Industrially relevant traits of Yarrowia lipolytica, like high growth rate, capacity to grow at high cell density or to synthesize biomolecules with high productivities, strongly rely on sufficient oxygen provision. Although the impact of oxygen availability (OA) on the physiology of Y. lipolytica has been already studied, its influence on recombinant protein (rProt) synthesis and secretion has been largely neglected to date. With the aim to fill this gap, a fluorescent reporter protein (yellow fluorescent protein [YFP]) was used herein as a proxy to follow simultaneously rProt synthesis and secretion in Y. lipolytica under different OAs. This study covers the analysis of the reporter gene expression through reverse transcription quantitative polymerase chain reaction, polypeptide synthesis and its retention‐to‐secretion ratio using flow cytometry and fluorymetry during shake flasks and bioreactor cultivations under different OA. The results gathered demonstrate that OA has a dramatic impact on the kinetics of intracellular and extracellular YFP accumulation. Higher rProt production and secretion were favoured under high OA, and were largely related to OA and not to cell growth. Our observations also suggest the existence of some upper limit of secretory protein accumulation inside the cells above which massive secretion is initiated. Moreover, at low OA, the first bottleneck in rProt synthesis occurs as early as at transcription level, which could results from a lower availability of transcriptional machinery elements. Finally, using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis.

中文翻译：

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在基于解脂耶氏酵母的蛋白质生产平台中，氧气可用性对异源基因表达和多肽分泌动力学的影响。

Yarrowia lipolytica 的工业相关特性，如高增长率、在高细胞密度下生长的能力或以高生产力合成生物分子的能力，强烈依赖于充足的氧气供应。尽管已经研究了氧气可用性 (OA) 对解脂耶氏酵母生理机能的影响，但其对重组蛋白 (rProt) 合成和分泌的影响迄今为止在很大程度上被忽视了。为了填补这一空白，本文使用荧光报告蛋白（黄色荧光蛋白 [YFP]）作为代理同时跟踪解脂耶氏酵母中的rProt 合成和分泌在不同的OA下。本研究涵盖了在不同 OA 下的摇瓶和生物反应器培养过程中，使用流式细胞术和荧光法通过逆转录定量聚合酶链反应、多肽合成及其保留分泌比分析报告基因的表达。收集的结果表明 OA 对细胞内和细胞外 YFP 积累的动力学有显着影响。在高 OA 下有利于更高的 rProt 产生和分泌，并且在很大程度上与 OA 相关，而不是与细胞生长有关。我们的观察还表明，细胞内存在一些分泌蛋白积累的上限，高于该上限，大量分泌就开始了。此外，在低 OA 时，rProt 合成的第一个瓶颈早在转录水平就出现了，这可能是由于转录机制元件的可用性较低造成的。最后，使用流式细胞术和生物反应器培养，我们强调卵形细胞在 rProt 合成方面通常更有效。