A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris |
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| Affiliation: | 1. Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, P.O. Box 91000, Portland, OR 97291-1000, USA;2. Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands;3. Institute for Microbial and Biochemical Technology, Forest Products Laboratory, US Department of Agriculture, One Gifford Pinchot Drive, Madison, WI 53705-2398, USA;1. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;2. National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;3. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;1. College of Science and Engineering, James Cook University, Townsville 4811, Qld, Australia;2. Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Townsville 4811, Qld, Australia;3. The University of Queensland, Protein Expression Facility, Brisbane 4072, Qld, Australia;4. College of Public Health, Medical and Veterinary Science, James Cook University, Townsville 4811, Qld, Australia;5. Department of Biology, Hong Kong Baptist University, Hong Kong |
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| Abstract: | In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5′ end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61–65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using β-lactamase as a reporter, we show that the FLD1 promoter (PFLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of β-lactamase produced under control of PFLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). Thus, PFLD1 is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain. |
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