Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter

Looser, Verena; Lüthy, Dominik; Straumann, Marcel; Hecht, Katrin; Melzoch, Karel; Kovar, Karin

doi:10.21256/zhaw-4803

Please use this identifier to cite or link to this item: https://doi.org/10.21256/zhaw-4803

Publication type:	Article in scientific journal
Type of review:	Peer review (publication)
Title:	Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter
Authors:	Looser, Verena Lüthy, Dominik Straumann, Marcel Hecht, Katrin Melzoch, Karel Kovar, Karin
DOI:	10.21256/zhaw-4803 10.1007/s00253-017-8123-x
Published in:	Applied Microbiology and Biotechnology
Volume(Issue):	101
Issue:	8
Page(s):	3163
Pages to:	3176
Issue Date:	27-Jan-2017
Publisher / Ed. Institution:	Springer
ISSN:	0175-7598 1432-0614
Language:	English
Subjects:	Secretion; Pichia pastoris; Methanol-free; CALB
Subject (DDC):	570: Biology
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−1 in glycerol-limited fedbatch cultures, basal levels of qp > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated qp occurred 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−1 and vice versa. Changes in qp were 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.
URI:	https://digitalcollection.zhaw.ch/handle/11475/10842
Fulltext version:	Published version
License (according to publishing contract):	CC BY 4.0: Attribution 4.0 International
Departement:	Life Sciences and Facility Management
Organisational Unit:	Institute of Chemistry and Biotechnology (ICBT)
Appears in collections:	Publikationen Life Sciences und Facility Management

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Looser, V., Lüthy, D., Straumann, M., Hecht, K., Melzoch, K., & Kovar, K. (2017). Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter. Applied Microbiology and Biotechnology, 101(8), 3163–3176. https://doi.org/10.21256/zhaw-4803

Looser, V. et al. (2017) ‘Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter’, Applied Microbiology and Biotechnology, 101(8), pp. 3163–3176. Available at: https://doi.org/10.21256/zhaw-4803.

V. Looser, D. Lüthy, M. Straumann, K. Hecht, K. Melzoch, and K. Kovar, “Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter,” Applied Microbiology and Biotechnology, vol. 101, no. 8, pp. 3163–3176, Jan. 2017, doi: 10.21256/zhaw-4803.

LOOSER, Verena, Dominik LÜTHY, Marcel STRAUMANN, Katrin HECHT, Karel MELZOCH und Karin KOVAR, 2017. Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter. Applied Microbiology and Biotechnology. 27 Januar 2017. Bd. 101, Nr. 8, S. 3163–3176. DOI 10.21256/zhaw-4803

Looser, Verena, Dominik Lüthy, Marcel Straumann, Katrin Hecht, Karel Melzoch, and Karin Kovar. 2017. “Effects of Glycerol Supply and Specific Growth Rate on Methanol-Free Production of CALB by P. Pastoris : Functional Characterisation of a Novel Promoter.” Applied Microbiology and Biotechnology 101 (8): 3163–76. https://doi.org/10.21256/zhaw-4803.

Looser, Verena, et al. “Effects of Glycerol Supply and Specific Growth Rate on Methanol-Free Production of CALB by P. Pastoris : Functional Characterisation of a Novel Promoter.” Applied Microbiology and Biotechnology, vol. 101, no. 8, Jan. 2017, pp. 3163–76, https://doi.org/10.21256/zhaw-4803.