Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.

中文翻译：

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通过使用驱动毕赤酵母共翻译易位的分泌信号，可以显着提高生物反应器规模的细胞性能和蛋白质产量

毕赤酵母 (Komagataella spp.) 已成为生产具有生物技术意义的异源蛋白质的最重要宿主生物之一，其中许多是细胞外蛋白质。蛋白质分泌途径已被认为是一个限制过程，其中许多障碍已被查明。最近，我们发现了 ER 易位水平的瓶颈。在早期的探索性研究中，通过使用改进的嵌合分泌信号驱动蛋白质通过共翻译易位途径，可以在很大程度上克服这一限制。在这里，我们在生物反应器规模上进一步测试了由前 Ost1 信号序列组成的改进的分泌信号，它通过共翻译易位驱动蛋白质，其次是来自酿酒酵母α因子交配信息素分泌信号的pro区。为了比较，测试了常用的全长 α 因子分泌信号，该信号通过翻译后易位驱动蛋白质。这两种分泌信号融合到三种不同的模型蛋白中：四聚体红色荧光蛋白 E2-Crimson，可用于可视化分泌途径中的障碍；来自热链芽孢杆菌 (BTL2) 的脂肪酶 2；和米根霉脂肪酶 (ROL)。在分批培养中测试所有菌株以研究获得的不同生长参数。携带改善的分泌信号的菌株显示感兴趣的蛋白质的最终产量增加。有趣的是，与携带常规分泌信号的同类相比，它们能够以显着更高的最大比生长速率生长。这些结果在 5 L 分批补料培养中得到证实，最终产品浓度和体积生产率也得到了提高。