Severino, Angelica (2024) EXPANDING PSEUDOALTERMONAS HALOPLANKTIS TAC125 AS HOST FOR RECOMBINANT PRODUCTION OF DIFFICULT PROTEINS: STRAIN ENGINEERING AND DEVELOPMENT OF GENOME EDITING TOOLS. [Tesi di dottorato]

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Abstract

Much of life on Earth has evolved to colonize cold environments, which are the most abundant environments on the surface of our planet and microorganisms have developed specific finely tuned strategies to adapt to these harsh conditions. Pseudoalteromonas haloplanktis TAC125 (PhTAC125), isolated from the Antarctic coastal sea waters, is a Gram-negative psychrophilic bacterium that has emerged as a promising biotechnological tool in its exploitation as a non-conventional host for recombinant production. In this study, we focused on the industrial implementation of PhTAC125 as an effective cell-factory for the recombinant production of "difficult proteins”. By combining two genetic strategies previously developed for this bacterium, we paired homologous recombination using a suicide vector with the PTasRNA gene silencing technology to construct the novel PhTAC125 KrPL2 strain, cured from the endogenous pMEGA plasmid. The applied curing strategy streamlined the genetic background of the bacterium without altering growth behavior or the metabolic profile, assessing the stability and robustness of KrPL2 as a non-conventional host. Towards its industrial exploitation, a CRISPR-Cas9 genome editing system was developed tailored for PhTAC125. By harnessing two Cas9 variants, namely SpCas9 from Streptococcus pyogenes and Sth1ACas9 from Streptococcus thermophilus, we employed a single-plasmid CRISPR-Cas9 tool demonstrating its high cleavage activity at the temperature of 20 °C in vivo in PhTAC125. Using a CRISPR-SpCas9-based genome editing tool, we demonstrated effective genome editing using homologous recombination (HR) and CRISPR-SpCas9 counterselection for targeted gene knockouts, generating a PhTAC125 Δlon mutant.

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