The Biosynthesis of Spinosyn in Saccharopolyspora spinosa:  Synthesis of the Cross-Bridging Precursor and Identification of the Function of SpnJ

Abstract

Spinosyns are glycosylated polyketide-derived macrolides possessing a perhydro-as-indacene core that is presumably formed via a series of intramolecular cross-bridging reactions. The unusual structure of the spinosyn aglycone suggests an intriguing biosynthetic pathway for its formation, which is expected to be initiated by the oxidation of the 15-OH group of the mature polyketide precursor and may involve a "Diels-Alder" type [4+2] cycloaddition reaction. Three possible routes, which differ in the order of oxidation and cyclization events, can be envisioned for the biosynthesis of the core structure. Sequence analysis of the spinosyn biosynthetic gene cluster led to the speculation of spnJ as the possible oxidase gene. To explore the early stage of intramolecular ring formation, we cloned and expressed the spnJ gene and purified the SpnJ protein which shows the characteristics of flavoproteins. Two possible substrates for SpnJ, the linear mature polyketide precursor and the corresponding cyclized macrolactone, were also synthesized. TLC and HPLC analysis of the incubation mixture of these compounds with SpnJ revealed that only the synthesized macrolactone could be converted to the corresponding ketone. This result clearly indicated that macrolactone formation proceeds 15-OH oxidation since the linear polyketide is not a substrate for SpnJ. In summary, the experiments described herein detail a convergent synthesis of spinosyn macrolactone and validate the catalytic function of SpnJ as a flavin-dependent oxidase. More significantly, we have established the spinosyn macrolactone as the immediate precursor of the tricyclic nucleus of spinosyns.Spinosyns A (1) and D (2) are polyketide-derived macrolides produced by Saccharopolyspora spinosa. 1 The combination of 1 and 2 serves as the active ingredient in a commercial insecticide, Naturalyte. This mixture exhibits excellent insecticidal activity with low mammalian toxicity and little detrimental environmental effects. 2 Structurally, the Spinosyns consist of a 22-membered macrolactone ring fused to a perhydro-as-indacene core scaffold. In addition, they are glycosylated with tri-O-methylrhamnose and forosamine at C-9 and C-17, respectively. The aglycone portion of Spinosyns is unusual among polyketide-derived secondary metabolites due to the presence of three intramolecular carbon-carbon bonds that constitute the as-indacene skeleton. The unusual nature of the Spinosyn aglycone suggests an intriguing biosynthetic pathway which has generated much recent attention. 3,4The Spinosyn (spn) biosynthetic gene cluster was cloned from S. spinosa. 3 Sequence analysis coupled with gene disruption experiments suggested that macrolactone 4, which is derived from the linear polyketide precursor 3, is a likely intermediate to form the as-indacene aglycone (9), 3,4 and the proteins encoded by spnF, spnJ, spnL, spnM could be involved in the cross- If 4 is indeed a precursor of the polycyclic aglycone and the cross-bridging reactions are all post-cyclization events, oxidation ...

Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in <b><i>Streptomyces</i></b> Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes

“…The entire synthesis elements are located in a 74-kb gene cluster ( spn cluster, spinosyn biosynthetic gene cluster) [Waldron et al, 2000]. In brief, spinosyn is synthesized initially with a propionic acid molecule and catalyzed by a type I polyketide synthase (PKS) to generate a 21-carbon chain which is later cyclized and modified to form aglycone (AGL) [Kim et al, 2007]. Rhamnose is the first sugar attached to AGL, which is then tri-O -methylated to yield the intermediate pseudoaglycone (PSA).…”

The spinosyn family of insecticides: realizing the potential of natural products research

“…[46][47][48] A subsequent investigation showed that SpnJ is an oxidase that converts the 15-hydroxyl group of the post-PKS monocyclized macrolactone (5) to a 15-keto group (6), but without any intramolecular cyclization (Figure 4). 49 Elucidating the detailed nature of the intramolecular cyclization steps leading to the aglycone (7) remains the object of further analyses.…”

A Type I/Type III Polyketide Synthase Hybrid Biosynthetic Pathway for the Structurally Unique ansa Compound Kendomycin

“…[60] SpnJ shows higher sequence homology to Ken19 (48 % identity and 60 % homology) and is involved in spinosad biosynthesis, which includes the formation of three intramolecular carbon-carbon bonds to build the A C H T U N G T R E N N U N G unusual as-indacene skeleton. Recent studies have shown that SpnJ is responsible for the oxidation of a hydroxyl group (C15) after TE-mediated macrolactone formation; [61] this was suggested earlier by Martin et al according to evidence from spinosyn PKS heterologous expression experiments. [62] Oxidation is required for the subsequent cyclizations (possibly through a Diels-Alder mechanism) and thus the involvement of SpnJ in the cross-bridging reactions cannot be excluded (although there is no evidence for this based on the in vitro data).…”