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Cited by 4Fungal and yeast genetics researchEnzyme Production and CharacterizationEnzyme Catalysis and Immobilization

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Methanol addition after glucose depletion improves rPOXA 1B production under the pGap in P. pastoris X33: breaking the habit

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Ardila-Leal, Leidy D., Poutou-Piñales, Raul A., Morales-Álvarez, Edwin D., Rivera-Hoyos, Claudia M., Pedroza-Rodríguez, Aura M., Quevedo-Hidalgo, Balkys E., Pérez-Flórez, Alejandro·2021·SN Applied Sciences
DOI 10.1007/s42452-020-04093-z
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Abstract

The purpose of this study was to demonstrate that methanol addition after glucose depletion has a positive effect on improving rPOXA 1B production under the control of pGap in P. pastoris.Four different culture media (A, B, C and D) were used to culture P. pastoris X33/pGapZα A-LaccPost-Stop(clone 1), containing a previously optimized POXA 1B synthetic gene coding for P. ostreatus laccase, which after glucose depletion was supplemented or not with methanol. Enzyme activity in culture media without methanol (A, B, C and D) was influenced by media components, presenting activity of 1254.30 ± 182.44, 1373.70 ± 182.44, 1343.50 ± 40.30 and 8771.61 ± 218.79 U L−1, respectively. In contrast, the same culture media (A, B, C and D) with methanol addition 24 h after glucose depletion attained activity of 4280.43 ± 148.82, 3339.02 ± 64.36, 3569.39 ± 68.38 and 14,868.06 ± 461.58 U L−1 at 192 h, respectively, representing an increase of approximately 3.9-, 2.4-, 3.3- and 1.6-fold compared with culture media without methanol. Methanol supplementation had a greater impact on volumetric enzyme activity in comparison with biomass production. We demonstrated what was theoretically and biochemically expected: recombinant protein production under pGap control by methanol supplementation after glucose depletion was successful, as a feasible laboratory production strategy of sequential carbon source addition, breaking the habit of utilizing pGap with glucose.

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  • pGAP has been successfully used for fungal laccases production.

    “pGAP (the constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase, E.C. 1.2.1.12) has successfully used for fungal laccases production [44,162,204,232].”

    From A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases (2021) · cites this paper as [44]

  • Laccases have been expressed in hosts including Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Kluyveromyces lactis, Aspergillus niger, Aspergillus sojae, and Trichoderma reesei.

    “Laccases have expressed in hosts as Saccharomyces cerevisiae [228], Yarrowia lipolytica [229], Pichia pastoris [44,204,230,231,232], Kluyveromyces lactis [233] and even filamentous fungi such as Aspergillus niger [234], Aspergillus sojae [235], and Trichoderma reesei [236].”

    From A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases (2021) · cites this paper as [44]

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Authors
Ardila-Leal, L. D., Poutou-Piñales, R. A., Morales-Álvarez, E. D., Rivera-Hoyos, C. M., Pedroza-Rodríguez, A. M., Quevedo-Hidalgo, B. E., & Pérez-Flórez, A.
Affiliations
  • Pontificia Universidad Javeriana (CO)
  • Universidad de Caldas (CO)
ORCID
Venue
SN Applied Sciences
Publisher
Springer Science and Business Media LLC
Published
2021-01
Volume
3
Issue
1
ISSN
  • 2523-3963 (ISSN-L)
  • 2523-3971
DOI
10.1007/s42452-020-04093-z
OpenAlex
W3120493625
Cited by
4
References
2
Citations by year
2024: 1 · 2023: 1 · 2021: 2
FWCI
0.31
Type
journal article
Language
en
Open access
Licence
Locations
SN Applied Sciences (publishedVersion, cc-by)
Funders
Pontificia Universidad Javeriana (00006337, 00007885)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
glucose depletion, recombinant protein expression, laccase, recombinant laccase

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