Copyright and reuse:The Warwick Research Archive Portal (WRAP) makes this work by researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available.Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. A note on versions:The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher's version. Please see the 'permanent WRAP URL' above for details on accessing the published version and note that access may require a subscription. AbstractThe over--expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition. Moreover, the expression of other plasmid--encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth.In this work, we have developed a second generation of an Escherichia coli expression system with an antibiotic--free plasmid maintenance mechanism based on an auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression has been minimized by tuning the expression levels of a repressor protein (LacI) and glyA using a selected library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. We apply our engineered antibiotic--free expression 2 system to the FucA over--production, showing increased production levels.Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity. Acknowledgements:This work was supported by the project "Novel Alternatives for Microbial Production of Enzymes and Multienzymatic stereoselective Synthesis (EnzProSyn)". M. P. acknowledges the Universitat Autònoma de Barcelona for the pre--doctoral grant.

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To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, capacity to efficiently synthesize the desired product and the use of selection markers that are acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of engineered strains—with different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA)—in high cell density cultures to evaluate their suitability to be used in bioprocess conditions that resemble industrial production. Results revealed that the first generation of engineered strain showed a 50 % reduction in fuculose-1-phosphate aldolase (FucA) production compared to the reference system (165 ± 13 mg FucA·g–1 DCW and 806 ± 12 AU·g–1 DCW) which is commercially available from QIAGEN and uses an antibiotic selection marker. The over-transcription glyA was found to be a major factor responsible for the metabolic burden leading to the decrease in FucA yield. The second- and third-generation of E. coli strains presented an increase in FucA production, being 202 ± 18 mg–1 FucA·g–1 DCW and 1176 ± 19 AU·g–1 DCW, and 1322 ± 19 AU·g–1 DCW and 245 ± 13 mg–1 FucA·g–1 DCW, respectively. Both strains presented a fitness improvement after the tuning of glyA expression levels and the deletion of the ampicillin resistance gene (bla) from the plasmid were carried out. The third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and is capable to produce FucA at similar levels compared to the original commercial expression system. Hence, our expression system possesses advantageous features compared to the commercial one and proved to have the potential to become an attractive platform for the production of recombinant proteins in a wide range of industrial biotechnology applications.