Calvanese, Marzia (2023) Cold-adapted bacteria response to temperature changes: from physiology to exploitation of their biotechnological potential. [Tesi di dottorato]

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Abstract

Psychrophilic organisms possess several evolutionary adaptations which allow them to thrive at low temperatures. However, the information on the acclimation properties of these microorganisms remains very limited. In this study, the cold- adaptations of a model microorganism, PhTAC125, were assessed by integrating transcriptomic and metabolomic data at 15 °C and 0 °C. These analyses highlighted the metabolic robustness of the PhTAC125 due to its capability to modulate the expression of numerous metabolic genes, producing similar profiles of metabolites involved in the PhTAC125 central pathways at two temperatures. Furthermore, the extracellular metabolic analysis allowed us to identify potential nutrient limitations in the growth medium, named GG. Indeed, a deeper extracellular metabolic analysis and the determination of the extracellular ferric ion concentration revealed that the significant changes in the lactate and 2-oxoglutarate profiles could be an adaptive response of the bacterium in dealing with Fe-limitation. In addition, the integration of transcriptomic and metabolic data suggested that the carbon, nitrogen, and iron sources could be unbalanced during the PhTAC125 growth at 15 °C. The next studies will focus on setting up an optimized growth medium starting from these considerations. PhTAC125 is also considered one of the most interesting unconventional hosts for difficult-to-express protein production. In the last years, numerous studies have focused on the development of an efficient gene expression technology in PhTAC125, however, other bottlenecks are limiting the overall efficiency of the recombinant production system, as an example is the low copy number of the psychrophilic vectors. For this purpose, the replication origin (OriR) was mutated randomly, and the collection of OriR-sequences was cloned into a psychrophilic expression vector containing the gene coding for a fluorescent protein (GFP). The library was transferred into the PhTAC125 and subjected to several rounds of selection by FACS, allowing the isolation and characterization of seven clones at higher PCN than the wild-type one, among which one of the selected clones allowed to achieve a two-order of magnitude enhancement in plasmid copy number. The improvements developed in the last years allowed PhTAC125 to be the only prokaryotic host able to produce the full length of Human Cyclin-dependent kinase-like 5 (hCDKL5), which is characterized mainly by a long unstructured region, except for its N-terminal catalytic domain. Despite the successful recombinant production of hCDKL5 in PhTAC125, the human protein expression still highlights some limiting factors related to its solubility. Some peculiar features make us think that the apparent insolubility of hCDLK5 was not associated with the formation of inclusion bodies (never observed in PhTAC125) but with the formation of liquid-like condensates. The condensation properties of this protein were investigated in vivo during the recombinant expression of a fluorescent protein fused to the N-terminal of hCDKL5 into PhTAC125. Furthermore, several rationally designed hCDLK5 mutants were constructed to uncover the contribution of each domain to its condensation. The findings demonstrated that the hCDKL5 forms intracellular clusters in the psychrophilic bacterium, and a region of the catalytic domain drives the protein assembly into condensates.

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