Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter.
A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.
Resina
David
D
Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Serrano
Alícia
A
Valero
Francisco
F
Ferrer
Pau
P
D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D005990:Glycerol / Q000378:metabolism; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D009584:Nitrogen / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013012:Sorbitol / Q000378:metabolism
D008744:Methylamines; D011994:Recombinant Proteins; D013012:Sorbitol; C027451:methylamine; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D008049:Lipase; D009584:Nitrogen; D005990:Glycerol; D000432:Methanol