Analysis of Specific Mutants in the Lasalocid Gene Cluster: Evidence for Enzymatic Catalysis of a Disfavoured Polyether Ring Closure

Abstract

Lasalocid is a highly atypical polyether ionophoric antibiotic, firstly because it contains a type of aromatic ring normally associated with fungal polyketides, and secondly because the formation of its tetrahydropyran ring appears to contravene Baldwin's rules, which predict the kinetically preferred routes for cyclisation reactions in organic chemistry. The lasalocid biosynthetic gene cluster has been cloned from Streptomyces lasaliensis, and the las locus (73,533 bp) was found to contain seven modular polyketide synthase (PKS) genes, including all the activities necessary for the synthesis of the aromatic moiety. Specific deletion from the gene cluster of the flanking lasC gene, which is predicted to encode a flavin-linked epoxidase, abolished production both of lasalocid and of the minor cometabolite iso-lasalocid without leading to accumulation of an identifiable intermediate; this suggests that oxidative cyclisation to form the polyether rings takes place on the PKS before release of the full-length polyketide product. Meanwhile, a mutant in which the adjacent epoxide hydrolase lasB had been deleted produced iso-lasalocid only. Iso-lasalocid differs from lasalocid in the replacement of the tetrahydropyran ring by a tetrohydrofuran ring and represents the kinetically favoured product of cyclisation. The LasB epoxide hydrolase is therefore directly implicated in control of the stereochemical course of polyether ring formation during lasalocid biosynthesis.

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

“…The formation of the characteristic ether bonds in polyethers are catalyzed by epoxidases and cognate epoxide hydrolases, and the epoxidases are found to be conserved and could be used to clone homologous genes (21,39). In order to design an efficient pair of primers for the amplification of putative epoxidase genes, the amino acid sequences of four available epoxidases, i.e., MonCI (27), NanO (37), NigCI (15), and LasC (35), were aligned to identify highly conserved blocks (Fig. 2).…”

“…4B) and annotated as 3-hydroxybutyryl-CoA dehydrogenase and 3-oxoacyl-(acyl carrier protein) synthase III genes, respectively. Genes orf11 and orf12 are thought to be involved in the dedicated supply of the butyrate extender unit for salinomycin biosynthesis (25,35). Genes orf11 and orf12 were individually replaced with aac(3)IV-oriT, and the salinomycin yields of the orf11 mutant JCY39 and the orf12 mutant JCY28 were found to be 10% and 36% of the wild type, respectively ( Fig.…”

“…So far, five polyether biosynthesis gene clusters have been identified and characterized for the biosynthesis of monensin (27), nanchangmycin (37), lasalocid (35), nigericin (15), and tetronomycin (9). Based on in silico and experimental analysis, the polyene backbones, assembled by a type I polyketide synthase (PKS), could undergo oxidative cyclization catalyzed by an epoxidase and one or more epoxide hydrolases to generate the characteristic pattern of ether bonds.…”

Characterization of an Epoxide Hydrolase from the Florida Red Tide Dinoflagellate, Karenia brevis

“…They Leadlay's in vivo studies on the production of lasalocid in the producing organism and a mutant lacking lsd19 clearly demonstrated that Lsd19 changes the regioselectivity of polyether ring formation. They found when Lsd19 is not present, the formation of the second ring proceeds the kinetically favored pathway to form isolasalocid, instead of lasalocid 65 . Latter studies on Lsd19 revealed it is composed two subunits, Lsd19A responsible for furan ring formation, and Lsd19B, taking roles in pyran ring formation, which are linked by a short loop and are oriented in a head-to-tail fashion.…”

“…Gradient annealing temperatures (65,63,61,59,57,55,53, 51 ºC) were used to find optimize condition. A touchdown reaction with annealing temperature decrease from 65 to 51 ºC in first eight cycles was also tested.…”