Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

中文翻译：

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通过内含子和CRISPR-Cas9提高黑曲霉中人促红细胞生成素的产量。

由于黑曲霉具有高表达和分泌能力，它已被用于生产异源蛋白。然而，人类蛋白质的表达水平是适度的。我们对研究黑曲霉是否可以高产量表达和分泌人促红细胞生成素（HuEPO）感兴趣。我们的策略是将内含子与CRISPR-Cas9结合使用以增加重组蛋白的产量。epo基因经过密码子优化，其表达由PmbfA启动子驱动。epo的另一个版本包含来自果糖1，，6-双磷酸酶（fbp）基因的内含子。根据对抗生素的抗性并且因为它们显示出不同于亲本菌株的蛋白质谱，所以选择了两个重组克隆uME12（无内含子）和uME23（有内含子），如SDS-PAGE所示。通过RT-PCR在两个菌落中证实了epo的表达，但仅通过uME23中的Western blot检测到重组EPO蛋白（rHUEPO）。ELISA估计来自uME23的rHuEPO产量约为1.8 mg L-1，表明内含子的存在导致较高的产量，可能是通过赋予mRNA更大的稳定性来实现的。另一方面，作为我们策略的一部分，我们决定在uME23菌株中使以下基因vps，prtT，algC和och1失活，这些基因与CRISPR-一起参与了黑曲霉蛋白质分泌，蛋白酶表达和蛋白质糖基化的调控。 Cas9，产生muPS20转化子。muPS20是不含蛋白酶的菌株，其rHuEPO生产水平提高了41.1倍。此外，其分子量约为27 kDa，表明上述基因中的突变可改善分泌，