Branched chain amino acid biosynthesis genes and regulators in Aspergillus nidulans

The branched chain amino acids (BCAA) leucine, isoleucine, and valine are important precursors for biosynthesis of proteins and secondary metabolites. The BCAA biosynthesis pathway is well characterized in Saccharomyces cerevisiae. However, recent work on BCAA pathway enzymes in Aspergillus revealed differences in the number of genes for several steps. The genes for the final two steps of leucine biosynthesis, catalyzed by β-isopropylmalate dehydrogenase (β-IDH) and BCAA aminotransferase (BAT), have not yet been characterized in the Aspergilli. The BATs also catalyze the final step of isoleucine and valine production. In S. cerevisiae, there is one β-IDH gene and two BAT genes. Using protein sequence similarity we identified two β-IDH genes in A. nidulans, leuD and leuE. We show that deletion of leuD, but not leuE, causes leaky leucine auxotrophy. The leuD∆ leuE∆ double mutant is a strict leucine auxotroph indicating that both genes encode functional enzymes. Quantitative RT-PCR reveals that leuE up-regulation compensates for loss of leuD. We identified, using protein sequence similarity, six A. nidulans BAT genes, batA-F. Deletion of these six genes separately does not confer BCAA auxotrophy. However, the double deletion mutant lacking the two most highly expressed BAT genes is a BCAA auxotroph, suggesting that these two genes encode the predominant biosynthetic enzymes and the other BAT genes may have evolved new roles. Two of the other BAT genes lie in the aspercryptins secondary metabolism gene cluster and likely catalyze biosynthesis of unusual BCAA components of aspercryptins. We have characterized the regulation of leucine biosynthesis pathway genes by the transcription factor LeuB. The leuB∆ mutant is a leaky leucine auxotroph. We have identified a LeuB paralog, LeuR, by sequence similarity. Deletion of leuR does not confer leucine auxotrophy. However, the leuB∆ leuR∆ double mutant is a strict leucine auxotroph, indicating that LeuR also regulates leucine biosynthesis.