NOVEL BIOACTIVE PRODUCTS FROM ANTARCTIC BACTERIA

Marine bacteria have considerable importance as sources of biologically active products. Marine microorganisms that live in cold regions have been largely underexplored, and may be endowed with interesting chemical repertoire. The microorganisms that thrive in these cold environments are referred to as psychrophiles or cold-adapted bacteria and are able to produce a large number of bioactive compounds, such as antimicrobial, anti-fouling and various pharmaceutically-relevant activities. In this contest, the aim of my PhD project was the research of new bioactive compounds of biotechnological interest from Polar marine bacteria. In particular, I focused my attention on three classes of molecules: I. Antimicrobial volatile organic compounds (VOCs); II. Anti-biofilm molecules; III. Cryoprotectant compounds. In order to explore the Pseudoalteromonas haloplanktis TAC125 (P.haloplanktis TAC125) chemical diversity as source of bioactive compounds, a suitable synthetic growth medium was developed, containing D-gluconate and L-glutamate as carbon, nitrogen and energy sources (GG medium). The definition of a synthetic medium is necessary for the scale up of P. haloplanktis TAC125 growth in automatic bioreactors. Moreover, a defined “minimum” medium could enhance the secondary metabolites production, and it surely makes their purification easier. Preliminary studies demonstrated that some Antarctic marine bacteria are able to produce volatile organic compounds (VOCs) that specifically inhibit the growth of Burkholderia cepacia complex (Bcc) strains. Amongst the tested Antarctic marine bacteria, P.haloplanktis TAC125 was further investigated. It is known that the P.haloplanktis TAC125 production of VOCs changes with growth medium composition. With the aim to identify the anti-Bcc VOCs, a suitable capture trap for volatile compounds was developed. A bioactive compound was identified, the methylamine, and its anti-Bcc activity was demonstrated by defining the Minimum Volatile Inhibitory Concentration (MVIC) on a panel of Bcc strains. Anti-biofilm molecules may have interesting biomedical applications in targeting adhesive properties of several insidious human pathogens. Previous results showed that the cell-free supernatant of P.haloplanktis TAC125 grown in static condition strongly inhibited bacterial adhesion. In particular, Staphylococcus epidermidis showed the highest susceptibility to the treatment. During this part of my PhD project the best conditions in which P.haloplanktis TAC125 produces the anti-biofilm compound/s were searched and a preliminary purification scheme was set up. In particular, the effect of growth mode, culture medium composition, growth phase and temperature was explored. The best production conditions were set as a benchmark for the scale-up of P.haloplanktis TAC125 anti-biofilm molecule/s in bioreactor. Marine cold-adapted microorganisms may be also source of another interesting class of chemical compounds, known as cryoprotectors, as they are able to avoid ice crystal formation inside living cells. Freeze-thaw cycles are quite common in the cold regions, especially in Polar one. Cold-adapted microorganisms are accustomed to being frozen within their habitats. Such organisms are also expected to have evolved adaptations to survive repeated freezing and thawing, as these processes tend to damage living cells and attenuate cell viability. The cold-adapted bacterium Colwellia psychrerythraea strain 34H (C. psychrerythraea 34H), attracted particular attention because it was reported to physically interact with sea ice crystals and secrete cryoprotectants of polysaccharidic nature in culture medium as a survival strategy. During my project, it was demonstrated that C. psychrerythraea 34H cells are covered by a capsula: the determination of chemical composition of purified capsular material revealed a novel polysaccharidic structure. Indeed the capsula was made by a linear tetrasaccharide repeating unit containing two amino sugars and two uronic acid, one of which is amidated by a threonine. The presence of an amminoacidic decoration of the capsular polysaccharide is quite uncommon in marine bacteria, but more intriguing is the decoration with Thr residues, as glycosilated Thr residues are essential for the interaction of anti-freeze glycoproteins (AFGPs) with ice crystals. In line with this indirect observation, in vitro assays demonstrated that the C. psychrerythraea 34H capsular polysaccharide is endowed with ice re-crystallization inhibition activity

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

Sweeter and stronger: Enhancing sweetness and stability of the single chain monellin MNEI through molecular design

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interactio

Biotechnological synthesis of succinic acid by actinobacillus succinogenes by exploitation of lignocellulosic biomass

