Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains.
Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains.
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葡萄糖淀粉酶是食品和生物燃料工业中最需要的工业酶之一。尼日尔是一种常用的生产商业葡糖淀粉酶的细胞工厂。几十年来，遗传操作促进了工业真菌菌株工程的重大进展，并深入了解其遗传特征。然而，基因工程在葡糖淀粉酶产生的工业菌株A中更费力。尼日尔N1和O 1的分生孢子数少（N1）或具有产孢能力（O 1）的真菌特性使其难以转化。尼日尔N1和O 1，并成功开发出高效转化工具。我们还使用自主复制质粒构建了成簇规则间隔短回文重复序列（CRISPR）/Cas9编辑无标记系统，以表达Cas9蛋白并引导RNA和选择标记。通过使用这里开发的遗传工具，我们产生了9个白化病缺失突变体。在非选择性条件下进行三轮继代培养后，白化病缺失株丢失了自主复制质粒。上述方法和优化过程为工程菌的改造提供了参考，不仅对这两株高产葡糖淀粉酶的工程菌的进一步改造，而且对其他工程菌的改造也有一定的借鉴意义。丝状真菌尼日尔被发酵工业广泛用于生产酶，特别是葡糖淀粉酶。虽然各种遗传技术已成功地用于野生型A。尼日尔，转化具有很少分生孢子的工业上使用的菌株（例如，A.尼日尔N1）或甚至是乌头状的（例如，A.尼日尔O 1）仍然费力。为此，我们建立了农杆菌介导法和原生质体介导法两种遗传转化方法。使用绿色荧光蛋白作为报告标记的尼日尔菌株N1和O 1。通过对菌丝体释放原生质体的各种因素进行优化，N1和O 1的转化效率分别达到89.3%（25/28）和82.1%（32/39）。助理N1和O 1的转化效率分别为98.2%（55/56）和43.8%（28/64）。我们还开发了一种无标记的CRISPR/Cas9基因组编辑系统，使用基于AMA 1的质粒来表达Cas9蛋白和sgRNA。在22个转化子中，在A.使用原生质体介导的转化方法和这里开发的无标记CRISPR/Cas9系统，本文改进的基因组编辑方法将加速阐明这些工业真菌中葡糖淀粉酶高产的机制，并将有助于在其他工业菌株中使用高效的靶向突变。尼日尔.
Glucoamylase is one of the most needed industrial enzymes in the food and biofuel industries. Aspergillus niger is a commonly used cell factory for the production of commercial glucoamylase. For decades, genetic manipulation has promoted significant progress in industrial fungi for strain engineering and in obtaining deep insights into their genetic features. However, genetic engineering is more laborious in the glucoamylase-producing industrial strains A. niger N1 and O1 because their fungal features of having few conidia (N1) or of being aconidial (O1) make them difficult to perform transformation on. In this study, we targeted A. niger N1 and O1 and successfully developed high-efficiency transformation tools. We also constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 editing marker-free system using an autonomously replicating plasmid to express Cas9 protein and to guide RNA and the selectable marker. By using the genetic tools developed here, we generated nine albino deletion mutants. After three rounds of sub-culturing under nonselective conditions, the albino deletions lost the autonomously replicating plasmid. Together, the tools and optimization process above provided a good reference to manipulate the tough working industrial strain, not only for the further engineering these two glucoamylase-hyperproducing strains, but also for other industrial strains. The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.
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影响因子：
46.9
作者：
de Groot, MJA;Bundock, P;Beijersbergen, AGM
通讯作者：
Beijersbergen, AGM
影响因子：
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作者：
Kovalenko, GA;Komova, OV;Rudina, NA
通讯作者：
Rudina, NA
影响因子：
2.8
作者：
Fang, Weiguo;Pei, Yan;Bidochka, Michael J.
通讯作者：
Bidochka, Michael J.
影响因子：
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作者：
Amey, RC;Athey-Pollard, A;Foster, GD
通讯作者：
Foster, GD
影响因子：
2.4
作者：
Duarte, R. T. D.;Staats, C. C.;Furlaneto, M. C.
通讯作者：
Furlaneto, M. C.