A significant problem during recombinant protein production is proteolysis. One of the most common preventive strategies is the addition of protease inhibitors, which has drawbacks, such as their short half-life and high cost, and their limited prevention of extracellular proteolysis. Actinomycetes produce the most commonly used inhibitors, which are non-ribosomal small aldehydic peptides. Previously, an unprecedented biosynthetic route involving a condensation-minus non-ribosomal peptide synthetase (NRPSs) and a tRNA utilizing enzyme (tRUE) was shown to direct the synthesis of one of these inhibitor peptides, livipeptin. Here, we show that expression of the livipeptin biosynthetic pathway encoded by the genes in CHO cells resulted in the production of this metabolite with cysteine protease inhibitory activity, implying that mammalian tRNAs were recruited by the lvp system. CHO cells transiently expressing the biosynthetic pathway produced livipeptin without affecting cell growth or viability. Expression of the system in CHO cells producing two model proteins, secreted alkaline phosphatase (hSeAP) and a monoclonal antibody, resulted in higher specific productivity with reduced proteolysis. We show for the first time that the expression of a bacterial biosynthetic pathway is functional in CHO cells, resulting in the efficient, low-cost synthesis of a protease inhibitor without adverse effects on CHO cells. This expands the field of metabolic engineering of mammalian cells by expressing the overwhelming diversity of actinomycetes biosynthetic pathways and opens a new option for proteolysis inhibition in bioprocess engineering.

中文翻译：

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产生小非核糖体肽醛的细菌生物合成途径在 CHO 细胞中的表达可防止重组蛋白的蛋白水解

重组蛋白生产过程中的一个重要问题是蛋白水解。最常见的预防策略之一是添加蛋白酶抑制剂，但其存在半衰期短、成本高、对细胞外蛋白水解的预防有限等缺点。放线菌产生最常用的抑制剂，它们是非核糖体小醛肽。此前，涉及缩合非核糖体肽合成酶 (NRPS) 和 tRNA 利用酶 (tRUE) 的前所未有的生物合成途径被证明可以指导其中一种抑制肽 Livipeptin 的合成。在这里，我们表明 CHO 细胞中基因编码的 Livipeptin 生物合成途径的表达导致了这种具有半胱氨酸蛋白酶抑制活性的代谢物的产生，这意味着哺乳动物 tRNA 是由 lvp 系统招募的。瞬时表达生物合成途径的 CHO 细胞产生利维肽而不影响细胞生长或活力。该系统在 CHO 细胞中的表达产生两种模型蛋白：分泌型碱性磷酸酶 (hSeAP) 和单克隆抗体，导致更高的比生产率和减少的蛋白水解。我们首次证明细菌生物合成途径的表达在 CHO 细胞中发挥作用，从而实现蛋白酶抑制剂的高效、低成本合成，而不会对 CHO 细胞产生不利影响。这通过表达放线菌生物合成途径的压倒性多样性扩展了哺乳动物细胞的代谢工程领域，并为生物过程工程中的蛋白水解抑制开辟了新的选择。