FK506 (tacrolimus) is a 23-membered macrolide of poliketide origin with a powerful immunosuppressive activity produced by several Streptomyces spp. It is a clinically important therapeutic drug used for preventing rejection of transplanted organs and in combating inflammatory skin diseases. FK506 can causes serious side effects primarily nephrotoxicity, neurotoxicity and arterial hypertension. Firstly, we have used the FK506-producing strain Streptomyces tsukubaensis NRRL 18488 to develop robust bioproces for FK506 production at the shaker level. However, the goal of this study was to develop a chemobiosinthetic process for production of a novel FK506 analogue by using allR inactivated mutant strain of S. tsukubaensis. The allR deletion mutant produced neither FK506, nor FK520 or propyl-FK506. The non-natural synthetic unit propargylmalonyl-SNAC was supplemented to the cultivation broth during the fermentation process of ∆allR mutant strain, which resulted in production of the new FK506 analogue propargyl-FK506. The presence of a new analogue propargyl-FK506 was confirmed by HPLC and MS. However, production of propargyl-FK506 was low as compared to the production of FK506 in wild type strain (S. tsukubaensis NRRL 18488) and production of FK506 after chemobiosinthesis with natural extender unit allylmalonly-SNAC when using the ∆allR mutant strain. Therefore, we tested different proparglymalonyl-SNAC feeding regimes. We observed significant inhibitory effect of proparglymalonyl-SNAC on the production of FK506 in wild type strain. We demonstrated that the most effective feeding regime was when supplementation of extender unit proparglymalonyl-SNAC was carried at the final concentration 0, 5 g/L at 24 °C.