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 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.

Figure 1

Cell growth of Y. lipolytica RIY368 (triangles) and P. pastoris RIY311 (squares) during culture in bioreactor. Cells were grown for 72 h in YSPGE and in YSPSM medium, respectively. Displayed values correspond to the means of independent duplicate experiments. Standard deviations were less than 4.9% for Y. lipolytica and 7.4% for P. pastoris.

Figure 2

Lipase activities of Y. lipolytica RIY368 (triangles) and P. pastoris RIY311 (squares) during bioreactor cultivations. Cells were grown for 72 h in YSPGE and in YSPSM medium, respectively. Displayed values correspond to the means and standard deviations of independent duplicate experiments.

Figure 3

Specific EGFP extracellular fluorescence of strain RIY313 (pAOX1-αMF-GFP) during culture in presence (dark grey) or absence (light grey) of proteasome inhibitor MG-132. Fluorescence was determined by spectrophotometry after 32 h and 48 h of culture in YSPSM medium. Fluorescence values were normalized to biomass values at the corresponding time. Displayed values correspond to the means of independent triplicate experiments. Standard deviations were less than 19.0% in presence and 5.3% in absence of MG-132, respectively.

Figure 4

Relative expression level of genes HAC1, DOA1 and RPN4 in strains RIY311 (pAOX1-CalB, panel A) and RIY313 (pAOX1-αMF-EGFP, panel B). Expression levels were normalised according to that of actin and compared to that of strain RIY283 (MutS). Samples were collected after 24 h (light grey) and 48 h (dark grey) of growth in YSPSM medium. Displayed values correspond to means and standard deviations of independent triplicate experiments.