Ermenegilda Parrilli

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview.

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.

Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics

Both conventional and innovative biomedical approaches require cost-effective protein drugs with high therapeutic potency, improved bioavailability, biocompatibility, stability and pharmacokinetics. The growing longevity of the human population, the increasing incidence and prevalence of age-related diseases and the better comprehension of genetic-linked disorders prompt to develop natural and engineered drugs addressed to fulfill emerging therapeutic demands. Conventional microbial systems have been for long time exploited to produce biotherapeutics, competing with animal cells due to easier operation and lower process costs. However, both biological platforms exhibit important drawbacks (mainly associated to intracellular retention of the product, lack of post-translational modifications and conformational stresses), that cannot be overcome through further strain optimization merely due to physiological constraints. The metabolic diversity among microorganisms offers a spectrum of unconventional hosts, that, being able to bypass some of these weaknesses, are under progressive incorporation into production pipelines. In this review we describe the main biological traits and potentials of emerging bacterial, yeast, fungal and microalgae systems, by comparing selected leading species with well established conventional organisms with a long run in protein drug production.

The cold-active Lip1 lipase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a member of a new bacterial lipolytic enzyme family

The genome of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 was searched for the presence of genes encoding ester-hydrolysing enzymes. Amongst the others, the gene PSHAa0051 coding for a putative secreted esterase/lipase was selected. The psychrophilic gene was cloned, functionally over-expressed in P. haloplanktis TAC125, and the recombinant product (after named PhTAC125 Lip1) was purified. PhTAC125 Lip1 was found to be associated to the outer membrane and exhibited higher enzymatic activity towards synthetic substrates with long acyl chains. A structural model was constructed using the structure of carboxylesterase Est30 from Geobacillus stearothermophilus as template. The model covered the central part of the protein with the exceptions of PhTAC125 Lip1 N- and C-terminal regions, where the psychrophilic protein displays extra-domains. The constructed model showed a typical α/β-hydrolase fold, and confirmed the presence of a canonical catalytic triad consisting of Ser, Asp and His. The sequence analysis showed that PhTAC125 Lip1 is distantly related to other lipolytic enzymes, but closely related to other putative psychrophilic esterases/lipases. The aligned proteins share common features, such as: (1) a conserved new active-site pentapeptide motif (LGG(F/L/Y)STG); (2) the likely extra-cytoplasmic localization, (3) the absence of a typical calcium-binding pocket, and (4) the absence of a canonical lid. These observations strongly suggest that aligned proteins constitute a novel lipase family, typical of psychrophilic marine γ-proteobacteria, and PhTAC125 Lip1 could be considered the first characterised member of this family.

Influence of Growth Temperature on Lipid and Phosphate Contents of Surface Polysaccharides from the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC 125

The chemical structural variations induced by different growth temperatures in the lipooligosaccharide and exopolysaccharide components extracted from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125 are described. The increase in phosphorylation with the increase in growth temperature seems to be general, because it happens not only for the lipooligosaccharide but also for the exopolysaccharide. Structural variations in the lipid components of lipid A also occur. In addition, free lipid A is found at both 25 and 4 degrees C but not at 15 degrees C, which is the optimal growth temperature, suggesting a incomplete biosynthesis of the lipooligosaccharide component under the first two temperature conditions.

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

BackgroundIn 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.ResultsP. 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.ConclusionHere 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 growth temperature on the production of antibody Fab fragments in different microbes: A host comparative analysis

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady‐state conditions in carbon‐limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.

Anti-biofilm activity of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

Considering the increasing impact of bacterial biofilms on human health, industrial and food-processing activities, the interest in the development of new approaches for the prevention and treatment of adhesion and biofilm formation capabilities has increased. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the rapid appearance of escape mutants. It is known that marine bacteria belonging to the genus Pseudoalteromonas produce compounds of biotechnological interest, including anti-biofilm molecules. Pseudoalteromonas haloplanktis TAC125 is the first Antarctic Gram-negative strain whose genome was sequenced. In this work the anti-biofilm activity of P. haloplanktis supernatant was examined on different staphylococci. Results obtained demonstrated that supernatant of P. haloplanktis, grown in static condition, inhibits biofilm of Staphylococcus epidermidis. In order to define the chemical nature of the biofilm-inhibiting compound, the supernatant was subject to various treatments. Data reported demonstrated that the biologically active component is sensible to treatment with sodium periodate suggesting its saccharidic nature.

The role of a 2-on-2 haemoglobin in oxidative and nitrosative stress resistance of Antarctic Pseudoalteromonas haloplanktis TAC125

The 2-on-2 haemoglobins, previously named truncated, are monomeric, low-molecular weight oxygen-binding proteins that share the overall topology with vertebrate haemoglobins. Although several studies on 2-on-2 haemoglobins have been reported, their physiological and biochemical functions are not yet well defined, and various roles have been suggested. The genome of the psychrophilic Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is endowed with three genes encoding 2-on-2 haemoglobins. To investigate the function played by one of the three trHbs, PhHbO, a PhTAC125 genomic mutant strain was constructed, in which the encoding gene was knocked-out. The mutant strain was grown under controlled conditions and several aspects of bacterium physiology were compared with those of wild-type cells when dissolved oxygen pressure in solution and growth temperature were changed. Interestingly, inactivation of the PhHbO encoding gene makes the mutant bacterial strain sensitive to high solution oxygen pressure, to H(2)O(2), and to a nitrosating agent, suggesting the involvement of PhHbO in oxidative and nitrosative stress resistance.

Secretion of alpha-amylase from Pseudoalteromonas haloplanktis TAB23: two different pathways in different hosts.

Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for alpha-amylase secretion in Escherichia coli. The occurrence of two different secretion pathways in different hosts is proposed.

Highly Phosphorylated Core Oligosaccaride Structures from Cold‐Adapted Psychromonas arctica

Many cold habitats contain plenty of microorganisms that represent the most abundant cold-adapted life forms on earth. These organisms have developed a wide range of adaptations that involve the cell wall of the microorganism. In particular, bacteria enhance the synthesis of unsaturated fatty acids of membrane lipids to maintain the membrane fluidity, but very little is known about the adaptational changes in the structure of the lipopolysaccharides (LPSs), the main constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. The aim of this study was to investigate the chemical structure of these LPSs for insight into the temperature-adaptation mechanism. For this objective, the cold-adapted Psychromonas arctica bacterium, which lives in the arctic sea-water near Spitzbergen (Svalbard islands, Arctic) was cultivated at 4 degrees C. The lipooligosaccharides (LOSs) were isolated and analysed by means of chemical analysis and electrospray ionisation high-resolution Fourier transform mass spectrometry. The LOS was then degraded either by mild hydrazinolysis (O-deacylation) or with hot 4 M KOH (N-deacylation). Both products were investigated in detail by using 1H and 13C NMR spectroscopy and mass spectrometry. The core consists of a mixture of species that differ because of the presence of nonstoichiometric D-fructose and/or D-galacturonic acid units.