Document Detail

Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations.
MedLine Citation:
PMID:  24326738     Owner:  NLM     Status:  Publisher    
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.
Johanna Panula-Perälä; Antti Vasala; Janne Karhunen; Heikki Ojamo; Peter Neubauer; Anu Mursula
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-12-12
Journal Detail:
Title:  Bioprocess and biosystems engineering     Volume:  -     ISSN:  1615-7605     ISO Abbreviation:  Bioprocess Biosyst Eng     Publication Date:  2013 Dec 
Date Detail:
Created Date:  2013-12-11     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101088505     Medline TA:  Bioprocess Biosyst Eng     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, 90014, Oulu, Finland,
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