Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed.

Key Points

• Eight expression systems, producing different reporter proteins were analyzed.

• The cells were maintained in steady-state by continuous chemostat culturing.

• Protein- and promoter-specific effects were observed.

重组分泌蛋白的过量生产会引发许多生理扰动。取决于给定的异源蛋白质特性，生产细胞面临不同的挑战，导致其生理学和靶蛋白生产速率方面的变化。在本研究中，我们使用稳态维持解脂耶氏酵母细胞以研究不同异源蛋白对宿主细胞生理行为的影响。这种方法可以使特定蛋白质过量生产的影响与生长阶段或由分析种群的异质性引起的现象脱钩。总共对八株变异株进行了恒化培养，这些变异株分别高产了不同分子量（27-65 kDa）和报道活性（酶促和荧光）的异源蛋白。根据底物利用率，生物量和代谢产物的产生以及报告蛋白的合成分析了维持状态的细胞。简化了各个产品之间碳和氮的分配，并对异源基因进行表达分析。此处获得的数据表明，使用更具转录活性的启动子会导致更多的C通量流向目标蛋白质，从而以较高的生物量积累为代价，显着提高目标多肽的比量和生产率，而对生物量却没有明显影响多元醇生产。报道蛋白翻译后修饰的程度，即二硫键数量和糖基化模式，强烈影响合成过程。观察到关于蛋白质形成动力学，基因表达水平和转录物-蛋白质线性的特异性反应。此处获得的数据表明，使用更具转录活性的启动子会导致更多的C通量流向目标蛋白质，从而以较高的生物量积累为代价，显着提高目标多肽的比量和生产率，而对生物量却没有明显影响多元醇生产。报道蛋白翻译后修饰的程度，即二硫键数量和糖基化模式，强烈影响合成过程。观察到关于蛋白质形成动力学，基因表达水平和转录物-蛋白质线性的特异性反应。此处获得的数据表明，使用更具转录活性的启动子会导致更多的C通量流向目标蛋白质，从而以较高的生物量积累为代价，显着提高目标多肽的比量和生产率，而对生物量却没有明显影响多元醇生产。报道蛋白翻译后修饰的程度，即二硫键数量和糖基化模式，强烈影响合成过程。观察到关于蛋白质形成动力学，基因表达水平和转录物-蛋白质线性的特异性反应。报道蛋白翻译后修饰的程度，即二硫键数量和糖基化模式，强烈影响合成过程。观察到关于蛋白质形成动力学，基因表达水平和转录物-蛋白质线性的特异性反应。报道蛋白翻译后修饰的程度，即二硫键数量和糖基化模式，强烈影响合成过程。观察到关于蛋白质形成动力学，基因表达水平和转录物-蛋白质线性的特异性反应。

关键点

•分析了产生不同报道蛋白的八个表达系统。

•通过连续的恒化器培养将细胞保持在稳态。

•观察到蛋白质和启动子特异性作用。