Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production Of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli . This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carba moylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in comple te medium. enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitr ogen source and the presence of inducer, 2-thiouracil, in the growth medium . Growth with (NH4)(2)SO4 as the nitrogen source repressed the production o f both enzymes (nitrogen repression) and also resulted in a rapid, but reve rsible loss of hydantoinase activity in induced cells (ammonia shock). Muta nt strains with inducer-independent production of the enzymes and/or altere d response to nitrogen control were isolated. Of greatest importance for in dustrial application was strain RU-ORPN 1F9, in which hydantoinase and NCAA H enzyme activity was inducer-independent and no longer sensitive to nitrog en repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterolog ous expression systems.