Transcriptional profiling of a NAGA kinase (nag5) mutant in Yarrowia lipolyitica

In the fungus Yarrowia lipolytica the transcription of the genes encoding the enzymes of the N-acetylgucosamine (NAGA) pathway (NAG genes) is regulated by the presence of NAGA in the culture medium. In a wild-type strain, those genes are expressed at a very low level during growth in glucose while their expression increases 20 to 40 fold in cultures grown in NAGA. However, a deletion of the gene YlNAG5, encoding the NAGA kinase, abolishes this regulation and renders the transcription of those genes independent of the presence of NAGA in the medium (Flores and Gancedo 2015). A possibility to explain this result is that the protein Nag5 is a moonlighting protein with two functions: one as a metabolic kinase, the other as a regulator implicated in the control of the expression of the genes of the NAGA catabolic pathway. An alternative explanation would be that the transcriptional deregulation of the NAG genes in the mutant is triggered by an intracellular NAGA accumulation derived from chitin turnover. Our objective in this work was to decide between these two possibilities. To this end we studied the regulation of the expression of the NAG genes in a double mutant Ylnag5 ngt1 that cannot internalize NAGA and in a Ylnag5 mutant expressing mutated versions of YlNag5 or heterologous NAGA kinases. Additionally, we performed RNA-seq experiments to monitor the effects of the Ylnag5 mutation on the expression of genes not related to the NAGA pathway. Our results are consistent with the idea of NAGA kinase being a moonlighting protein with two roles, one as metabolic enzyme and other as regulatory protein in the transcription of some genes. Six samples are analyzed, corresponding to wild type (strain 660), a Ylnag5 deletant (strain 753) and the mutant strain carrying a plamid that harbors the YlNAG5 gene (886). Two biological replicas were made.

在解脂耶氏酵母（Yarrowia lipolytica）中，编码N-乙酰葡糖胺（N-acetylgucosamine, NAGA）代谢通路酶类的基因（即NAG基因，NAG genes）的转录受培养基中NAGA的存在状态调控。在野生型菌株中，此类基因在葡萄糖培养基中生长时表达量极低，而在NAGA培养基中培养时，其表达量可上调20至40倍。然而，编码NAGA激酶（NAGA kinase）的YlNAG5基因发生缺失后，这种调控效应会被消除，使得此类基因的转录不再受培养基中NAGA的影响（Flores与Gancedo，2015）。针对该实验结果，一种可能的解释是Nag5蛋白属于兼职蛋白（moonlighting protein），具备双重功能：其一为代谢激酶，其二为参与调控NAGA分解代谢通路基因表达的调控因子。另一种备选解释则为，突变体中NAG基因的转录失调，是由几丁质周转所产生的胞内NAGA积累所诱发的。本研究的目标便是在这两种解释中做出抉择。为此，我们针对无法内化NAGA的双突变体Ylnag5 ngt1，以及表达突变型YlNag5或异源NAGA激酶的Ylnag5突变体，开展了NAG基因表达调控的相关研究。此外，我们还开展了RNA测序（RNA-seq）实验，以探究Ylnag5突变对非NAGA通路相关基因表达的影响。本研究结果支持NAGA激酶作为兼职蛋白具备双重功能的假说：其一为代谢酶，其二为调控部分基因转录的调控因子。本研究共分析6份样本，分别对应野生型菌株（660菌株）、Ylnag5基因缺失突变体（753菌株）以及携带携载YlNAG5基因质粒的突变菌株（886菌株），且设置了2次生物学重复。