PDF(Pubmed)
Abstract：
Filamentous fungi can produce raw-starch-degrading enzyme, however, regulation of production of raw-starch-degrading enzyme remains poorly understood thus far. Here, two novel transcription factors raw-starch-degrading enzyme regulator D (RsrD) and raw-starch-degrading enzyme regulator E (RsrE) were identified to participate in the production of raw-starch-degrading enzyme in Penicillium oxalicum. Individual knockout of rsrD and rsrE in the parental strain Δku70 resulted in 31.1%-92.9% reduced activity of raw-starch-degrading enzyme when cultivated in the presence of commercial starch from corn. RsrD and RsrE contained a basic leucine zipper and a Zn2Cys6-type DNA-binding domain, respectively, but with unknown functions. RsrD and RsrE dynamically regulated the expression of genes encoding major amylases over time, including raw-starch-degrading glucoamylase gene PoxGA15A and α-amylase gene amy13A. Interestingly, RsrD and RsrE regulated each other at transcriptional level, through binding to their own promoter regions; nevertheless, both failed to bind to the promoter regions of PoxGA15A and amy13A, as well as the known regulatory genes for regulation of amylase gene expression. RsrD appears to play an epistatic role in the module RsrD-RsrE on regulation of amylase gene expression. This study reveals a novel regulatory pathway of fungal production of raw-starch-degrading enzyme.IMPORTANCETo survive via combating with complex extracellular environment, filamentous fungi can secrete plant polysaccharide-degrading enzymes that can efficiently hydrolyze plant polysaccharide into glucose or other mono- and disaccharides, for their nutrients. Among the plant polysaccharide-degrading enzymes, raw-starch-degrading enzymes directly degrade and convert hetero-polymeric starch into glucose and oligosaccharides below starch gelatinization temperature, which can be applied in industrial biorefinery to save cost. However, the regulatory mechanism of production of raw-starch-degrading enzyme in fungi remains unknown thus far. Here, we showed that two novel transcription factors raw-starch-degrading enzyme regulator D (RsrD) and raw-starch-degrading enzyme regulator E (RsrE) positively regulate the production of raw-starch-degrading enzyme by Penicillium oxalicum. RsrD and RsrE indirectly control the expression of genes encoding enzymes with amylase activity but directly regulate each other at transcriptional level. These findings expand diversity of gene expression regulation in fungi.
摘要：
丝状真菌能产生原淀粉降解酶,然而,迄今为止，对生淀粉降解酶的生产调节仍知之甚少。这里,确定了两个新的转录因子原淀粉降解酶调节因子D（RsrD）和原淀粉降解酶调节因子E（RsrE）参与了草酸青霉中原淀粉降解酶的产生。当在玉米的商业淀粉存在下培养时，亲本菌株Δku70中rsrD和rsrE的单独敲除导致生淀粉降解酶活性降低31.1％-92.9％。RsrD和RsrE包含碱性亮氨酸拉链和Zn2Cys6型DNA结合域，分别,但功能未知。随着时间的推移，RsrD和RsrE动态调节编码主要淀粉酶的基因的表达，包括生淀粉降解葡糖淀粉酶基因PoxGA15A和α-淀粉酶基因amy13A。有趣的是,RsrD和RsrE在转录水平上相互调控，通过与它们自己的启动子区域结合；尽管如此，两者都未能与PoxGA15A和amy13A的启动子区域结合，以及已知的调节淀粉酶基因表达的调节基因。RsrD似乎在模块RsrD-RsrE中对淀粉酶基因表达的调节起重要作用。这项研究揭示了真菌生产生淀粉降解酶的新调控途径。重要通过与复杂的细胞外环境对抗而生存，丝状真菌可以分泌植物多糖降解酶，可以有效地将植物多糖水解为葡萄糖或其他单糖和二糖，为了他们的营养。在植物多糖降解酶中，生淀粉降解酶在淀粉糊化温度以下直接降解并转化为葡萄糖和寡糖，可应用于工业生物炼制以节省成本。然而,到目前为止，真菌中产生原淀粉降解酶的调节机制仍然未知。这里,我们表明，两个新的转录因子原淀粉降解酶调节因子D（RsrD）和原淀粉降解酶调节因子E（RsrE）正调节草酸青霉对原淀粉降解酶的产生。RsrD和RsrE间接控制编码具有淀粉酶活性的酶的基因的表达，但在转录水平上直接调节彼此。这些发现扩大了真菌基因表达调控的多样性。
