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Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations

The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle(®) T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a...

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Autores principales: Schwab, Karen, Lauber, Jennifer, Hesse, Friedemann
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597275/
https://www.ncbi.nlm.nih.gov/pubmed/28952595
http://dx.doi.org/10.3390/bioengineering3040032
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author Schwab, Karen
Lauber, Jennifer
Hesse, Friedemann
author_facet Schwab, Karen
Lauber, Jennifer
Hesse, Friedemann
author_sort Schwab, Karen
collection PubMed
description The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle(®) T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS) models were built based on the fluorometric data recorded during the EnPresso(®) B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP) of 0.19 g·L(−1) and 0.08 g·L(−1), as well as for the prediction of the optical density (RMSEP 0.24). In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10(−4) µg w/w) and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min(−1)·µg(−1)) proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems.
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spelling pubmed-55972752017-09-21 Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations Schwab, Karen Lauber, Jennifer Hesse, Friedemann Bioengineering (Basel) Article The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle(®) T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS) models were built based on the fluorometric data recorded during the EnPresso(®) B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP) of 0.19 g·L(−1) and 0.08 g·L(−1), as well as for the prediction of the optical density (RMSEP 0.24). In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10(−4) µg w/w) and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min(−1)·µg(−1)) proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems. MDPI 2016-11-21 /pmc/articles/PMC5597275/ /pubmed/28952595 http://dx.doi.org/10.3390/bioengineering3040032 Text en © 2016 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Schwab, Karen
Lauber, Jennifer
Hesse, Friedemann
Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations
title Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations
title_full Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations
title_fullStr Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations
title_full_unstemmed Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations
title_short Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations
title_sort fluorometric in situ monitoring of an escherichia coli cell factory with cytosolic expression of human glycosyltransferase galnact2: prospects and limitations
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597275/
https://www.ncbi.nlm.nih.gov/pubmed/28952595
http://dx.doi.org/10.3390/bioengineering3040032
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