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

Abstract

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.