TY - JOUR
T1 - Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter
AU - Resina, David
AU - Maurer, Michael
AU - Cos, Oriol
AU - Arnau, Carolina
AU - Carnicer, Marc
AU - Marx, Hans
AU - Gasser, Brigitte
AU - Valero, Francisco
AU - Mattanovich, Diethard
AU - Ferrer, Pau
N1 - Funding Information:
Research in Vienna was supported by the Austrian Research Promotion Agency (programme FHplus) and the Austrian Exchange Service.
Funding Information:
This work was supported by the Projects CTQ2004-00300 and CTQ2007-60347 and the integrated action HU2005-0001 of the Spanish Ministry of Science and Innovation, and by the grant No. 2005-SGR-00698 from the AGAUR, Generalitat de Catalunya. DR was recipient of a doctoral fellowship from the Spanish Ministry of Science and Technology. The Department of Chemical Engineering of the UAB constitutes the Biochemical Engineering Unit of the Reference Network in Biotechnology of the Generalitat de Catalunya.
PY - 2009/9/15
Y1 - 2009/9/15
N2 - The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding a β-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h-1. It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/Δgas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.
AB - The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding a β-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h-1. It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/Δgas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.
KW - BiP
KW - Fed-batch cultivation
KW - Formaldehyde dehydrogenase promoter
KW - Gas1
KW - Hac1
KW - Pichia pastoris
KW - Rhizopus oryzae lipase
KW - Unfolded protein response
UR - http://www.scopus.com/inward/record.url?scp=70350547284&partnerID=8YFLogxK
U2 - 10.1016/j.nbt.2009.01.008
DO - 10.1016/j.nbt.2009.01.008
M3 - Article
C2 - 19552885
AN - SCOPUS:70350547284
SN - 1871-6784
VL - 25
SP - 396
EP - 403
JO - New Biotechnology
JF - New Biotechnology
IS - 6
ER -