Characterisation of the gene cluster for l-rhamnose catabolism in the yeast Scheffersomyces (Pichia) stipitis.

In Scheffersomyces (Pichia) stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species. Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are physically clustered. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogues in L-rhamnose utilising yeasts we analysed the function of one of the paralogues, LRA41 by heterologous expression and biochemical characterization. Lra41p has similar catalytic properties as the Lra4p but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterised in S. stipitis. We expressed the L-rhamnonate dehydratase, Lra3p, in Saccharomyces cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate.