Studies on the biosynthesis of clavulanic acid. III. Incorporation of DL-[3,4-13C2]glutamic acid.

The role of glutamate in clavulanic acid biosynthesis was investigated by feeding DL-[3,4-13C2]glutamate to a Streptomyces clavuligerus fermentation. The DL-[3,4-13C2]glutamate was synthesised by reacting [2-13C]diethylmalonate with O-tosyl-N-benzoyl-[3-13C]dehydroserine ethyl ester, which in turn was synthesised by condensing [13C]ethylformate with N-benzoylglycine ethyl ester. 13C NMR examination of the benzyl clavulanate derived from the fermentation revealed the predicted labelling of carbons 2 and 8 with accompanying 13C-13C spin-spin coupling. Other enrichments and couplings were observed which could be explained by metabolism of the labelled glutamate via the tricarboxylic acid cycle to give further clavulanic acid precursors. These results confirm that glutamate provides the oxazolidine carbon skeleton as predicted by previous experiments.