A Novel Biosynthetic Pathway Providing Precursors for Fatty Acid Biosynthesis and Secondary Metabolite Formation in Myxobacteria

Abstract

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using 13 C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.Fatty acids (FA) 1 are important building blocks of cell membranes. The various groups of prokaryotes differ remarkably in the structure and synthesis of fatty acid-derived lipids, which serve as reliable systematic marker molecules in chemotaxonomy (1). Unsaturated and branched chain (or iso-) FA increase the fluidity of the membrane and fulfill the function of thermal adaptation. The higher the content of these FA, the lower is the solid-to-liquid phase transition temperature of lipids.The biosynthesis of FA has been elucidated in detail, revealing that the difference between the biosynthetic pathways to FA and iso-FA lies only in the respective acylated primer acyl carrier proteins and in the condensing enzymes, which prefer modified primer acyl chains for elongation instead of the "normal" acetyl primer (1, 2). The modified primers used for iso-FA biosynthesis are the coenzyme A esters of isovaleric acid (IVA, resulting in iso-odd FA with an uneven number of carbon atoms), isobutyric acid (IBA, resulting in iso-even FA with an even number of carbon atoms), or 2-methyl butyric acid (2MBA, resulting in ante-iso FA, in which the methyl branch is located on the third carbon atom counted from the -end of the chain), which can be generated via the degradation pathway from branched chain amino acids or from intermediates of the biosyntheses of these essential amino...

3-Hydroxy-3-Methylglutaryl-Coenzyme A (CoA) Synthase Is Involved in Biosynthesis of Isovaleryl-CoA in the Myxobacterium Myxococcus xanthus during Fruiting Body Formation

“…The alternative pathway to IV-CoA in myxobacteria was confirmed by incorporation of pathway intermediates into the compounds of interest, and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) was proposed as the central branching point between isoprenoid and IV-CoA biosynthesis ( Fig. 1) (16,17). The key step in the proposed new pathway is the decarboxylation/dehydration of HMG-CoA to 3,3-dimethylacrylyl-CoA or 3-methylbut-3-enoyl-CoA, which we were able to show previously in vitro (17).…”

“…In all myxobacteria investigated so far, isoodd fatty acids are the dominant fatty acid family and can make up to 75% of all fatty acids, as shown for Myxococcus xanthus (2,3,7). Analysis of bkd mutants of the myxobacteria M. xanthus and Stigmatella aurantiaca showed that iso-odd fatty acids and IV-CoA-derived secondary metabolites are still produced in these mutants although at a reduced level (16,23).…”

“…Further feeding experiments with 13 C-labeled acetate revealed that the IV-CoA in the residual iso-odd FAs or secondary metabolites is derived from three acetate units by following an incorporation pattern also found in the mevalonate-dependent biosynthesis of isoprenoids (16,17). The alternative pathway to IV-CoA in myxobacteria was confirmed by incorporation of pathway intermediates into the compounds of interest, and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) was proposed as the central branching point between isoprenoid and IV-CoA biosynthesis ( Fig.…”

Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

“…We speculate that the transporter mutant is unable to utilize BCAAs, preventing growth, and it therefore displays a deficiency in predation. Moreover, it has been shown that BCAAs, such as leucine, serve as the precursor for fatty acid biosynthesis and also as starter molecules for polyketide synthases (41,42). Reduced levels of available BCAAs due to disrup-tion of the ABC transporter could result in an altered specialized metabolite or fatty acid profile and generate deficits in growth on otherwise-suitable prey.…”

“…One of the most frequent transposon insertion sites (11 independent isolates) occurred in MXAN_5713, encoding an ABC transporter/permease predicted to be involved in branched-chain amino acid (BCAA) transport. BCAAs are essential to M. xanthus and function as precursors for fatty acid synthesis as well as specialized metabolite production (31,41,42). All 11 transposon insertion mutations in MXAN_5713 displayed an LOF phenotype (see Fig.…”