A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

BACKGROUND: The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. RESULTS: A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. CONCLUSION: Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger

Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic α-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity. Results: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera. Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium

Influence of production process design on inclusion bodies protein: the case of an Antarctic flavohemoglobin

<p>Abstract</p> <p>Background</p> <p>Protein over-production in <it>Escherichia coli </it>often results in formation of inclusion bodies (IBs). Some recent reports have shown that the aggregation into IBs does not necessarily mean that the target protein is inactivated and that IBs may contain a high proportion of correctly folded protein. This proportion is variable depending on the protein itself, the genetic background of the producing cells and the expression temperature. In this paper we have evaluated the influence of other production process parameters on the quality of an inclusion bodies protein.</p> <p>Results</p> <p>The present paper describes the recombinant production in <it>Escherichia coli </it>of the flavohemoglobin from the Antarctic bacterium <it>Pseudoalteromonas haloplanktis </it>TAC125. Flavohemoglobins are multidomain proteins requiring FAD and heme cofactors. The production was carried out in several different experimental setups differing in bioreactor geometry, oxygen supply and the presence of a nitrosating compound. In all production processes, the recombinant protein accumulates in IBs, from which it was solubilized in non-denaturing conditions. Comparing structural properties of the solubilized flavohemoglobins, i.e. deriving from the different process designs, our data demonstrated that the protein preparations differ significantly in the presence of cofactors (heme and FAD) and as far as their secondary and tertiary structure content is concerned.</p> <p>Conclusions</p> <p>Data reported in this paper demonstrate that other production process parameters, besides growth temperature, can influence the structure of a recombinant product that accumulates in IBs. To the best of our knowledge, this is the first reported example in which the structural properties of a protein solubilized from inclusion bodies have been correlated to the production process design.</p

Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

<p>Abstract</p> <p>Background</p> <p>In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in <it>Pseudoalteromonas haloplanktis </it>TAC125. This system makes use of the psychrophilic α-amylase from <it>P. haloplanktis </it>TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, <it>Pseudoalteromonales </it>are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a <it>P. haloplanktis </it>TAC125 mutant strain with reduced extra-cellular proteolytic activity.</p> <p>Results</p> <p><it>P. haloplanktis </it>TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of <it>P. haloplanktis </it>TAC125 in which the <it>gspE </it>gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera.</p> <p>Conclusion</p> <p>Here we report a cell engineering approach to the construction of a <it>P. haloplanktis </it>TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the <it>gspE </it>mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that <it>P. haloplanktis </it>TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.</p

Acetate: friend or foe? Efficient production of a sweet protein in Escherichia coli BL21 using acetate as a carbon source

Escherichia coli is, to date, the most used microorganism for the production of recombinant proteins and biotechnologically relevant metabolites. High density cell cultures allow efficient biomass and protein yields. However, their main limitation is the accumulation of acetate as a by-product of unbalanced carbon metabolism. Increased concentrations of acetate can inhibit cellular growth and recombinant protein production, and many efforts have been made to overcome this problem. On the other hand, it is known that E. coli is able to grow on acetate as the sole carbon source, although this mechanism has never been employed for the production of recombinant proteins

Pentadecanal and pentadecanoic acid coatings reduce biofilm formation of Staphylococcus epidermidis on PDMS

Staphylococcus epidermidis is well known to be one of the major causes of infections related to medical devices, mostly due to its strong capacity to form device-associated biofilms. Nowadays, these infections represent a severe burden to the public health system and the necessity of novel antibacterial strategies for the treatment of these difficult-to-eradicate infections is urgent. The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 was found to be able to produce an anti-biofilm molecule, the pentadecanal, active against S. epidermidis. In this work, we modified one of the most widely used silicone-based polymers, polydimethylsiloxane (PDMS), by adsorption of pentadecanal and its most promising derivative, pentadecanoic acid, on the PDMS surface. The biofilm formation of S. epidermidis RP62A on both untreated and modified PDMS was performed in a parallel plate flow chamber system, demonstrating the capability of the proposed anti-biofilm coatings to strongly reduce the biofilm formation. Furthermore, drug-release capacity and long-term efficacy (21 days) were also proven for the pentadecanoic acid coating

Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4

Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by (1)H and (13)C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid

Antarctic Marine Bacteria as a Source of Anti-Biofilm Molecules to Combat ESKAPE Pathogens

: The ESKAPE pathogens, including bacteria such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, pose a global health threat due to their ability to resist antimicrobial drugs and evade the immune system. These pathogens are responsible for hospital-acquired infections, especially in intensive care units, and contribute to the growing problem of multi-drug resistance. In this study, researchers focused on exploring the potential of Antarctic marine bacteria as a source of anti-biofilm molecules to combat ESKAPE pathogens. Four Antarctic bacterial strains were selected, and their cell-free supernatants were tested against 60 clinical ESKAPE isolates. The results showed that the supernatants did not exhibit antimicrobial activity but effectively prevented biofilm formation and dispersed mature biofilms. This research highlights the promising potential of Antarctic bacteria in producing compounds that can counteract biofilms formed by clinically significant bacterial species. These findings contribute to the development of new strategies for preventing and controlling infections caused by ESKAPE pathogens

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis