The formation of thienamycin is thought to occur through a different pathway from classic β-lactams (penicillins, cephalosporins).  Production of classic β-lactams in both fungi and bacteria occur through two steps: First, the condensation of -cysteine, -valine, and -α-aminoadipic acid by ACV synthetase (ACVS, a nonribosomal peptide synthetase) and then cyclization of this formed tripeptide by isopenicillin N synthetase (IPNS).
The gene cluster (thn) for the biosynthesis of thienamycin of S. cattleya was identified and sequenced in 2003, lending insight into the biosynthetic mechanism for thienamycin formation. The biosynthesis is thought to share features with the biosynthesis of the simple carbapenems, beginning with the condensation of malonyl-CoA with glutamate-5-semialdehyde to form the pyrroline ring. The β-lactam is then formed by a β-lactam synthetase, which makes use of ATP, providing a carbapenam. At some later point, oxidation to the carbapenem and ring inversions must occur.
The hydroxyethyl side chain of thienamycin is thought to be a result of two separate methyl transfers from S-adenosyl methionine. According to the proposed gene functions, ThnK, ThnL, and ThnP could catalyze these methyl-transfer steps. A β-lactam synthetase (ThnM) is thought to catalyze the formation of the β-lactam ring fused to the five-membered ring. How the cysteaminyl side-chain is incorporated is largely unknown, although ThnT, ThnR, and ThnH are involved in the processing of CoA to cysteamine for use in the pathway. Various oxidations complete the biosynthesis.