Transcription factor reveals interconnected regulation of carbon source utilization and carbon catabolite repression in an oleaginous yeast

Cells must sense and respond to available nutrients to survive. Microbes evolved mechanisms to activate genes necessary to utilize a carbon source specifically when it is present. To efficiently grow in mixed carbohydrate environments, microbes repress genes necessary to utilize carbon sources that require substantial resources to catabolize when a simpler carbon source is present, known as carbon catabolite repression. We investigated nutrient utilization in the basidiomycete, oleaginous yeast Rhodotorula (Rhodosporidium) toruloides. R. toruloides is a saprophytic fungus that utilizes the building blocks of complex polysaccharides found in plant cell walls. A transcription factor homologous to the cellulose degradation regulator CLR-2/ClrB in Ascomycete filamentous fungi, which we named Cbr1, is required for cellobiose utilization in R. toruloides. Although the role of CLR-2/ClrB is limited to regulating expression of genes involved in cellulose and hemicellulose utilization, Cbr1 is also involved in fucose and tricarboxylic acid cycle intermediate utilization. Additionally, Cbr1 inhibits carbon catabolite repression specifically during utilization of glucose-glucose disaccharides, which may have evolved to limit the glucose-mediated repression of genes encoding proteins that cleave disaccharides into glucose. In fungi, it is thought carbon source-specific transcription factors activate genes necessary for carbon source utilization, while carbon catabolite repression is regulated by transcription factors that broadly repress all nonpreferred carbon source utilization genes when a preferred carbon source is present. Our data provide evidence for a noncanonical carbon catabolite repression mechanism and suggest transcriptional mechanisms regulating carbon catabolite repression may be less broadly conserved than previously thought. Overall design: 3'mRNA-seq profiling of Rhodotorula (Rhodosporidium) toruloides wild type and cbr1 deletion strains exposed to fucose as the sole carbon source for 8h, wild type, cbr1 deletion, and bgl1bgl2 double deletion strains exposed to cellobiose as the sole carbon source for 4 hours, and wild type, cbr1 deletion, and tct1 deletion exposed to citrate as a sole carbon source for 24 hours. Reads were counted using a GFF3 file generated by the peaks2utr software v. 1.4.1 in order to improve annotation of 3'UTRs. Wild type BAM files from the above experiments, as well as wild type cells exposed to 1% succinate as the sole carbon source for 24 hours, and wild type cells exposed to Vogel's minimal media with NH4Cl and 2% glucose as the sole nitrogen and carbon sources, respectively, were combined and used as the input for peaks2utr. The GFF3 file generated by peaks2utr is included as a supplementary file. Each experiment was performed in biological triplicate.

细胞必须感知并响应可获取的营养物质以维持存活。微生物演化出相应调控机制，仅在特定碳源存在时，激活利用该碳源所需的基因。为在混合碳水化合物环境中高效生长，当更简易的碳源存在时，微生物会阻遏分解代谢需消耗大量资源的碳源利用相关基因，该过程被称为碳分解代谢物阻遏（carbon catabolite repression）。

我们针对担子菌门产油酵母解脂红冬孢酵母（Rhodotorula (Rhodosporidium) toruloides，以下简称R. toruloides）的营养利用情况展开研究。R. toruloides是一种腐生真菌，可利用植物细胞壁中复杂多糖的结构单元。本研究中，我们将一个与子囊菌门丝状真菌纤维素降解调控因子CLR-2/ClrB同源的转录因子命名为Cbr1，其为解脂红冬孢酵母利用纤维二糖所必需。尽管CLR-2/ClrB的功能仅局限于调控纤维素与半纤维素利用相关基因的表达，但Cbr1同时参与岩藻糖与三羧酸循环中间产物的利用。此外，Cbr1会特异性地在以葡萄糖-葡萄糖二糖为碳源时抑制碳分解代谢物阻遏，这一演化机制或用于限制葡萄糖介导的、对编码将二糖裂解为葡萄糖的蛋白的基因的阻遏。

在真菌中，学界普遍认为碳源特异性转录因子会激活对应碳源利用所需的基因，而碳分解代谢物阻遏由转录因子调控：当优先碳源存在时，这类转录因子会广泛阻遏所有非优先碳源的利用基因。本研究数据为一种非经典的碳分解代谢物阻遏机制提供了实验证据，并表明调控碳分解代谢物阻遏的转录机制的保守性可能低于此前的认知。

整体实验设计：对以岩藻糖为唯一碳源培养8小时的解脂红冬孢酵母野生型与cbr1缺失菌株开展3'mRNA-seq测序；对以纤维二糖为唯一碳源培养4小时的野生型、cbr1缺失与bgl1bgl2双缺失菌株开展3'mRNA-seq测序；对以柠檬酸盐为唯一碳源培养24小时的野生型、cbr1缺失与tct1缺失菌株开展3'mRNA-seq测序。

测序读段通过peaks2utr软件v1.4.1生成的GFF3文件进行计数，以优化3'非翻译区（3'UTR）的注释。将上述实验中的野生型BAM文件，以及以1%琥珀酸盐为唯一碳源培养24小时的野生型细胞、以Vogel基本培养基添加氯化铵作为唯一氮源、2%葡萄糖作为唯一碳源培养的野生型细胞的测序数据合并，作为peaks2utr的输入数据。peaks2utr生成的GFF3文件已作为补充文件提供。所有实验均设置三次生物学重复。