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Background A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. Results Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. Conclusions Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce “intractable” proteins, especially those containing large intrinsically disordered regions.

Marine bacteria that have adapted to thrive in extreme environments, such as Pseudoalteromonas haloplanktis TAC125 (PhTAC125), offer a unique biotechnological potential. The discovery of an endogenous megaplasmid (pMEGA) raises questions about its metabolic impact and functional role in that strain. This study aimed at streamlining the host genetic background by curing PhTAC125 of the pMEGA plasmid using a sequential genetic approach. We combined homologous recombination by exploiting a suicide vector, with the PTasRNA gene-silencing technology interfering with pMEGA replication machinery. This approach led to the construction of the novel PhTAC125 KrPL2 strain, cured of the pMEGA plasmid, which exhibited no significant differences in growth behavior, though showcasing enhanced resistance to oxidative stress and a reduced capacity for biofilm formation. These findings represent a significant achievement in developing our understanding of the role of the pMEGA plasmid and the biotechnological applications of PhTAC125 in recombinant protein production. This opens up the possibility of exploiting valuable pMEGA genetic elements and further advancing the genetic tools for PhTAC125.

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood–brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.

Background Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis , a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey. Results We have used recombinant L. lactis NZ9000 carrying the nisin inducible pNZ8148 vector to produce MNEI, a small sweet protein derived from monellin, with potential for food industry applications as a high intensity sweetener. We have been able to sustain this production using a medium based on the cheese whey from the production of ricotta cheese, with minimal pre-treatment of the waste. As a proof of concept, we have also tested these conditions for the production of MMP-9, a protein that had been previously successfully obtained from L. lactis cultures in standard growth conditions. Conclusions Other than presenting a new system for the recombinant production of MNEI, more compliant with its potential applications in food industry, our results introduce a strategy to valorize dairy effluents through the synthesis of high added value recombinant proteins. Interestingly, the possibility of using this whey-derived medium relied greatly on the choice of the appropriate codon usage for the target gene. In fact, when a gene optimized for L. lactis was used, the production of MNEI proceeded with good yields. On the other hand, when an E. coli optimized gene was employed, protein synthesis was greatly reduced, to the point of being completely abated in the cheese whey-based medium. The production of MMP-9 was comparable to what observed in the reference conditions.

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

hCDKL5 refers to the human cyclin-dependent kinase like 5 that is primarily expressed in the brain. Mutations in its coding sequence are often causative of hCDKL5 deficiency disorder, a devastating neurodevelopmental disorder currently lacking a cure. The large-scale recombinant production of hCDKL5 is desirable to boost the translation of preclinical therapeutic approaches into the clinic. However, this is hampered by the intrinsically disordered nature of almost two-thirds of the hCDKL5 sequence, making this region more susceptible to proteolytic attack, and the observed toxicity when the enzyme is accumulated in the cytoplasm of eukaryotic host cells. The bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is the only prokaryotic host in which the full-length production of hCDKL5 has been demonstrated. To date, a system-level understanding of the metabolic burden imposed by hCDKL5 production is missing, although it would be crucial for upscaling of the production process. Here, we combined experimental data on protein production and nutrients assimilation with metabolic modelling to infer the global consequences of hCDKL5 production in PhTAC125 and to identify potential overproduction targets. Our analyses showed a remarkable accuracy of the model in simulating the recombinant strain phenotype and also identified priority targets for optimised protein production.

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K+ and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future.Key points• Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125.• Two order of magnitude improvement of OriR-derived psychrophilic expression systems.• Almost twenty times enhancement in Green fluorescent protein production.

Pseudoalteromonas haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood-brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.