or aspartate residue via adenylylation. The N-terminal amine formed by the B protein (protease) then reacts with this activated side chain to form the macrocycle-forming isopeptide bond. The exact steps and reaction intermediates in lasso peptide biosynthesis remain unknown due to experimental difficulties associated with the proteins. Commonly, the B protein is referred to as the lasso protease, and the C protein is referred to as the lasso cyclase.
Some lasso peptide biosynthetic gene clusters also require an additional protein of unknown function for biosynthesis. Additionally, lasso peptide gene clusters usually include an ABC transporter (D protein) or an isopeptidase, although these are not strictly required for lasso peptide biosynthesis and are sometimes absent.  No X-ray crystal structure is yet known for any lasso peptide biosynthetic protein.
The biosynthesis of lasso peptides is particularly interesting due to the inaccessibility of the threaded-lasso topology to chemical peptide synthesis.