Monensin B

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.

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.

This paper reports the first comparative study of the gas-phase fragmentation chemistry of monensin in negative ion mode electrospray and nanoelectrospray tandem mass spectrometry. The fragmentation was observed to occur at lower energies in nanoelectrospray than electrospray. The major product ions are proposed to be formed via an initial neutral elimination of methanol followed be subsequent fragmentation. The low-mass product ions were observed at the same m/z for both monensin A and B.