Bethlehem, Lukas: Design & generation of an efficient E. coli cell-factory for the overproduction of the compatible solutes ectoine & hydroxyectoine. - Bonn, 2020. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-58216
@phdthesis{handle:20.500.11811/8429,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-58216,
author = {{Lukas Bethlehem}},
title = {Design & generation of an efficient E. coli cell-factory for the overproduction of the compatible solutes ectoine & hydroxyectoine},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2020,
month = jul,

note = {The compatible solutes ectoine and hydroxyectoine are produced by halophilic or halotolerant bacteria in order to balance and adapt to osmotic alterations in their habitat. Besides this, the potent ability of these compounds to stabilize protein and cellular structures has led to significant interest in the scientific and industrial community over the last two decades. Successful marketing of especially ectoine in various skin-care and medical products has been achieved and the field of application is further growing through new implementations in critical medical fields like inflammatory bowel disease (IBD) or Alzheimer’s disease.
The industrial production of these compatible solutes, however, remains inefficient and costly, due to the limitations of the natural producer strain Halomonas elongata. The world-wide effort to develop an industrially relevant and cost-effective heterologous overproduction strain, has been moderately successful so far. The low productivities of these strains could be related to a poor activity of the employed halophilic enzymes to produce ectoine and hydroxyectoine in their non-halophilic heterologous host systems.
To overcome these obstacles, I transferred the hydroxyectoine gene cluster from the non-halophilic Acidiphilium cryptum into Escherichia coli. The design of an optimized expression plasmid led to very high product titers and exceptional productivities in shake flask experiments. Overproduction could be performed at low salt conditions (0% NaCl) leading to the natural excretion of > 99% of the produced ectoines into the culture medium. The scale-up of the production in bioreactor experiments, however, revealed an insuperable halt in production after only 10 hours, hence limiting ectoine titers. By qPCR and mass-spectrometry analysis we observed a rapid loss of ectoine biosynthesis enzymes over time, caused by plasmid instability in combination with the high productivity of the heterologous strain. We were able to solve this issue by the addition of a β-lactam inhibitor into the culture medium, ultimately resulting in high product titers (10 g/L) and the most effective heterologous ectoine overproduction process described in literature to date (3 gectoine/gdcw). The low salt conditions of this cell factory facilitate downstream processes and reduce the costs of waste-water treatment and equipment maintenance. By performing detailed 13C-flux analysis of the strain’s metabolic activity we further identified potential bottle-necks of the here established protocol, eventually allowing for additional metabolic engineering processes that could even enhance strain productivity and efficiency.},

url = {http://hdl.handle.net/20.500.11811/8429}
}

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