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.

FIGURE 1.

The structures of FK506, FK520, and dihydro-FK506 (FK506D) differ only in the side chain bound to the carbon C21. Incorporation of the allylmalonyl-SNAC extender unit analog (compound 10) into the structure of FK506 is marked (gray areas).

FIGURE 2.

Schematic representation of gene clusters encoding FK506 and FK520 biosynthetic pathways. a, in FK506-producing species Streptomyces sp. MA6548, the PKS core region (above) sequence consists of fkbC, fkbB, fkbO, fkbP, and fkbA PKS genes (13). b, the FK520 biosynthetic cluster PKS core architecture with additional genes encoding biosynthesis of methoxymalonyl-CoA and ethylmalonyl-CoA extender units (12). c, schematic representation of the FK506 biosynthetic cluster from S. tsukubaensis NRRL 18488 with a conserved area of methoxymalonyl-CoA biosynthetic genes and a unique sequence of genes, designated as all subcluster on the left fringe, encoding biosynthesis of five-carbon extender unit leading to the allyl group in position C21. The size and the location of the deleted portion of the allK gene are marked.

FIGURE 3.

Schematic representation of the genes encoding an unusual PKS-related diketide synthase system at the left side of the all subcluster (a) and comparison with the more complex architectures of homologous systems found in Burkholderia spp. (gi:83650017) (b) and B. marina (gi:87287237) (c). AT, acyltransferase; KR, ketoreductase.

FIGURE 4.

HPLC analysis of FK506 and FK520 with the retention time of ∼9 and 8 min, respectively. Wild type strain (a), allK in-frame deletion mutant strain (b), allK in-frame deletion mutant strain fed with allylmalonyl-SNAC thioester (compound 10) (c), and the same strain fed with allylmalonyl-diSNAC double thioester (compound 11) (d). Similar incubation conditions and times (given under “Experimental Procedures”) were used for all experiments.

FIGURE 5.

a, proposed biosynthetic pathway for the five-carbon extender unit leading to the allyl group at position C21 of FK506. Firstly, either propionyl-CoA or acryloyl-CoA (compound 4) is condensed with malonyl-CoA (compound 5) by the action of diketide synthase system encoded by allA and allK yielding a five-carbon intermediate most probably bound to ACP. In the next step the 3-keto derivative (compound 6) is reduced to 3-hydroxy compound (compound 7) by the product of allS gene. The dehydration step in the next stage is likely to be performed by a generic dehydratase, not encoded in the cluster. In the last step, the 2-pentenoyl derivative (compound 8) is reduced and carboxylated by the crotonyl-CoA carboxylase/reductase, encoded in allR, to propylmalonyl-CoA or allylmalonyl-CoA (compound 9). The precise moment of transesterification from ACP to CoA and generation of the terminal double bond cannot be established at this time. b, chemical structures of allylmalonyl-SNAC monoester (compound 10) and allylmalonyl-SNAC double ester (compound 11) synthetic analogs of the five-carbon extender unit (compound 9), included into the growing polyketide chain by module 4 of the FK506 PKS. When added to the growth medium, compound 11 probably passes through cell membranes and is then partially hydrolyzed to compound 10, which is incorporated into the growing polyketide chain.