Abstract

Urocanic acid (UA), an intermediate in L-histidine catabolism, is a unique heterocyclic compound with various bioactivities. This study demonstrates UA production from glucose as a carbon source using engineered Corynebacterium ammoniagenes. The wild-type strain C. ammoniagenes NBRC 12071 was subjected to disruption of hutU, which encodes urocanate hydratase. After 7 days of cultivation with a minimum medium containing 2% glucose, the resulting strain MM1 produced 2.0 ± 0.1 mg/L UA with 0.29 ± 0.00 mg/g-cells/day specific production rate, while the wild-type produced a trace amount of UA. This indicated that the disruption arrested UA consumption. MM1 was spontaneously mutated with N-methyl-N’-nitro-N-nitrosoguanidine, forming 3-amino-1,2,4-triazole-resistant strains, some of which produced 0.16–0.29 g/L UA from 2% glucose. This indicates that the disruption of hutU was useful for constructing spontaneous mutants that produce UA. To improve UA production, hutH, which encodes histidine ammonia lyase, was overexpressed in MM1 using the strong C. ammoniagenes promoter Prpl21. After 7 days of cultivation, the resulting strain MM5 produced 7.7 ± 0.3 mg/L UA with 1.73 ± 0.12 mg/g-cells/day specific production rate. This indicates that overexpression strengthened the L-histidine catabolism. The use of a semi-synthetic medium would help improve the growth of engineered strains. To the best of our knowledge, this is the first report on UA production by engineered C. ammoniagenes.

Key words: Genetic engineering, L-Histidine, Corynebacterium ammoniagenes, Urocanic acid, Spontaneous mutation