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 so-called 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.
Highlights
The production of recombinant proteins at industrial scale is of increasing economic importance
We have recently developed a novel set of expression vectors based on the promoter of EYK1 gene encoding erythrulose kinase[10,11]
Its pro-region was codon optimized and synthesized during previous work[11]. It was cloned in expression vectors specific for Y. lipolytica (JMP4266, promoter pEYK1-A3B, URA3 marker) and P. pastoris
Summary
The production of recombinant proteins (rProt) at industrial scale is of increasing economic importance. 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, allowing growth on raw materials or industrial by-products[1,2]. MutS strains have often been demonstrated to produce higher rProt yields than Mut+ strains[15,16,17], which comes with the added advantage of requiring lower levels of methanol as inducer The latter is an important consideration since the use of methanol on large-scale presents drawbacks such as toxicity and flammability, high oxygen consumption and high heat release.
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