METHOD FOR PRODUCING RECOMBINANT STRAIN CONTAINING BIOSYNTHETIC CLUSTER ENCODING GENETICALLY MODIFIED POLAMETIDE SYNTHASE OF RAPAMICIN

摘要

1. A method for producing a recombinant strain that contains a biosynthetic cluster encoding a genetically modified rapamycin polyketide synthase, where the methylmalonyl-CoA-specific AT domain of module 10 is replaced with malonyl-CoA-specific AT domain. ! 2. The method according to claim 1, where the methylmalonyl-CoA-specific AT domain of module 10 is replaced by the malonyl-CoA-specific AT domain of one of the following clusters: rapamycin, monensin, FK506, erythromycin, FK520, amphotericin, angolamycin, tylosin, Hig FK523, Meridamycin, Antascomycin, FK525 and Tsukubamycin. ! 3. The method according to claim 2, where the malonyl-CoA-specific AT domain is selected from one of the following clusters: rapamycin, monensin and FK506. ! 4. The method according to claim 3, where the AT domain is selected from the group consisting of the AT domain of rapamycin module 2, the AT domain of monensin module 3, the AT domain of monensin module 6, the AT domain of monensin module 8, and the AT domain of the module 3 FK506 and AT domain of module 7 FK506. ! 5. The method according to claim 4, where malonyl-CoA-specific AT domain is derived from rapamycin module 2. ! 6. The method according to any one of claims 1 to 5, where the method includes additional steps:! (a) isolating the AT to be introduced as a single DNA fragment with suitable flanking restriction enzyme binding sites,! (b) amplification and isolation of regions of the DNA sequence homologous to the flanking sequences of the AT target using suitable restriction enzyme binding sites,! (c) ligating together the three DNA fragments described in (a) and (b) to obtain, in the reading frame, a left-handed (LHS) homology sequence followed by an AT donor domain followed by a right-handed (RHS) homology,! (d) the introduction of a complete sequence