title: Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations.
abstract: Recombinant protein synthesis in Pichia pastoris is generally controlled by the strong methanol inducible AOX1 promoter which is repressed by glucose and glycerol. In shake flasks, commonly one or two methanol pulses are added per day for induction. Such pulse feeding procedure leads to carbon starvation phases, which may enhance proteolytic activities and, therefore, cause product losses. Starvation between the methanol pulses could be avoided with a continuous enzymatic feed of glucose from a glucose-based polymer. The amount of glucose was low enough to prevent AOX1 repression by glucose. Energy and carbon were continuously supplied for cell maintenance resulting in significantly increased cell densities and product activities, as shown here at the example of a fungal lipase expressed in P. pastoris. A threefold improvement in measured product activity was obtained by applying enzymatic glucose feed and a further improvement was achieved by applying a defined mixture of ammonium compounds. The strategy described here simplifies the general procedure in shaken cultures by allowing the direct continuation of the cultivation from glucose to the methanol-based production phase without a medium change. It is easily applicable to multiwell plates and thus beneficial for high throughput applications.
authors: Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, 90014, Oulu, Finland, johanna.panula-perala@oulu.fi.
authors: Johanna
authors: J
authors: Panula-Perälä
authors: Antti
authors: A
authors: Vasala
authors: Janne
authors: J
authors: Karhunen
authors: Heikki
authors: H
authors: Ojamo
authors: Peter
authors: P
authors: Neubauer
authors: Anu
authors: A
authors: Mursula
mesh_terms: D000143:Acids / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D064751:Ammonium Compounds / Q000737:chemistry; D001709:Biotechnology / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry; D013997:Time Factors
chemical_list: D000143:Acids; D064751:Ammonium Compounds; D005656:Fungal Proteins; D011108:Polymers; D000429:Alcohol Oxidoreductases; D008049:Lipase; D005947:Glucose; D000432:Methanol