Monensin B

Furanone derivatives from terrestrial Streptomyces spp.[Pubmed:23074908]

Nat Prod Commun. 2012 Sep;7(9):1199-202.

Chemical investigation of the terrestrial Streptomyces sp. isolates GT2005/020 and ANK148 led to the isolation of two microbial furanone derivatives, 5-hydroxy-4-methylnaphtho[1,2-b]furan-3-one (1) and 4-hydroxy-5-methyl-furan-3-one (2), respectively, which have some similarity to quorum sensing molecules of the AI-2 type. In addition, the known compounds chalcomycin, ferulic acid, indole-3-acetic acid, uracil, thymine, 2'-deoxy-thymidin, Monensin B (3), phencomycin, and 1-acetyl-beta-carboline were isolated. The structures of 1 and 2 were deduced from extensive studies of NMR (1D and 2D) and mass spectra. Additionally, the complete NMR shift assignments for Monensin B (3) using H-H COSY, HMQC and HMBC experiments are reported here for the first time. We are describing the taxonomy and fermentation of the producing strains, the structure elucidation of the new metabolites and their bioactivity.

Crotonyl-coenzyme A reductase provides methylmalonyl-CoA precursors for monensin biosynthesis by Streptomyces cinnamonensis in an oil-based extended fermentation.[Pubmed:15470123]

Microbiology (Reading). 2004 Oct;150(Pt 10):3463-72.

It is demonstrated that crotonyl-CoA reductase (CCR) plays a significant role in providing methylmalonyl-CoA for Monensin Biosynthesis in oil-based 10-day fermentations of Streptomyces cinnamonensis. Under these conditions S. cinnamonensis L1, a derivative of a high-titre producing industrial strain C730.1 in which ccr has been insertionally inactivated, produces only 15 % of the monensin yield. Labelling of the coenzyme A pools using [3H]-beta-alanine and analysis of intracellular acyl-CoAs in the L1 and C730.1 strains demonstrated that loss of ccr led to lower levels of the monensin precursor methymalonyl-CoA, relative to coenzyme A. Expression of a heterologous ccr gene from Streptomyces collinus fully restored monensin production to the L1 mutant. Using C730.1 and an oil-based extended fermentation an exceptionally efficient and comparably intact incorporation of ethyl [3,4-13C2]acetoacetate into both the ethylmalonyl-CoA- and methylmalonyl-CoA-derived positions of monensin was observed. No labelling of the malonyl-CoA-derived positions was observed. The opposite result was observed when the incorporation study was carried out with the L1 strain, demonstrating that ccr insertional inactivation has led to a reversal of carbon flux from an acetoacetyl-CoA intermediate. These results dramatically contrast similar analyses of the L1 mutant in glucose-soybean medium which indicate a role in providing ethylmalonyl-CoA but not methylmalonyl-CoA, thus causing a change in the ratio of monensin A and Monensin B analogues, but not the overall monensin titre. These results demonstrate that the relative contributions of different pathways and enzymes to providing polyketide precursors are thus dependent upon the fermentation conditions. Furthermore, the generally accepted pathways for providing methylmalonyl-CoA for polyketide production may not be significant for the S. cinnamonensis high-titre monensin producer in oil-based extended fermentations. An alternative pathway, leading from the fatty acid catabolite acetyl-CoA, via the CCR-catalysed reaction is proposed.

Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization.[Pubmed:12940979]

Mol Microbiol. 2003 Sep;49(5):1179-90.

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in Monensin Biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in Monensin Biosynthesis.

MeaA, a putative coenzyme B12-dependent mutase, provides methylmalonyl coenzyme A for monensin biosynthesis in Streptomyces cinnamonensis.[Pubmed:11222607]

J Bacteriol. 2001 Mar;183(6):2071-80.

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 B12-dependent mutases. Plasmid-based expression of meaA from the ermE* 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.

Precursor supply for polyketide biosynthesis: the role of crotonyl-CoA reductase.[Pubmed:11162231]

Metab Eng. 2001 Jan;3(1):40-8.

Crotonyl-CoA reductase (CCR), which catalyzes the reduction of crotonyl-CoA to butyryl-CoA, is common to most streptomycetes and appears to be inducible by either lysine or its catabolites in Streptomyces cinnamonensis grown in chemically defined medium. A major role of CCR in providing butyryl-CoA from acetate for monensin A biosynthesis has been demonstrated by the observation of a change in the monensin A/Monensin B ratio in the parent C730.1 strain (50/50) and a ccr (encoding CCR) disruptant (12:88) of S. cinnamonensis in a complex medium. Both strains produce significantly higher monensin A/Monensin B ratios in a chemically defined medium containing valine as a major carbon source than in either complex medium or chemically defined medium containing alternate amino acids. This observation demonstrates that under certain growth conditions valine catabolism may have a more significant role than CCR in providing butyryl-CoA. Such a process most likely involves an isomerization of the valine catabolite isobutyryl-CoA, catalyzed by the coenzyme B(12)-dependent isobutyryl-CoA mutase. Monensin labeling experiments using dual (13)C-labeled acetate in the ccr-disrupted S. cinnamonensis indicate the presence of an additional coenzyme B(12)-dependent mutase linking branched and straight-chain C(4) compounds by a new pathway.

