Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria.
Department of Biotechnology, Institute of Microbiology and Microbial Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Curr Genet. 2021 Aug;67(4):641-661. doi: 10.1007/s00294-021-01165-4. Epub 2021 Mar 16.
The transcription factors Cat8 and Sip4 were described in Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.
转录因子 Cat8 和 Sip4 在酿酒酵母和乳酸克鲁维酵母中被描述为具有非常相似的 DNA 结合结构域，并通过与碳源响应元件 (CSREs) 结合，在非发酵生长条件下对各种基因的去阻遏是必需的。甲醇营养型酵母毕赤酵母（又名巴斯德毕赤酵母）有两个转录因子 (TFs)，它们基于序列相似性被认为是 Cat8 的假定同源物，分别称为 Cat8-1 和 Cat8-2。目前尚不清楚它们参与了哪些细胞过程，也不清楚其中一个是否是 Sip4 的同源物。为了研究 Cat8 同源物在毕赤酵母中的作用，我们生成了这两个 TF 的过表达或缺失菌株。测试了这些突变菌株在不同碳源上的生长能力，并定量了碳代谢中选定基因的转录水平。我们的实验表明，TFs 是毕赤酵母利用 C2 碳源生长所必需的，但不是葡萄糖、甘油或甲醇。Cat8-1 缺失的毕赤酵母在乙酸上的生长受到损害，而 Cat8-1 和 Cat8-2 都参与了毕赤酵母在乙醇上的生长。相应地，这两个 TF 都参与了 ADH2、ALD4 和 ACS1 的激活，这三个基因编码了乙醇同化过程中重要的酶。与酿酒酵母和乳酸克鲁维酵母不同，Cat8-1 不调节毕赤酵母中假定的 Sip4 家族成员 Cat8-2 的转录。此外，Cat8-1 对于乙醛酸循环基因的激活是必需的，而 Cat8-2 对于肉碱穿梭系统基因的激活是必需的。Cat8-1 和 Cat8-2 都不是糖异生基因激活所必需的。最后，CAT8-2 基因在葡萄糖上被 Mig1-2 转录因子抑制，并且在所有测试的碳源上被 Cat8-2 蛋白自身抑制。我们的研究确定了毕赤酵母 Cat8-1 和 Cat8-2 参与 C2 代谢，并强调了它们与其他酵母物种同源物的相似性和差异。
