Yeasts are widely used cell factories for commercial heterologous protein production, however, specific productivities are usually tightly coupled to biomass formation. This greatly impacts production processes, which are commonly not run at the maximum growth rate, thereby resulting in suboptimal productivities. To tackle this issue, we evaluated transcriptomics datasets of the yeast Pichia pastoris (syn. Komagataella phaffii), which is known for its high secretory efficiency and biomass yield. These showed a clear downregulation of genes related to protein translation with decreasing growth rates, thus revealing the yeast translation machinery as cellular engineering target. By overexpressing selected differentially expressed translation factors, translation initiation was identified to be the main rate-limiting step. Specifically, overexpression of factors associated with the closed-loop conformation, a structure that increases stability and rates of translation initiation before start codon scanning is initiated, showed the strongest effects. Overexpression of closed-loop factors alone or in combination increased titers of different heterologous proteins by up to 3-fold in fed-batch processes. Furthermore, translation activity, correlating to the obtained secreted recombinant protein yields, selected transcript levels and total protein content were higher in the engineered cells. Hence, translation factor overexpression, globally affects the cell. Together with the observed impact on the transcriptome and total protein content, our results indicate that the capacity of P. pastoris for protein production is not at its limit yet.

酵母是广泛用于商业异源蛋白质生产的细胞工厂，然而，特定的生产力通常与生物质形成紧密相关。这极大地影响了生产过程，这些生产过程通常不会以最大增长率运行，从而导致生产力欠佳。为了解决这个问题，我们评估了酵母毕赤酵母（同义词Komagataella phaffii）的转录组学数据集，以其高分泌效率和生物质产量而闻名。这些表明与蛋白质翻译相关的基因随着生长速率的降低而明显下调，从而揭示了酵母翻译机制作为细胞工程目标。通过过度表达选定的差异表达翻译因子，翻译起始被确定为主要的限速步骤。具体而言，与闭环构象相关的因素的过度表达，一种在开始密码子扫描之前增加稳定性和翻译起始率的结构，显示出最强的效果。在分批补料过程中，单独或组合闭环因子的过表达使不同异源蛋白质的滴度增加多达 3 倍。此外，翻译活动，与获得的分泌重组蛋白产量相关，工程细胞中选定的转录水平和总蛋白含量更高。因此，翻译因子过度表达会全局影响细胞。连同观察到的对转录组和总蛋白含量的影响，我们的结果表明，用于蛋白质生产的毕赤酵母尚未达到其极限。