Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

Abstract

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.