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首页> 外文期刊>Applied Microbiology >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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Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

机译：丝状真菌米曲霉通过破坏液泡蛋白分选受体基因Aovps10来增强异源蛋白的生产和分泌

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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, Δ Aovps10 mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the Δ Aovps10 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.
机译：丝状真菌因其高分泌能力和真核翻译后修饰而作为异源蛋白质生产的宿主受到关注。但是，尽管具有这些积极的属性，但转录后加工中的瓶颈限制了蛋白质的产量。液泡蛋白分选基因VPS10编码用于识别和传递几种酵母液泡蛋白的分选受体。尽管它还可以靶向重组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。在这项研究中，克隆并测序了来自丝状真菌米曲霉的AoVps10的cDNA。显微镜观察表达与增强的绿色荧光蛋白融合的AoVps10的转化体，发现融合蛋白位于高尔基体和前真空区室。此外，Aovps10基因的破坏导致液泡羧肽酶AoCpyA丢失并分泌到培养基中，表明将液泡蛋白分类为液泡需要AoVps10。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的最大细胞外产生水平分别提高了3倍和2.2倍。细胞外异源蛋白质的蛋白质印迹分析也显示出生产率的提高。这些结果表明，AoVps10在米曲霉中异源蛋白分泌的调节中起作用，并且可能参与通过高尔基体的液泡蛋白降解。

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