A unique metabolic gene cluster regulates lactose and galactose metabolism in the yeast <i>Candida intermedia</i>

Lactose assimilation is a relatively rare trait in yeasts, and <i>Kluyveromyces</i> yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast <i>Candida intermedia</i>. Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved <i>LAC</i> cluster (<i>LAC12</i>, <i>LAC4</i>) for lactose uptake and hydrolysis, the conserved <i>GAL</i> cluster (<i>GAL1</i>, <i>GAL7</i>, and <i>GAL10</i>) for galactose catabolism through the Leloir pathway, and a "<i>GALLAC</i>" cluster containing the transcriptional activator gene <i>LAC9</i>, second copies of <i>GAL1</i> and <i>GAL10</i>, and a <i>XYL1</i> gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the <i>GALLAC</i> cluster is unique to <i>C. intermedia</i> and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for <i>C. intermedia's</i> growth on lactose and galactose. We also show that the regulatory network in <i>C. intermedia</i>, governed by Lac9 and Gal1 from the <i>GALLAC</i> cluster, differs significantly from the galactose and lactose regulons in <i>Saccharomyces cerevisiae</i>, <i>Kluyveromyces lactis</i>, and <i>Candida albicans</i>. Moreover, although lactose and galactose metabolism are closely linked in <i>C. intermedia</i>, our results also point to important regulatory differences.IMPORTANCEThis study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast <i>C. intermedia</i>. Notably, the unique <i>GALLAC</i> cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how <i>C. intermedia</i> has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of <i>C. intermedia</i> as a cell-factory for conversion of lactose-rich whey into value-added products.