Origin of the Allyl Group in FK506 Biosynthesis

Abstract

FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex.FK506 (tacrolimus) and its structural analogs FK520 (ascomycin) and rapamycin (sirolimus) are secondary metabolites belonging to a class of macrolactone compounds of great medicinal importance. They are currently registered for use as immunosuppressants after organ transplantation as well as for treatment of inflammatory skin diseases and eczema (1-3). In recent clinical studies, these compounds have also shown great promise for treatment of various forms of cancer, as well as for neurological disorders (4, 5). FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase (PKS) 2 /nonribosomal peptide synthetase systems. The allyl group at carbon 21 of FK506 is replaced by an ethyl group in FK520, this being the only structural difference between the FK506 and FK520 compounds (see Fig. 1). A common feature of these complex polyketides is the involvement of large polyfunctional polyketide synthases comprising multifatty acid synthase-like domains arranged in sets of modules (6). Each module is responsible for the recognition and incorporation of a specific carboxylic acid-derived extender unit into the growing polyketide chain as well as for subsequent reductive reactions (7,8). Compounds such as FK506/520 and rapamycin are formed by incorporation of lysine-derived pipecolic acid by the nonribosomal peptide synthase-like domain through the cyclization step, resulting in the fo...

Roles of fkbN in Positive Regulation and tcs7 in Negative Regulation of FK506 Biosynthesis in Streptomyces sp. Strain KCTC 11604BP

“…strain ATCC 55098 (MA6858) (26), Streptomyces sp. strain ATCC 53770 (MA6548) (25), and S. tsukubaensis NRRL 18488 (8). Recently, the sequences of the entire FK506 biosynthetic gene clusters from Streptomyces sp.…”

Annotation of the Modular Polyketide Synthase and Nonribosomal Peptide Synthetase Gene Clusters in the Genome of Streptomyces tsukubaensis NRRL18488

“…As described previously, a seed culture was used for the inoculation of 250-ml Erlenmeyer flasks containing 50 ml of PG3 production medium (9), and cultivation was carried out at 28°C at 220 rpm for 62 h. A 2-ml sample was collected and mixed thoroughly with 4 ml of RNAlater RNA stabilization reagent (Qiagen). After 5 min of incubation at room temperature, the mixture was stored at Ϫ80°C.…”

“…Culture conditions for S. tsukubaensis NRRL18488 were described previously (9). The isolation and manipulation of genomic DNA were carried out according to standard methods (15,23).…”

“…Streptomyces tsukubaensis NRRL18488 is a natural producer of the medically important immunosuppressant FK506, the biosynthesis of which is encoded by a large biosynthetic cluster containing three modular PKS genes (9,20). S. tsukubaensis NRRL18488 is also the wild-type progenitor of most industrially used FK506-producing strains (16).…”

FK506 Maturation Involves a Cytochrome P450 Protein-Catalyzed Four-Electron C-9 Oxidation in Parallel with a C-31O-Methylation

“…5A) (12,13). During the macrolide-forming process, the PKSs for FK506 incorporate two unusual extender units, methoxymalonyl (MOM) (also common for FK520) and allylmalonyl (AM) (unique for FK506), to afford two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, respectively (7,(14)(15)(16). Genetic enhancement of the in vivo supply of MOM and AM improved FK506 production (17), therefore providing a strategy potentially helpful for increasing the yields of other secondary metabolites that contain pathway-specific building blocks.…”