Regulation of D ‐amino acid oxidase expression in the yeast Rhodotorula gracilis

Rhodotorula gracilis is a oleaginous yeast which utilizes D ‐amino acids as a source of carbon and/or nitrogen. D ‐amino acid oxidase (DAAO), which converts D ‐amino acids in the corresponding α‐keto acids and ammonia, is the first enzyme involved in the catabolism of D ‐amino acids. DAAO activity is induced by the presence of D ‐alanine, but the presence of the L ‐isomer prevents induction by inhibiting the transport of D ‐alanine into cells. To understand how DAAO expression is regulated, R. gracilis cells were grown on media containing different nitrogen and/or carbon sources. As a general rule, the level of DAAO mRNA reached a maximum after 15 h growth and preceded by ∼6 h the maximum level of DAAO activity. The inducer D ‐alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to de novo synthesis. The presence of a supplemental carbon source (e.g. succinate or glucose) does not repress DAAO expression. Ammonium sulphate appears to have a negative effect on DAAO mRNA translation and on the expression of DAAO activity: DAAO is only partially active when the yeast is grown in the presence of D ‐alanine and ammonium sulphate. The best expression of DAAO activity was obtained by growing the cells for 12 h at 30 °C in the presence of glucose and D ‐alanine using cells pre‐cultured for 10 h on glucose and L ‐alanine (0.99 U/mg protein, corresponding to ∼1.0% total proteins in the crude extract). Under these growth conditions a six‐fold increase in DAAO production was achieved. Copyright © 2003 John Wiley & Sons, Ltd.