Role of crotonyl coenzyme A reductase in determining the ratio of polyketides monensin A and monensin B produced by Streptomyces cinnamonensis.[Pubmed:10542184]

J Bacteriol. 1999 Nov;181(21):6806-13.

The ccr gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from Streptomyces cinnamonensis C730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of Streptomyces collinus and Streptomyces coelicolor. A ccr-disrupted mutant, S. cinnamonensis L1, was constructed by inserting the hyg resistance gene into a unique BglII site within the ccr coding region. By use of the ermE* promoter, the S. collinus ccr gene was expressed from plasmids in S. cinnamonensis C730. 1/pHL18 and L1/pHL18. CCR activity in mutant L1 was shown to decrease by more than 90% in both yeast extract-malt extract (YEME) medium and a complex fermentation medium, compared to that in wild-type C730.1. Compared to C730.1, mutants C730.1/pHL18 and L1/pHL18 exhibited a huge increase in CCR activity (14- and 13-fold, respectively) in YEME medium and a moderate increase (3.7- and 2. 7-fold, respectively) in the complex fermentation medium. In the complex fermentation medium, S. cinnamonensis L1 produced monensins A and B in a ratio of 12:88, dramatically lower than the 50:50 ratio observed for both C730.1 and C730.1/pHL18. Plasmid (pHL18)-based expression of the S. collinus ccr gene in mutant L1 increased the monensin A/Monensin B ratio to 42:58. Labeling experiments with [1, 2-(13)C(2)]acetate demonstrated the same levels of intact incorporation of this material into the butyrate-derived portion of monensin A in both C730.1 and mutant C730.1/pLH18 but a markedly decreased level of such incorporation in mutant L1. The addition of crotonic acid at 15 mM led to significant increases in the monensin A/Monensin B ratio in C730.1 and C730.1/pHL18 but had no effect in S. cinnamonensis L1. These results demonstrate that CCR plays a significant role in providing butyryl-CoA for monensin A biosynthesis and is present in wild-type S. cinnamonensis C730.1 at a level sufficient that the availability of the appropriate substrate (crotonyl-CoA) is limiting.

Biosynthesis of monensins A and B: the role of isoleucine.[Pubmed:3957157]

Folia Microbiol (Praha). 1986;31(1):8-14.

Isoleucine added to the cultivation medium of Streptomyces cinnamonensis C-100-5 induced a relative increase of the production of Monensin B at the expense of monensin A. U-14C-Isoleucine was found not to be a specific Monensin B precursor. The incorporation of 1-13C-2-methylbutyrate into monensins A and B showed the label to be evenly incorporated in both products at carbon atoms originating from C(1) of propionate. In regulatory mutants insensitive to 2-amino-3-chlorobutyrate isoleucine influenced the production of monensins only slightly but strains resistant to 2-aminobutyrate and norleucine decreased their total production by 2-12% in the presence of isoleucine which was associated with a decrease of monensin A content by 14-52%. The inhibitory effect of isoleucine on the biosynthesis of valine, a specific precursor of the butyrate unit of monensin A, is discussed.

Effect of precursors on biosynthesis of monensins A and B.[Pubmed:3979923]

Folia Microbiol (Praha). 1985;30(1):30-3.

Precursors of monensins (acetate, propionate, butyrate, isobutyrate) affect the total production and the relative proportion of monensins A and B. Addition of propionate into the fermentation medium causes a prevalence of Monensin B whereas butyrate and isobutyrate stimulate the production of monensin A and suppress the production of Monensin B.

Isolation of novel antibiotics X-14667A and X-14667B from Streptomyces cinnamonensis subsp. urethanofaciens and their characterization as 2-phenethylurethanes of monensins B and A.[Pubmed:7309620]

J Antibiot (Tokyo). 1981 Oct;34(10):1248-52.

Antibiotics X-14667A (1) and X-14667B (2) are novel monovalent polyether antibiotics of the spiroketal type isolated from fermented cultures of Streptomyces cinnamonensis subsp. urethanofaciens together with monensin (3), its lower homolog, factor B (4) and 1,3-diphenethylurea (6). By a combination of microanalysis, mass spectrometry and 13C nmr, antibiotics X-14667A and B have been shown to be natural 2-phenethylurethanes of Monensin B and A respectively. Both structures have been confirmed by reacting the appropriate monensin with 2-phenethylisocyanate to yield semi-synthetic compounds that are identical to the natural products.