Lauro, Concetta (2021) Pseudoalteromonas haloplanktis TAC125 as a cell factory for the production of recombinant proteins: strain improvement and novel engineering technologies. [Tesi di dottorato]

Anteprima

Testo Lauro_Concetta_33.pdf Download (4MB) | Anteprima

Abstract

Pseudoalteromonas haloplanktis TAC125 (PhTAC125) represents a promising biological system for the recombinant production of high-quality proteins due to its profound differences in cellular physiochemical conditions in comparison to the commonly used mesophilic bacteria. The establishment of efficient constitutive and regulated gene expression systems, optimized culture media, mathematical metabolic models, and fermentative processes allowed the exploitation of this bacterium to produce complex eukaryotic proteins. In this scenario, this research project aimed to explore and extend the biotechnological capabilities of PhTAC125 as a cell factory. In the first part of my PhD project, I focused on the development of a mutant strain engineered to boost the performance of an IPTG-inducible expression system. The obtained strain, named KrPl lacY+, proved to be able to produce the E. coli lactose transporter and a truncated Lon protease devoid of its catalytic domain. The improvement in recombinant production derived from KrPl lacY+ was also demonstrated at low temperatures and encouraged further optimization toward cheaper and sustainable industrial processes. As described in the second chapter of this thesis, KrPl lacY+ was exploited for the recombinant production of the human partially IDP kinase CDKL5, unsuccessfully produced in other prokaryotic systems. Different strategies were applied to overcome the bottlenecks affecting the overall production yield, taking into account the translational efficiency, the optimization of the coding sequence and fusion partners, and the increase of expression plasmid copy number. The establishment of such an improved platform allowed the achievement of high production yields of CDKL5 in a full-length and active form, enabling its application for functional, structural as well as therapeutic studies. Finally, to further strengthen the exploitation of PhTAC125, an asRNA-mediated regulatory system was developed. The results described in the last chapter of the present study demonstrated the feasibility of conditional gene silencing in PhTAC125, opening new perspectives for manipulating marine psychrophilic bacteria in basic and applicative studies.

Downloads

Actions (login required)

Modifica documento