SYNTHESIS OF AROMATIC COMPOUNDS BY NEUROSPORA

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Abstract
A mutant Neurospora strain (arom) with multiple aromatic requirements was isolated following treatment of macroconidia with methyl-bis (B-chloroethyl)amine. The arom locus was in the right arm of linkage group II, distal to pe and proximal to ac, 21.7 units from the centromere. Feeding experiment indicated that the arom locus was involved in the production of shikimic acid which was a common precursor of PAB, tryptophan, phenylalanine and tyrosine. Dehydroquinic acid (DHQ) and dehydroshikimic acid (DHS), which are pre-cursors of shikimic acid in E. coli. were inactive in promoting growth of the arom mutant, indicating either a different situation in Neurospora or a block in the conversion of DHS to shikimic acid. To test these theories the arom strain was grown on shikimic acid or on the four aromatic substances, and the culture filtrates were tested for accumulation of compounds involved in aromatic synthesis by means of butanol -acetic acid-developed paper chromatograms. DHS was identified as and was believed to lie between DHQ and shikimic acid in the pathway of aromatic synthesis. DHS was accumulated at the rate of 1-3 [mu]gm. per ml of culture filtrate within 120 hrs. of growth, followed by a slow decrease in concentration. Protocatechuic acid (PCA) was also isolated from the arom strain in large amounts (up to 2 mg/20 ml) during the first 4 days of growth. These results indicated that the arom mutation blocked the conversion of DHS to shikimic acid and that DHS was converted to PCA. Further tests were made using C14-labeled sucrose and a double mutant arom-tryp-1, which required for growth shikimic acid (replaceable by PAB, phenylalanine and tyrosine) and tryptophan (replaceable by indole). The double mutant accumulated anthranilic acid only in the presence of shikimic acid. Anthranilic acid and PCA were isolated from the culture filtrate, and tyrosine and alanine were obtained from the mycelium. Isotope analyses indicated that PCA and alanine were uniformly labeled, whereas anthranilic acid was relatively unlabeled, supporting the theory that shikimic acid was converted to anthranilic acid and that PCA was produced from DHS. The relative activities of the isolated tyrosine and of the tyrosine carboxyl carbon indicated that the ring carbons were derived from shikimic acid and the side-chain carbons from other sources. The calculated ratio of activities of the carbon atoms of the side chain and ring of tyrosine (6.4) was essentially the same as the ratio between the activities of PCA and of anthranilic acid (7.1), in keeping with the proposed derivation of tyrosine and the assumption that the side-chain carbons were equally labeled.

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