MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

Abstract

The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B 12 -dependent mutases. Plasmid-based expression of meaA from the ermE‫ء‬ promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.Streptomycetes produce a large number of structurally diverse polyketide antibiotics by a process similar to long-chain fatty acid biosynthesis (20). Polyketide biosynthesis, catalyzed by polyketide synthases, uses carboxylated acyl thioesters, such as malonyl-coenzyme A (CoA), methylmalonyl-CoA, or ethylmalonyl-CoA, as extender units. These precursors form the polyketide carbon backbone and side chains, as seen in the examples of rifamycin (2), erythromycin (11), and monensin (6). Malonyl-CoA and ethylmalonyl-CoA are likely derived from the carboxylation of acetyl-CoA and butyryl-CoA, respectively (16, 35), while methylmalonyl-CoA can be produced from a variety of different pathways (6). As several methylmalonyl-CoA molecules are required to build a single polyketide (six for erythromycin, seven for monensin A, and eight for rifamycin), the levels of methylmalonyl-CoA under certain conditions may represent a limiting factor in production titers. For monensin biosynthesis, either ethylmalonyl-CoA or methylmalonyl-CoA can be used at ...

Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338

“…There is a single cluster of the adenosylcobalamin-dependent methylmalonyl-CoA mutase genes (SACE_5638-5640). S. erythraea, unlike many streptomycetes, has no homolog of crotonyl-CoA reductase or of adenosylcobalamin-dependent isobutyryl-CoA mutase, explaining its inability to furnish butyrate units for polyketide biosynthesis 38,39 ; it also has no homolog of meaA in S. coelicolor, which has been implicated in provision of methylmalonyl-CoA from acetoacetyl-CoA 40 . The availability of the complete genome sequence now provides the basis for systematic approaches to identify and manipulate such feeder pathways with the aim of increasing polyketide production.…”

Predicted highly expressed genes in the genomes of Streptomyces coelicolor and Streptomyces avermitilis and the implications for their metabolism

“…In Streptomyces, methylmalonyl-CoA and ethylmalonylCoA are two of the most common chain extender units for the biosynthesis of many polyketide antibiotics, and several pathways for their generation have been proposed (Hopwood & Sherman, 1990;Zhang & Reynolds, 2001 (Birch et al, 1993;Li et al, 2004). In addition, a meaA gene, which shares similarity with the large subunit of methylmalonyl-CoA mutase, was involved in an unknown pathway to methylmalonyl-CoA formation (Zhang & Reynolds, 2001).…”

“…However, in S. coelicolor and S. avermitilis all of the genes in the TCA cycle were predicted to be PHX genes, including citrate synthase, aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarate hydratase and malate dehydrogenase (in order of their action in the TCA cycle) (Table 3). One possible reason for the high presence of PHX genes in the TCA cycle genes of Streptomyces may be that members of this genus depend on the TCA cycle not only for ATP production, but also as a major source of carbon chain precursors to various primary and secondary metabolites, such as methylmalonyl-CoA Zhang & Reynolds, 2001). Several genes encoding cytochrome, electron-transfer flavoprotein genes and ATP synthase are also among the PHX in the two Streptomyces species.…”

“…In addition, a meaA gene, which shares similarity with the large subunit of methylmalonyl-CoA mutase, was involved in an unknown pathway to methylmalonyl-CoA formation (Zhang & Reynolds, 2001). Crotonyl-coenzyme A reductase and isobutyryl-CoA mutase were also involved in two pathways for production of ethylmalonyl-CoA, from valine degradation or acetoacetyl-CoA, respectively (ZerbeBurkhardt et al, 1998;Liu & Reynolds, 1999).…”

Crotonyl-coenzyme A reductase provides methylmalonyl-CoA precursors for monensin biosynthesis by Streptomyces cinnamonensis in an oil-based extended fermentation

“…In streptomycetes, malonyl-CoA and ethylmalonyl-CoA are considered to be generated by carboxylation of acetyl-CoA and butyryl-CoA, respectively (Liu & Reynolds, 1999;Rodríguez & Gramajo, 1999). Numerous pathways can contribute to providing methylmalonylCoA, and the role of these pathways in different polyketide biosynthetic processes has been of interest for many years (Hunaiti & Kolattukudy, 1984;Reynolds et al, 1988;Tang et al, 1994;Zhang & Reynolds, 2001;Zhang et al, 1999a).…”