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Bioengineering 2016, 3(4), 32; doi:10.3390/bioengineering3040032

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

Biberach University of Applied Sciences, Institute of Applied Biotechnology (IAB), 88400 Biberach, Germany
These authors contributed equally to this work.
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Academic Editor: Christoph Herwig
Received: 16 October 2016 / Revised: 13 November 2016 / Accepted: 16 November 2016 / Published: 21 November 2016
(This article belongs to the Special Issue Hybrid Modelling and Multi-Parametric Control of Bioprocesses)
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Abstract

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. View Full-Text
Keywords: E. coli SHuffle® T7; Glycosyltransferase GalNAcT2; In Situ monitoring; soft sensor; fluorescence spectroscopy E. coli SHuffle® T7; Glycosyltransferase GalNAcT2; In Situ monitoring; soft sensor; fluorescence spectroscopy
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MDPI and ACS Style

Schwab, K.; Lauber, J.; Hesse, F. Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations. Bioengineering 2016, 3, 32.

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