Structure and Biosynthesis of Myxofacyclines: Unique Myxobacterial Polyketides Featuring Varing and Rare Heterocycles^[]**

Abstract

A metabolome-guided screening approach in the novel myxobacterium Corallococcus sp. MCy9072 resulted in the isolation of the unprecedented natural product myxofacycline A, which features a rare isoxazole substructure. Identification and genomic investigation of additional producers alongside targeted gene inactivation experiments and heterologous expression of the corresponding biosynthetic gene cluster in the host Myxococcus xanthus DK1622 confirmed a noncanonical megaenzyme complex as the biosynthetic origin of myxofacycline A. Induced expression of the respective genes led to significantly increased production titers enabling the identification of six further members of the myxofacycline natural product family. Whereas myxofacyclines A-D display an isoxazole substructure, intriguingly myxofacyclines E and F were found to contain 4-pyrimidinole, a heterocycle unprecedented in natural products. Lastly, myxofacycline G features another rare 1,2-dihydropyrol-3-one moiety. In addition to a full structure elucidation, we report the underlying biosynthetic machinery and present a rationale for the formation of all myxofacyclines. Unexpectedly, an extraordinary polyketide synthase-nonribosomal peptide synthetase hybrid was found to produce all three types of heterocycle in these natural products.

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“…MCy9072 resulted in the isolation of myxofacycline A ( 31 ), featuring a rare isoxazole substructure. 125 Retro-biosynthetic analysis proposed that a non-canonical hybrid PKS/NRPS BGC ( iso ) containing an aminotransferase gene is responsible for the biosynthesis of 31 . The heterologous expression of the promoter-engineered iso in Myxococcus xanthus Mx x48 enabled to produce myxofacycline A as well as congeners myxofacyclines B–G ( 32–37 ).…”

“…The heterologous expression of the promoter-engineered iso in Myxococcus xanthus Mx x48 enabled to produce myxofacycline A as well as congeners myxofacyclines B–G ( 32–37 ). 125 Interestingly, a single BGC could afford three types of rare heterocycles: isoxazole in myxofacyclines A–D ( 31–34 ), 4-pyrimidinole ring in myxofacyclines E and F ( 35 and 36 ), and 1,2-dihydropyrrol-3-one in myxofacycline G ( 37 ). 125 In contrast to frequently found 1,3-oxazoles for NPs, the isoxazole motif (1,2-oxazole) is rarely reported.…”

“…125 Interestingly, a single BGC could afford three types of rare heterocycles: isoxazole in myxofacyclines A–D ( 31–34 ), 4-pyrimidinole ring in myxofacyclines E and F ( 35 and 36 ), and 1,2-dihydropyrrol-3-one in myxofacycline G ( 37 ). 125 In contrast to frequently found 1,3-oxazoles for NPs, the isoxazole motif (1,2-oxazole) is rarely reported. While 4-pyrimidinole was accessible by chemical synthesis, it has never been found in the paradigm of NPs before.…”

“…The isoxazole-containing linear fatty acid moiety of myxadazoles is actually myxofacyclines. 125 DMB is derived from the vitamin B 12 metabolism, as indicated by isotope labelling and precursor feeding. The convergence of two distinct metabolic pathways through a C–N coupling achieves an unprecedented hybridization mode within the scope of NPs, although it remains elusive if an anonymous enzyme mediates this process.…”

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“…Remarkably, the NRPS and PKS assembly lines found in myxobacteria often diverge from the canonical models found in other bacteria in terms of module architectures, biochemical behavior, building block selection and chain termination [ 215 ]. As a result, the final products of the pathways include in many cases highly unusual moieties, as illustrated by the alternative heterocycles featured by the myxofacyclines, including isoxazole, 4-pyrimidinolide and 1,2-dihydropyrol-3-one, all of them generated by the same PKS-NRPS system [ 216 ]. In addition to this, the molecular targets for myxobacterial compounds are in many cases rarely targeted by compounds produced by other microorganisms [ 211 ].…”