Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation^†

Abstract

SummaryThe 18-membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl-CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non-producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI-minus mutant (12-desnitrile-12-methylborrelidin) possessing a methyl group and that of the borJ-minus mutant (12-desnitrile-12-carboxylborrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12-desnitrile-12-methyl-borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12-desnitrile-12-methyl-borrelidin into borrelidin was also achieved in a heterologous system co-expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK , which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ . On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.

Bezerramycins A–C, Antiproliferative Phenoxazinones from Streptomyces griseus Featuring Carboxy, Carboxamide or Nitrile Substituents

“…[23] In borrelidin biosynthesis, it was postulated that the nitrile functional group derives from enzymatic oxidation of a carbon atom, with subsequent transamination, aldoxime formation, and dehydration. [24] In analogy, the nitrile nitrogen atom of 3 could be introduced by transamination of a C-1 formyl precursor of 2 or 5. The ensuing amine would undergo two oxidations to an N,N-dihydroxy species, followed by successive dehydration to the nitrile.…”

Strategies for the Design and Discovery of Novel Antibiotics using Genetic Engineering and Genome Mining

“…In particular, the inactivation of genes encoding enzymes participating in oxygenation reactions or in the production and attachment of deoxysugars has been highly prolific in the generation of novel compounds. Oxygenases such as cytochrome P450 s, FAD-dependent oxygenases, dioxygenases, epoxidases, or hydroxylases have been targeted in order to obtain derivatives from several antibiotics, e.g., erythromycin (Weber et al 1989), borrelidin (Olano et al 2004b), or tirandamycin (Carlson et al 2011). On the other hand, inactivation of genes involved in the production of deoxysugars or in glycosylation leads to mutants accumulating compounds with a different pattern of glycosylation such as those derived from erythromycin (Dhillon et al 1989), mithramycin (Fenández et al 1998;Remsing et al 2002), or hygromycin A (Palaniappan et al 2006).…”

Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

“…[8] In the case of borrelidin this might occur during post-PKS formation of the nitrile at C12, the mechanism of which remains to be fully elucidated. [25] Figure 1. Borrelidin is synthesised on a modular PKS.…”