Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter
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...
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ftdatacite:10.21256/zhaw-4803 2023-05-15T13:55:24+02:00 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 2017 application/pdf https://dx.doi.org/10.21256/zhaw-4803 https://digitalcollection.zhaw.ch/handle/11475/10842 en eng Springer Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/legalcode cc-by-4.0 CC-BY Secretion Pichia pastoris Methanol-free CALB 570 Biologie Text article-journal Journal Article ScholarlyArticle 2017 ftdatacite https://doi.org/10.21256/zhaw-4803 2021-11-05T12:55:41Z 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. Text Antarc* Antarctica DataCite Metadata Store (German National Library of Science and Technology) |
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English |
topic |
Secretion Pichia pastoris Methanol-free CALB 570 Biologie |
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Secretion Pichia pastoris Methanol-free CALB 570 Biologie Looser, Verena Lüthy, Dominik Straumann, Marcel Hecht, Katrin Melzoch, Karel Kovar, Karin Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter |
topic_facet |
Secretion Pichia pastoris Methanol-free CALB 570 Biologie |
description |
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. |
format |
Text |
author |
Looser, Verena Lüthy, Dominik Straumann, Marcel Hecht, Katrin Melzoch, Karel Kovar, Karin |
author_facet |
Looser, Verena Lüthy, Dominik Straumann, Marcel Hecht, Katrin Melzoch, Karel Kovar, Karin |
author_sort |
Looser, Verena |
title |
Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter |
title_short |
Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter |
title_full |
Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter |
title_fullStr |
Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter |
title_full_unstemmed |
Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter |
title_sort |
effects of glycerol supply and specific growth rate on methanol-free production of calb by p. pastoris : functional characterisation of a novel promoter |
publisher |
Springer |
publishDate |
2017 |
url |
https://dx.doi.org/10.21256/zhaw-4803 https://digitalcollection.zhaw.ch/handle/11475/10842 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_rights |
Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/legalcode cc-by-4.0 |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.21256/zhaw-4803 |
_version_ |
1766262007832510464 |