Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B

Abstract

The large-scale production of recombinant proteins (rProt) is becoming increasingly economically important. Among the different hosts used for rProt production, yeasts are gaining popularity. The socalled non-conventional yeasts, such as the methylotrophic Pichia pastoris and the dimorphic Yarrowia lipolytica, are popular choices due to their favorable characteristics and well-established expression systems. Nevertheless, a direct comparison of the two systems for rProt production and secretion was lacking. This study therefore aimed to directly compare Y. lipolytica and P. pastoris for the production and secretion of lipase CalB in bioreactor. Y. lipolytica produced more than double the biomass and more than 5-fold higher extracellular lipase than P. pastoris. Furthermore, maximal CalB production levels were reached by Y. lipolytica in half the cultivation time required for maximal production by P. pastoris. Conversely, P. pastoris was found to express 7-fold higher levels of CalB mRNA. Secreted enhanced green fluorescent protein -in isolation and fused to CalB-and protease inhibitor MG-132 were used in P. pastoris to further investigate the reasons behind such discrepancy. The most likely explanation was ultimately found to be protein degradation by endoplasmic reticulum-associated protein degradation preceding successful secretion. This study highlighted the multifaceted nature of rProt production, prompting a global outlook for selection of rProt production systems.The production of recombinant proteins (rProt) at industrial scale is of increasing economic importance. Among the different microbial chassis that have been developed for that purpose, yeasts are emerging as the preferred option for the production of recombinant enzymes and therapeutic proteins. The main advantage of yeasts over bacterial systems such as Escherichia coli is the possibility to obtain post-translational modified proteins in the culture supernatant at a gram per liter scale. Historically, Saccharomyces cerevisiae has been used as the reference eukaryotic chassis, however it suffers several drawbacks such as low protein productivity, overflow metabolism and hyperglycosylation of rProt. Moreover, it is less metabolically adapted to catabolize raw carbon and nitrogen sources, which are nowadays increasingly considered as feedstocks in bioprocesses, with the intention of reducing the process costs. Currently, non-conventional yeasts such as Pichia pastoris and Yarrowia lipolytica are considered as realistic alternatives to S. cerevisiae for rProt synthesis. Both species combine the advantages of growth at high cell density and production and secretion of rProt at high yields, with low nutritional requirements, thus allowing growth on raw materials or industrial by-products 1,2 .In most cases, the processes developed for rProt production are two-step systems involving a first phase of biomass generation under repressive or non-inducing conditions, followed by an induction phase during which rProt are synthetized and secreted into the cult...

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

“…It has been previously suggested that although P. pastoris is naturally capable of producing large amounts of proteins in response to methanol, it is not naturally prone to high-level secretion of proteins. In contrast, Y. lipolytica naturally secretes high levels of various native enzymes and hence its secretory pathway is naturally less prone to induce endoplasmic reticulum (ER) associated degradation (Theron, Vandermies, Telek, Steels, & Fickers, 2020). Although Y. lipolytica's native potential in this regard is highly competitive (alkaline extracellular protease can be secreted up to 2 g L −1 ; Le Dall, Nicaud, & Gaillardin, 1994;Ogrydziak, 2013), it is interesting to systematically study different parameters that enable further enhancement of this trait by both bioprocessing (Vandermies & Fickers, 2019) and genetic engineering approaches (Celi nska, Borkowska, Białas, Korpys, & Nicaud, 2018).…”

Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling

“…Three strains exhibiting no significant difference in the activity were selected for further studies. As demonstrated previously, such strategy enables selection of strains with only a single expression cassette integrated in a neutral site in the genome, ensuring negligible inter-clone variation [9,41].…”

“…Taking advantage of the progress within this field, it is now feasible to generate a number of variants of complex genetic constructions to carefully study and optimize their design, so that expression of target genes is adjusted to the requirements of a specific bioprocess. Due to unique native metabolic properties, like intrinsically high production of lipids [6], polyols [7], organic acids [8], and recently evidenced superior capacity for heterologous proteins production over a typical workhorse in this regard -Komagataella phaffi (P. pastoris) [9], Y. lipolytica stains are frequently subjected to genetic modifications broadening its scope of utilized substrates, with the aim to improve economic feasibility of production processes [10]. For example, Y. lipolytica strains has been endowed with artificial ability to grow on sucrose [11], inulin [12], galactose [13], starch [14] or even cellulose [15,16].…”

“…Out of the pre-screened pool, three representative strains demonstrating negligible differences in starch consumption rate were subjected to further analyses. Such preselection strategy has been recently successfully used [9] and demonstrated that the following interclone variation was negligible.…”

“…Three sub-clones with negligible variability in the analyzed trait, were subjected to further studies. According to previous findings, such strategy enables selection of clones with a single copy of heterologous cassette, integrated in neutral site in the genome, ensuring minimal variance in these parameters [9,48]. The reference Po1h strain was each time cultured simultaneously in the same media formulations, but supplemented with uracil (up to 2 mg/L).…”