title: Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter.
abstract: As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (qp) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l-1) and at a growth rate close to μmax(0.15 h-1). Between 0.017 and 0.115 h-1in glycerol-limited fedbatch cultures, basal levels of qp> 0.4 mg g-1h-1CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h-1, an elevated qpoccurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the qp(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h-1and vice versa. Changes in qpwere related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.
authors.affiliation: Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences (ZHAW), Campus Grüental, 8820, Wädenswil, Switzerland.
authors.forename: Verena
authors.initials: V
authors.lastname: Looser
authors.affiliation: Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences (ZHAW), Campus Grüental, 8820, Wädenswil, Switzerland.
authors.forename: Dominik
authors.initials: D
authors.lastname: Lüthy
authors.affiliation: Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences (ZHAW), Campus Grüental, 8820, Wädenswil, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Straumann
authors.affiliation: Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences (ZHAW), Campus Grüental, 8820, Wädenswil, Switzerland.
authors.forename: Katrin
authors.initials: K
authors.lastname: Hecht
authors.affiliation: Department of Biotechnology, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Melzoch
authors.affiliation: Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences (ZHAW), Campus Grüental, 8820, Wädenswil, Switzerland. koka@zhaw.ch.
authors.forename: Karin
authors.initials: K
authors.lastname: Kovar
mesh_terms: D061249:Batch Cell Culture Techniques; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D000432:Methanol / Q000032:analysis*; D010843:Pichia / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica; D005990:Glycerol; D000432:Methanol
keywords: Candida antarctica lipase B; Methanol-free; Pichia pastoris; Product formation kinetics; Secretion; Specific productivity