Constitutive expression of Botrytis aclada laccase in Pichia pastoris.
The heterologous expression of laccases is important for their large-scale production and genetic engineering--a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL(-1)) and the AOX1 system (495 mgL(-1)) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg(-1) GAP, 14.2 Umg(-1) AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h).
Food Biotechnology Laboratory, Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
Roman
R
Kittl
Christoph
C
Gonaus
Christian
C
Pillei
Dietmar
D
Haltrich
Roland
R
Ludwig
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D020171:Botrytis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D007700:Kinetics; D042845:Laccase / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic
D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D042845:Laccase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases