Many of cryptophycins are promising antitumor agents against a wide variety of tumors but exhibit dose-limiting neurotoxicity in Phase II clinical trials. Recently, new methods and reagents have been developed to prepare cryptophycin analogs possibly bearing beneficial physiochemical properties and reduced side effects. In particular, a chemoenzymatic strategy employing thioesterase (CrpTE) domain and P450 expoxidase in cryptophycin biosynthesis has successfully generated many cryptophycin analogs. As a follow-up, we have developed a novel chemoenzymatic route relying on a multifunctional biosynthetic enzyme, CrpD. CrpD is a bimodular non-ribosomal peptide synthetase involved in the formation of cryptophycin macrocyclic core structure. Its second module (CrpD-M2) was heterologously overexpressed and purified in this study. Bioinformatic analysis has suggested that, contrary to previous prediction, CrpD-M2 adenylation (A) domain may activate α-ketoisocoproate or α-hydroxyisocoproate.  This was experimentally confirmed in the well-estabolished ATP/PPi exchange assay. The specificity of this domain was further probed using a series of substrate analogs. Interestingly, several non-natural substrates were recognized, thereby suggesting that CrpD-M2 has the potential to generate novel cryptophycin analogs. Furthermore, the catalytic activity of CrpD-M2 condensation domain was investigated utilizing three chemically synthesized substrates. The formation of seco-cryptophycin intermediates tethered to CrpD-M2 was tracked by FTMS analysis, demonstrating this condensation domain unusual ability to generate C-O bond. The intermediates were subesequently released and macrocyclized by in trans CrpTE to produce three cryptophycin analogs, which were confirmed by LC-MS analysis. This work represents the first example of a chemoenzymatic approach utilizing a single multifunctional enzyme to generate structurally diverse cryptophycin analogs.