Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load.

While overproduction of recombinant secretory proteins (rs-Prots) triggers multiple changes in the physiology of the producer cell, exposure to suboptimal growth conditions may further increase that biological response. The environmental conditions may modulate the efficiency of both the rs-Prot gene transcription and translation but also the polypeptide folding. Insights into responses elicited by different environmental stresses on the rs-Prots synthesis and host yeast physiology might contribute to a better understanding of fundamental biology processes, thus providing some clues to further optimise bioprocesses. Herein, a series of batch cultivations of Yarrowia lipolytica strains differentially metabolically burdened by the rs-Prots overproduction have been conducted. Combinations of different stress factors, namely pH (3/7) and oxygen availability (kLa 28/110 h-1), have been considered for their impact on cell growth and morphology, substrate consumption, metabolic activity, genes expression, and secretion of the rs-Prots. Amongst others, our data demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth. Moreover, the observed decrease in rs-Prot production under adverse environmental conditions rather results from the emergence of a less-producing cell subpopulation than from the decrease of the synthetic capacity of the whole cell population.

Impact of oxygen availability on heterologous geneexpression and polypeptide secretion dynamics in Yarrowia lipolytica-based protein production platforms.

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.