Regulation of <scp>D</scp>‐amino acid oxidase expression in the yeast <i>Rhodotorula gracilis</i>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Rhodotorula gracilis</jats:italic> is a oleaginous yeast which utilizes <jats:sc>D</jats:sc>‐amino acids as a source of carbon and/or nitrogen. <jats:sc>D</jats:sc>‐amino acid oxidase (DAAO), which converts <jats:sc>D</jats:sc>‐amino acids in the corresponding α‐keto acids and ammonia, is the first enzyme involved in the catabolism of <jats:sc>D</jats:sc>‐amino acids. DAAO activity is induced by the presence of <jats:sc>D</jats:sc>‐alanine, but the presence of the <jats:sc>L</jats:sc>‐isomer prevents induction by inhibiting the transport of <jats:sc>D</jats:sc>‐alanine into cells. To understand how DAAO expression is regulated, <jats:italic>R. gracilis</jats:italic> 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 <jats:sc>D</jats:sc>‐alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to <jats:italic>de novo</jats:italic> 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 <jats:sc>D</jats:sc>‐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 <jats:sc>D</jats:sc>‐alanine using cells pre‐cultured for 10 h on glucose and <jats:sc>L</jats:sc>‐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.</jats:p>