In the present study, we introduced point mutations into Ac_rapA which encodes a
polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109,
in order to construct a mutant with an inactivated enoylreductase (ER) domain, which
was able to synthesize a new rapamycin analog. Based on the homologous recombination
induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the
site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating
plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues
of the active center, Ala–Gly–Gly, were converted to Ala–Ser–Pro. The broth of the
mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum
to that of rapamycin was found. The sample of the new peak was prepared by solvent
extraction, column chromatography, and crystallization methods. The structure of new
compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS
and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction
(MLR). An ER domain–deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027,
was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure
was elucidated to be 35, 36-didehydro-27- O -demethylrapamycin. The biological activity
of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the
first ER domain of rapA, one of the modular polyketide synthase responsible for
macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a
novel rapamycin analog, 35, 36-didehydro-27- O -demethylrapamycin, demonstrating that
the enoylreductase domain was responsible for the reduction of the double bond between
C-35 and C-36 during rapamycin synthesis.