Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil.
Biotechnol Biofuels. 2013 Jun 25;6(1):91. doi: 10.1186/1754-6834-6-91.
Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans.
Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA::GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA::GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose.
Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.
尽管人们对木质纤维素酶的调控机制有了新的认识，但对于不同碳源的感知以及导致细胞代谢和水解酶分泌适应的信号级联反应，人们的了解仍然较少。因此，本研究在模式丝状真菌构巢曲霉中研究了非必需蛋白激酶（NPK）和磷酸酶（NPP）在碳源和/或能量状态感知中的作用。
鉴定出 11 种 NPK 和 7 种 NPP 参与了构巢曲霉的纤维素和某些情况下的半纤维素酶的产生。利用 CreA::GFP 融合蛋白，通过荧光显微镜确定了亲本菌株中 CreA 介导的碳分解代谢物阻遏（CCR）的调节。通过 RAS 信号通路感应磷酸化葡萄糖诱导 CreA 阻遏，而碳饥饿导致去阻遏。在纤维素上的生长代表碳饥饿和去阻遏条件。通过全基因组转录组学（GEO accession 47810）评估了鉴定出的 NPK 在纤维素诱导的反应和 CreA 去阻遏调节中的作用。在 NPK 缺陷背景下评估 CreA::GFP 定位和通过引入 ∆creA 突变恢复内切纤维素酶活性。schA 和 snfA 激酶的缺失均显著降低了纤维素诱导的转录反应，包括水解酶和转运蛋白的表达。确定了这两种 NPK 控制基因转录的机制，因为 NPK 缺陷突变体无法在去阻遏条件下（如碳饥饿或在纤维素上生长）解锁 CreA 介导的碳分解代谢物阻遏。
总之，本研究鉴定了多个参与碳源和/或能量状态感知的激酶和磷酸酶，同时表明 schA 和 snfA 在调节 CreA 去阻遏和构巢曲霉水解酶产生中的重叠协同作用。碳饥饿诱导的信号对 CreA 去阻遏、允许转录激活剂结合的重要性，对于水解酶分泌至关重要。
