MeaA, a Putative Coenzyme B12-Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

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 the same stage of elongation to generate monensin A or monensin B, respectively (14, 24). Thus, in this organism, changes in the levels of methylmalonyl-CoA affect not only the titers but also the ratio of monensin A to monensin B. This feature makes Streptomyces cinnamonensis an excellent organism for probing pathways that contribute in both minor and major ways to generating methylmalonyl-CoA for polyketide biosynthesis. Three generally accepted routes to methylmalonyl-CoA are (i) the isomerization of succinyl-CoA, catalyzed by the coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) (28, 43); (ii) carboxylation of propionyl-CoA, catalyzed by either propionyl-CoA carboxylase (PCC) (7, 35) or methylmalonyl-CoA transcarboxylase (MMT) (22); and (iii) a multistep oxidation of isobutyryl-CoA (34) (Fig. ​(Fig.1).1). The mutAB genes encoding MCM involved in the first of these pathways have been cloned from monensin A-producing S. cinnamonensis (6), as well as from rifamycin SV-producing Amycolatopsis mediterranei U32 (49) and other prokaryotic and mammalian sources (27, 47). A mutAB disruption has no effect on the monensins A and B total production and ratio in S. cinnamonensis cells (44). Plasmid-based overexpression of the mutAB genes in S. cinnamonensis, on the other hand, has been reported to yield both a slight increase in total monensin production and a decreased ratio of monensin A to monensin B (49). These observations suggest that under these conditions, methylmalonyl-CoA is a limiting factor in monensin biosynthesis and that the natural levels of MCM activity generated from the mutAB genes do not contribute significantly to this process. Genes encoding either PCC or MMT, enzymes capable of generating methylmalonyl-CoA from propionyl-CoA, have not yet been cloned from S. cinnamonensis, and thus their role in providing this precursor through the second pathway has not been evaluated. A PCC encoded by the pcc gene has been cloned and characterized for Streptomyces coelicolor A3(2) (7, 35) and erythromycin-producing Saccharopolyspora erythraea (12). Disruption of the pcc gene in S. erythraea has no effect on erythromycin production (12). However, even in this organism, the role of carboxylation of propionyl-CoA in methylmalonyl-CoA formation is not clear, as there are probably an MMT and additional acyl-CoA (or propionyl-CoA) carboxylases (6, 7, 18, 35). FIG. 1 (A) The structures of monensins A and B. Methylmalonyl-CoA positions in both monensins A and B are marked in bold, and the ethylmalonyl-CoA position in monensin A is hatched. (B) Proposed pathways for methylmalonyl-CoA formation in S. cinnamonensis. The ... The third pathway from methylmalonyl-CoA, clearly established from numerous biosynthetic studies with monensin (34) and tylosin (30) and other polyketide antibiotic producing organisms, is oxidation of isobutyryl-CoA (Fig. ​(Fig.1).1). Isobutyryl-CoA can be formed either from valine catabolism or from butyryl-CoA by carbon skeleton rearrangement catalyzed by coenzyme B12-dependent isobutyryl-CoA mutase (ICM) (34). The icm genes encoding S. cinnamonensis ICM were recently cloned and sequenced, and insertional inactivation of icm has been shown to have no detectable effect on monensin production (33, 44, 48). Thus, it appears that either ICM or MCM activities can be removed from S. cinnamonensis without significantly affecting the pools of methylmalonyl-CoA for monensin biosynthesis. Methylmalonyl-CoA in these strains might thus be obtained directly from the oxidation of valine-derived isobutyryl-CoA (44) or from the carboxylation of propionyl-CoA. A third possibility was raised by the unexpected observation that [1,3-13C2]acetoacetyl-CoA can be converted intact into [1,2-13C2]methylmalonyl-CoA in S. cinnamonensis in the absence of either ICM or MCM. There are no other known pathways beyond that involving ICM which would explain such an intact interconversion, and these data suggested the presence of another coenzyme B12-dependent mutase and/or unidentified pathway(s) involved in methylmalonyl-CoA formation (44). A novel meaA gene encoding an MCM-like protein with unknown function has been found in both Streptomyces collinus (15) and Methylobacterium extorquens AM1 (9, 37). The predicted MeaA amino acid sequence contains the distinctive coenzyme B12-binding domain and exhibits high end-to-end homology to the large subunit of MCM and both subunits of ICM. The meaA gene has been shown to be involved in acetate assimilation in both of these organisms (9, 15, 37). In both S. collinus and S. coelicolor, the meaA gene was found to be 20 to 40 bp downstream of the crotonyl-CoA reductase (CCR)-encoding gene ccr, with the same transcriptional orientation. CCR plays a key role in the catalysis of the last reductive step in the biosynthesis of butyryl-CoA from acetyl-CoA in S. collinus (15) and a significant role in providing butyryl-CoA from monensin A biosynthesis in S. cinnamonensis (24). The genetic organization of meaA and ccr implies a possible role of meaA in the butyryl-CoA or methylmalonyl-CoA pathways. Our interest in the clarification of the role of various methylmalonyl-CoA formation pathways in polyketide-producing streptomycetes prompted us to further examine this novel mutase gene. In this study, we report the cloning and sequencing of meaA from monensin-producing S. cinnamonensis cells. Gene disruption, gene overexpression, and labeling studies clearly demonstrate that MeaA is involved in methylmalonyl-CoA formation, but that this process does not involve an acetoacetyl-CoA–butyryl-CoA intermediate. Furthermore, the generation and analysis of an meaA icm mutant of S. cinnamonensis has clearly demonstrated that under the tested conditions, the expression of these two genes is crucial for providing the majority of this methylmalonyl-CoA used for monensins A and B production. Surprisingly, valine catabolism does not contribute significantly to providing methylmalonyl-CoA in this mutant.