Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10
Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10
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DOI：
10.1128/aem.03087-09
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发表时间：
2010-07
影响因子：
4.4
通讯作者：
Jaewoo Yoon;Tuerxun Aishan;J. Maruyama;K. Kitamoto
中科院分区：
文献类型：
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作者：
Jaewoo Yoon;Tuerxun Aishan;J. Maruyama;K. Kitamoto
丝状真菌由于其高分泌能力和真核翻译后修饰而成为异种蛋白生产的宿主。然而，尽管有这些积极的属性，转录后加工的瓶颈限制了蛋白质的产量。液泡蛋白分选基因VPS10编码一个分选受体，用于识别和传递几种酵母液泡蛋白。虽然它也可以针对重组蛋白和异常蛋白进行液泡降解，但对其破坏对异源蛋白生产的影响的了解有限。本研究对丝状真菌米曲霉（Aspergillus oryzae）的AoVps10基因进行了克隆和测序。显微镜下观察到表达AoVps10的转化体与增强的绿色荧光蛋白融合，发现融合蛋白定位于高尔基体和泡前区室。此外，Aovps10基因的破坏导致空泡羧肽酶AoCpyA的错选和分泌到培养基中，这表明Aovps10是空泡蛋白分选到空泡中所必需的。为了研究异源蛋白的细胞外生产水平，构建了表达牛凝乳酶（CHY）或人溶菌酶（HLY）的ΔAovps10突变体。有趣的是，ΔAovps10突变使CHY和HLY的最大胞外生成水平分别提高了3倍和2.2倍。细胞外异种蛋白的Western blot分析也显示了生产力的提高。这些结果表明，AoVps10在水稻芽孢杆菌中发挥调控外源蛋白分泌的作用，并可能通过高尔基体参与液泡蛋白的降解。
ABSTRACT Filamentous fungi have received attention as hosts for heterologous protein production because of their high secretion capability and eukaryotic posttranslational modifications. However, despite these positive attributes, a bottleneck in posttranscriptional processing limits protein yields. The vacuolar protein sorting gene VPS10 encodes a sorting receptor for the recognition and delivery of several yeast vacuolar proteins. Although it can also target recombinant and aberrant proteins for vacuolar degradation, there is limited knowledge of the effect of its disruption on heterologous protein production. In this study, cDNA encoding AoVps10 from the filamentous fungus Aspergillus oryzae was cloned and sequenced. Microscopic observation of the transformant expressing AoVps10 fused with enhanced green fluorescent protein showed that the fusion protein localized at the Golgi and prevacuolar compartments. Moreover, disruption of the Aovps10 gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA into the medium, indicating that AoVps10 is required for sorting of vacuolar proteins to vacuoles. To investigate the extracellular production levels of heterologous proteins, ΔAovps10 mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the ΔAovps10 mutation increased the maximum extracellular production levels of CHY and HLY by 3- and 2.2-fold, respectively. Western blot analysis of extracellular heterologous proteins also demonstrated an improvement in productivity. These results suggest that AoVps10 plays a role in the regulation of heterologous protein secretion in A. oryzae and may be involved in the vacuolar protein degradation through the Golgi apparatus.