The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-β-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.

中文翻译：

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米曲霉中N-聚糖缺失的糖蛋白的分泌产生。

制药行业对糖蛋白的生产有很高的需求。糖蛋白的糖型对于药理活性至关重要。然而，一般而言，细胞产生具有异源糖型的糖蛋白，这不利于制备均匀，有效的治疗性蛋白。在这里，为了产生更多具有N-聚糖均一性的糖蛋白，我们应用了GlycoDelete策略，在该策略中，来自真菌性红景天菌（EndoT）的内切β-N-乙酰氨基葡糖苷酶（ENGase）在高尔基体膜上表达，以裂解N-聚糖。分泌的糖蛋白，到米曲霉细胞。首先，我们选择了候选的跨膜结构域，将EndoT靶向米曲霉细胞中的高尔基体膜，生成了表达跨膜融合的EndoT蛋白的构建体，并产生了四个潜在的AoGlycoDelete菌株。然后我们证实这些菌株产生的α-淀粉酶的分子量低于天然α-淀粉酶的分子量，而对生长没有影响。为了测试米曲霉α-淀粉酶蛋白是否已被EndoT切割，我们从AoGlycoDelete菌株中表达并纯化了带有HA标签的α-淀粉酶AmyB和葡糖淀粉酶GlaA蛋白。完整蛋白的MS和N-聚糖分析证实，从AoGlycoDelete菌株生产的AmyB-HA和GlaA-HA均不包含N-聚糖。最后，我们确定了由AoGlycoDelete菌株产生的淀粉酶的酶活性，这表明缺乏N-聚糖不会在测试条件下影响其活性。总的来说，