Recombinant protein production in Pseudoalteromonas haloplanktis TAC125 biofilm.

Biofilms have great potential for producing valuable products, and recent research has been performed on biofilms for the production of compounds with biotechnological and industrial relevance. However, the production of recombinant proteins using this system is still limited. The recombinant protein production in microbial hosts is a well-established technology and a variety of expression systems are available. Nevertheless, the production of some recombinant proteins can result in proteolyzed, insoluble, and non-functional forms, therefore it is necessary to start the exploration of non-conventional production systems that, in the future, could be helpful to produce some "difficult" proteins. Non-conventional production systems can be based on the use of alternative hosts and/or on non-conventional ways to grow recombinant cells. In this paper, the use of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 grown in biofilm conditions was explored to produce two fluorescent proteins, GFP and mScarlet. The best conditions for the production were identified by working on media composition, and induction conditions, and by building a new expression vector suitable for the biofilm conditions. Results reported demonstrated that the optimized system for the recombinant protein production in biofilm, although it takes longer than planktonic production, has the same potentiality as the classical planktonic approach with additional advantages since it needs a lower concentration of the carbon sources and doesn't require antibiotic addition. Moreover, in the case of mScarlet, the production in biofilm outperforms the planktonic system in terms of a better quality of the recombinant product.

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125.

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.

CATASAN Is a New Anti-Biofilm Agent Produced by the Marine Antarctic Bacterium Psychrobacter sp. TAE2020.

The development of new approaches to prevent microbial surface adhesion and biofilm formation is an emerging need following the growing understanding of the impact of biofilm-related infections on human health. Staphylococcus epidermidis, with its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in infections of medical devices. In the research of new anti-biofilm agents against S. epidermidis biofilm, Antarctic marine bacteria represent an untapped reservoir of biodiversity. In the present study, the attention was focused on Psychrobacter sp. TAE2020, an Antarctic marine bacterium that produces molecules able to impair the initial attachment of S. epidermidis strains to the polystyrene surface. The setup of suitable purification protocols allowed the identification by NMR spectroscopy and LC-MS/MS analysis of a protein-polysaccharide complex named CATASAN. This complex proved to be a very effective anti-biofilm agent. Indeed, it not only interferes with cell surface attachment, but also prevents biofilm formation and affects the mature biofilm matrix structure of S. epidermidis. Moreover, CATASAN is endowed with a good emulsification activity in a wide range of pH and temperature. Therefore, its use can be easily extended to different biotechnological applications.

Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

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.

Conditional gene silencing in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Since the release in 2005 of the genome sequence and annotation of the first Antarctic marine bacterium, the number of genomes of psychrophilic microorganisms in public databases has steadily increased. Unfortunately, the lack of effective molecular tools for the manipulation of these environmental strains still hampers our understanding of their peculiar strategies to thrive in freezing conditions, limiting the functional genomics approaches to differential analyses only. Over the past two decades, our research group established the first effective gene cloning/expression technology in the Antarctic Gram-negative marine bacterium Pseudoalteromonas haloplanktis TAC125. The setup of a genome mutagenesis technique (based on homologous recombination and counterselection events) further supported the use of this strain, which became an attractive model for studying microbial adaptations to freezing lifestyle. Moreover, to further extend the functional analyses to its essential genes, the set-up of a conditional gene silencing approach is desirable. In this paper, we report the development of an asRNA regulatory system in the Antarctic bacterium, testing the feasibility of Hfq-dependent and PTasRNA strategies previously developed in Escherichia coli. Stable and efficient silencing of two chromosomal genes was obtained by using PTasRNAs, reaching very high levels of downregulation.

Soluble Recombinant Protein Production in Pseudoalteromonas haloplanktis TAC125: The Case Study of the Full-Length Human CDKL5 Protein.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is an unconventional protein production host displaying a notable proficiency in the soluble production of difficult proteins, especially of human origin. Furthermore, the accumulation of recombinant products in insoluble aggregates has never been observed in this bacterium, indicating that its cellular physicochemical conditions and/or folding processes are rather different from those observed in mesophilic bacteria. The ability of this cell factory was challenged by producing a human protein, the cyclin-dependent kinase-like 5 (hCDKL5) in the bacterium cytoplasm at 0 °C. Human CDKL5 is a serine/threonine protein kinase characterized by the absence of a defined structure for the last two/third of its sequence, one of the largest intrinsically disordered regions so far observed in a human protein. This large unstructured domain makes difficult its production in most of the conventional hosts since the recombinant product accumulates as insoluble aggregates and/or is heavily proteolyzed. As the full-length hCDKL5 production is of great interest both for basic science and as protein drug for an enzyme replacement therapy, its production in the Antarctic bacterium was tested by combining the use of a regulated psychrophilic gene expression system with the use of a defined growth medium optimized for the host growth at subzero temperature. This is the first report of soluble and full-length recombinant production of hCDKL5 protein in a bacterium.

Modelling hCDKL5 Heterologous Expression in Bacteria.

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.

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering.

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.

Cold-adapted bacterial extracts as a source of anti-infective and antimicrobial compounds against Staphylococcus aureus.

Aim: The dramatic emergence of antibiotic resistance has directed the interest of research toward the discovery of novel antimicrobial molecules. In this context, cold-adapted marine bacteria living in polar regions represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire. The aim of this work was to identify new antimicrobials and/or antibiofilm molecules produced by cold-adapted bacteria. Materials & methods: Organic extracts obtained from polar marine bacteria were tested against Staphylococcus aureus. Most promising samples were subjected to suitable purification strategies. Results: Results obtained led to the identification of a novel lipopeptide able to effectively inhibit the biofilm formation of S. aureus. Conclusion: New lipopeptide may be potentially useful in a wide variety of biotechnological and medical applications.