Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica.
The sequences encoding the genes for endoglucanase II and cellobiohydrolase II from the fungus Trichoderma reesei QM9414 were successfully cloned and expressed in Yarrowia lipolytica under the control of the POX2 or TEF promoters, and using either the native or preproLip2 secretion signals. The expression level of both recombinant enzymes was compared with that obtained using Pichia pastoris, under the control of the AOX1 promoter to evaluate the utility of Y. lipolytica as a host strain for recombinant EGII and CBHII production. Extracellular endoglucanase activity was similar between TEF-preoproLip2-eglII expressed in Y. lipolytica and P. pastoris induced by 0.5 % (v/v) methanol, but when recombinant protein expression in P. pastoris was induced with 3 % (v/v) methanol, the activity was increased by about sevenfold. In contrast, the expression level of cellobiohydrolase from the TEF-preproLip2-cbhII cassette was higher in Y. lipolytica than in P. pastoris. Transformed Y. lipolytica produced up to 15 mg/l endoglucanase and 50 mg/l cellobiohydrolase, with the specific activity of both proteins being greater than their homologs produced by P. pastoris. Partial characterization of recombinant endoglucanase II and cellobiohydrolase II expressed in both yeasts revealed their optimum pH and temperature, and their pH and temperature stabilities were identical and hyperglycosylation had little effect on their enzymatic activity and properties.
Chulalongkorn University, Bangkok, Thailand. nassapat.boonvitthya@hotmail.com
Nassapat
N
Boonvitthya
Sophie
S
Bozonnet
Vorakan
V
Burapatana
Michael J
MJ
O'Donohue
Warawut
W
Chulalaksananukul
D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; C056373:endoglucanase 2; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase