Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, 1060, Wien, Austria.
Appl Microbiol Biotechnol. 2010 Sep;88(1):95-103. doi: 10.1007/s00253-010-2710-4. Epub 2010 Jun 22.
Fungal hydrophobins have potential for several applications because of their abilities to change the hydrophobicity of different surfaces. Yet because of their tendency for aggregation and attachment to interfacial areas only few production processes have so far been reported. Towards the development of a heterologous production system, we report here the expression of a class I hydrophobin DewA of Aspergillus nidulans in Hypocrea jecorina (Trichoderma reesei). Using the H. jecorina hfb2 (class II hydrophobin-encoding) promoter and lactose as a carbon source, only a minor fraction of the DewA remained cell-wall-bound and the majority of it secreted into the medium with up to 15% of the total secreted protein. N-terminal amino acid sequencing showed that it was correctly processed. In contrast, no DewA was produced under the cel7A (cellobiohydrolase I) promoter, although its mRNA was abundantly detected in the cells. This lack of secretion is not due to trapping in the cell wall or to its degradation because of the unfolded protein response. Recombinant DewA could be conveniently precipitated from the culture filtrate, and its bioactivity proven by its ability to stably bind to hydrophilic and hydrophobic surfaces (glass and Teflon, respectively). We thus consider H. jecorina as a promising host for further optimization of DewA production.
真菌疏水性蛋白由于能够改变不同表面的疏水性,因此具有多种应用潜力。然而,由于其易于聚集和附着在界面区域,迄今为止,只有少数生产工艺得到了报道。为了开发一种异源生产系统,我们在这里报告了 Aspergillus nidulans 类 I 疏水性蛋白 DewA 在 Hypocrea jecorina(Trichoderma reesei)中的表达。使用 H. jecorina hfb2(类 II 疏水性蛋白编码)启动子和乳糖作为碳源,只有一小部分 DewA 仍然与细胞壁结合,大部分分泌到培养基中,总分泌蛋白的比例高达 15%。N 端氨基酸测序表明它被正确加工。相比之下,在 cel7A(纤维素酶 I)启动子下没有产生 DewA,尽管在细胞中大量检测到其 mRNA。这种缺乏分泌不是由于被困在细胞壁中或由于未折叠蛋白反应而降解。重组 DewA 可以方便地从培养滤液中沉淀出来,并通过其能够稳定结合亲水和疏水表面(分别为玻璃和聚四氟乙烯)的能力证明其生物活性。因此,我们认为 H. jecorina 是进一步优化 DewA 生产的有前途的宿主。