4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) is incorporated into the process of their biosynthesis for FK506, FK520 and rapamycin as a starter unit. fkbO (in FK506/FK520 biosynthesis pathway) and rapK (in rapamycin biosynthesis pathway) are known to encode chorismatase which plays an important role in the conversion of chorismate to 4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid (DCDC). Then DCDC turns to DHCHC, which is enlongated by a series of condensation steps. The biosynthesis of these immunosuppressive agents can be completed by lysine-derived pipecolate attachment, cyclization, and post-PKS reactions.. We conducted mutational biosynthesis of a new FK506 analogue by feeding non-natural starter units to fkbO deletion mutant strain of Streptomyces sp. KCTC 11604BP. The new synthesized compound, 32-dehydroxy-FK506 showed reduced immunosuppressive activity compared to FK506 while maintaining similar neurite outgrowth activity to the parent compound FK506. These results provide the potential of 32-dehydroxy-FK506 as a novel neuroregenerative agent and demonstrate the effectiveness of this mutational biosynthetic approach.