Siebecker Benedikt, Schütze Tabea, Spohner Sebastian, Haefner Stefan, Meyer Vera
Chair of Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
BASF SE, Ludwigshafen, Germany.
Front Bioeng Biotechnol. 2023 Oct 6;11:1279146. doi: 10.3389/fbioe.2023.1279146. eCollection 2023.
, formerly known as , is used in industry to produce lignocellulolytic enzymes and heterologous proteins. However, the transcriptional network driving the expression of these proteins remains elusive. As a first step to systematically uncover this network, we investigated growth, protein secretion, and transcriptomic fingerprints of strains deficient in the cellulolytic transcriptional regulators Clr1, Clr2, and Clr4, respectively. The genes encoding Clr1, Clr2, and Clr4 were individually deleted using split marker or the CRISPR/Cas12a technology and the resulting strains as well as the parental strain were cultivated in bioreactors under chemostat conditions using glucose as the carbon source. During steady state conditions, cellulose was added instead of glucose to study the genetic and cellular responses in all four strains to the shift in carbon source availability. Notably, the and deletion strains were unable to continue to grow on cellulose, demonstrating a key role of both regulators in cellulose catabolism. Their transcriptomic fingerprints uncovered not only a lack of cellulase gene expression but also reduced expression of genes predicted to encode hemicellulases, pectinases, and esterases. In contrast, the growth of the deletion strain was very similar compared to the parental strain. However, a much stronger expression of cellulases, hemicellulases, pectinases, and esterases was observed. The data gained in this study suggest that both transcriptional regulators Clr1 and Clr2 activate the expression of genes predicted to encode cellulases as well as hemicellulases, pectinases, and esterases. They further suggest that Clr1 controls the basal expression of cellulases and initiates the main lignocellulolytic response to cellulose via induction of expression. In contrast, Clr4 seems to act as a repressor of the lignocellulolytic response presumably via controlling expression. Comparative transcriptomics in all four strains revealed potentially new regulators in carbohydrate catabolism and lignocellulolytic enzyme expression that define a candidate gene list for future analyses.
以前称为 ，在工业中用于生产木质纤维素分解酶和异源蛋白质。然而，驱动这些蛋白质表达的转录网络仍然不清楚。作为系统揭示该网络的第一步，我们分别研究了纤维素分解转录调节因子Clr1、Clr2和Clr4缺陷菌株的生长、蛋白质分泌和转录组指纹图谱。使用分裂标记或CRISPR/Cas12a技术分别缺失编码Clr1、Clr2和Clr4的基因，并将所得菌株以及亲本菌株在生物反应器中以葡萄糖作为碳源在恒化器条件下培养。在稳态条件下，添加纤维素代替葡萄糖以研究所有四种菌株对碳源可用性变化的遗传和细胞反应。值得注意的是， 和 缺失菌株无法继续在纤维素上生长，这表明这两种调节因子在纤维素分解代谢中都起着关键作用。它们的转录组指纹图谱不仅揭示了纤维素酶基因表达的缺乏，还降低了预测编码半纤维素酶、果胶酶和酯酶的基因的表达。相比之下， 缺失菌株的生长与亲本菌株非常相似。然而，观察到纤维素酶、半纤维素酶、果胶酶和酯酶的表达要强得多。本研究获得的数据表明，转录调节因子Clr1和Clr2都激活了预测编码纤维素酶以及半纤维素酶、果胶酶和酯酶的基因的表达。它们进一步表明，Clr1控制纤维素酶的基础表达，并通过诱导 表达启动对纤维素的主要木质纤维素分解反应。相比之下，Clr4似乎通过控制 表达而作为木质纤维素分解反应的抑制剂。所有四种菌株中的比较转录组学揭示了碳水化合物分解代谢和木质纤维素分解酶表达中潜在的新调节因子，这些调节因子定义了未来分析的候选基因列表。
