Liao Gui-Yan, Zhao Shuai, Zhang Ting, Li Cheng-Xi, Liao Lu-Sheng, Zhang Feng-Fei, Luo Xue-Mei, Feng Jia-Xun
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, 530004 Guangxi People's Republic of China.
Biotechnol Biofuels. 2018 Oct 8;11:276. doi: 10.1186/s13068-018-1276-8. eCollection 2018.
Perfect and low cost of fungal amylolytic and cellulolytic enzymes are prerequisite for the industrialization of plant biomass biorefinergy to biofuels. Genetic engineering of fungal strains based on regulatory network of transcriptional factors (TFs) and their targets is an efficient strategy to achieve the above described aim. produces integrative amylolytic and cellulolytic enzymes; however, the regulatory mechanism associated with the expression of amylase and cellulase genes in remains unclear. In this study, we screened for and identified novel TFs regulating amylase and/or cellulase gene expression in 1-95 through comparative transcriptomic and genetic analyses.
Comparative analysis of the transcriptomes from 1-95 grown on media in the presence and absence of glucose or soluble starch as the sole carbon source screened 33 candidate TF-encoding genes that regulate amylase gene expression. Thirty of the 33 genes were successfully knocked out in the parental strain ∆, with seven of the deletion mutants firstly displaying significant changes in amylase production as compared with the parental strain. Among these, ∆ (: ) showed the most significant decrease (81.5%) in amylase production, as well as a 57.7% reduction in filter paper cellulase production. Real-time quantitative reverse transcription PCR showed that dynamically regulated the expression of major amylase and cellulase genes during cell growth, and in vitro electrophoretic mobility shift assay revealed that TpRfx1 bound the promoter regions of genes encoding α-amylase (/), glucoamylase (/), cellobiohydrolase (/), β-glucosidase (/ and endo-β-1,4-glucanase (/). TpRfx1 protein containing a regulatory factor X (RFX) DNA-binding domain belongs to RFX family.
We identified a novel RFX protein TpRFX1 that directly regulates the expression of amylase and cellulase genes in , which provides new insights into the regulatory mechanism of fungal amylase and cellulase gene expression.
真菌淀粉酶和纤维素酶完美且低成本是植物生物质生物精炼为生物燃料实现工业化的前提条件。基于转录因子（TFs）及其靶标的调控网络对真菌菌株进行基因工程改造是实现上述目标的有效策略。里氏木霉能产生整合型淀粉酶和纤维素酶；然而，里氏木霉中淀粉酶和纤维素酶基因表达相关的调控机制仍不清楚。在本研究中，我们通过比较转录组学和遗传学分析，筛选并鉴定了里氏木霉1-95中调控淀粉酶和/或纤维素酶基因表达的新型转录因子。
对在以葡萄糖或可溶性淀粉作为唯一碳源的培养基上生长的里氏木霉1-95的转录组进行比较分析，筛选出33个调控淀粉酶基因表达的候选转录因子编码基因。这33个基因中的30个在亲本菌株Δ中成功敲除，其中7个缺失突变体与亲本菌株相比，淀粉酶产量首先出现显著变化。其中，Δ（：）的淀粉酶产量下降最为显著（81.5%），滤纸纤维素酶产量也降低了57.7%。实时定量逆转录PCR表明，在细胞生长过程中动态调控主要淀粉酶和纤维素酶基因的表达，体外电泳迁移率变动分析表明，TpRfx1与编码α-淀粉酶（/）、糖化酶（/）、纤维二糖水解酶（/）、β-葡萄糖苷酶（/和内切-β-1,4-葡聚糖酶（/）的基因启动子区域结合。含有调控因子X（RFX）DNA结合结构域的TpRfx1蛋白属于RFX家族。
我们鉴定了一种新型RFX蛋白TpRFX1，其直接调控里氏木霉中淀粉酶和纤维素酶基因的表达，这为真菌淀粉酶和纤维素酶基因表达的调控机制提供了新的见解。