Succinic acid is increasingly used in pharmaceutical industries, for the production of additives in food industries, in agriculture and in refinery processes as a precursor of many chemical compounds among which the most important is the succinate salt. It is also used as an ion chelator and surfactant, and for the biochemicals production. Currently, succinic acid is mainly produced through chemical petroleum-based processes, usually from n-butane using maleic anhydride. However, the use of petrochemical feedstocks raises serious environmental problems, due to the higher values of temperature and pressure required. The biotechnological production of succinic acid by microbial conversion of lignocellulosic biomass is attracting growing interest due to the environmental and economic advantages offered. This research is focused on the exploitation of Arundo donax (Giant reed) as a source of lignocellulosic biomass. Arundo donax is a perennial crop particularly suitable for energy production, as it offers high yields per hectare, even in partially fertile or polluted soils, not used for agriculture. Hydrolyzate of Arundo donax will be used as growth media for the Actinobacillus succinogenes 130Z, a bacterium typically found in the bovine rumen, that is recognized as one of the most promising for the biotechnological production of succinic acid, as it is able to produce higher concentrations of succinic acid. The experimental analysis is carried out to optimize the production of succinic acid taking into account the effect of the most critical parameters of the process (microbial biomass, pH, reducing sugars, volatile fatty acids, and succinic acid). Tests have shown that in 48h the sugars are completely biodegraded with a total production of bio-succinic acid of 5.9 g for 9.1 g of reducing sugars, an hourly production 0.12 g h-1 with a yield equal to 65%

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

Yield, quality, antioxidants and elemental composition of peanut as affected by plant density and harvest time

With the perspective of reintroducing peanut cultivation in southern Italy about six decades after its dismissal, research was carried out with the aim to identify the best performing farming management in terms of yield and quality. In this respect, the effect of the factorial combination between four plant densities (6.1, 7.8, 10.3, and 12.1 plants m-2) and two harvest times (100 and 110 days after planting) was assessed on pod and seed yield, as well as on seed quality, antioxidant activity, and elemental composition. The later harvest time determined a 26.9% dry weight increase, but a 14.3% decrease in the number of seeds per pod. Plant density significantly influenced all the yield and growth indices except for mean seed weight. Yield and growth of each plant were best affected by the lowest plant density, whereas the opposite trend was recorded for the same parameters referred to the surface area unit. The density of 12.1 plants m-2 resulted in a 32% reduction in pods per plant compared to 6.1 plants m-2, but had the greatest effect on seed production per m2 . The leaf area index was the highest with the density of 12.1 plants m-2. The total dry weight increased by 1.7-fold from 6.1 to 12.1 plants m-2. Compared to the first harvest time, in the second one the protein content decreased by 6.8%, and total polyphenols and antioxidant activity decreased by 11.2% and 7.6%, respectively. The second harvest time led to a depletion of N, P, and Mg, by 6.8%, 6.2%, and 6.8%, respectively, and a 7.1% Ca increase. The reintroduction of peanut cultivation in southern Italy is a realistic goal, though further studies regarding the crop system management are needed

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

Indirect daylight oxidative degradation of polyethylene microplastics by a bio-waste modified TiO2-based material

Microplastics are recognized as an emerging critical issue for the environment. Here an innovative chemical approach for the treatment of microplastics is proposed, based on an oxidative process that does not require any direct energy source (irradiation or heat). Linear low-density polyethylene (LLDPE) was selected as target commodity polymer, due to its widespread use, chemical inertness and inefficient recycling. This route is based on a hybrid material coupling titanium oxide with a bio-waste, rosin, mainly constituted by abietic acid, through a simple sol-gel synthesis procedure. The ligand-to-metal charge transfer complexes formed between rosin and Ti4+ allow the generation of reactive oxygen species without UV irradiation for its activation. In agreement with theorical calculations, superoxide radical ions are stabilized at ambient conditions on the surface of the hybrid TiO2. Consequently, an impressive degradation of LLDPE is observed after 1 month exposure in a batch configuration under indirect daylight, as evidenced by the products revealed by gas chromatography-mass spectrometry analysis and by chemical and structural modifications of the polymer surface. In a context of waste exploitation, this innovative and sustainable approach represents a promising cost-effective strategy for the oxidative degradation of microplastics, without producing any toxic by-products

Structural characterization of an all-aminosugar-containing capsular polysaccharide from Colwellia psychrerythraea 34H

Colwellia psychrerythraea strain 34H, a Gram-negative bacterium isolated from Arctic marine sediments, is considered a model to study the adaptation to cold environments. Recently, we demonstrated that C. psychrerythraea 34H produces two different extracellular polysaccharides, a capsular polysaccharide and a medium released polysaccharide, which confer cryoprotection to the bacterium. In this study, we report the structure of an additional capsular polysaccharide produced by Colwellia grown at a different temperature. The structure was determined using chemical methods, and one- and two-dimensional NMR spectroscopy. The results showed a trisaccharide repeating unit made up of only amino-sugar residues: N-acetyl-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (bacillosamine), and 2-acetamido-2-deoxyglucuronic acid with the following structure: →4)-β-d-GlcpNAcA-(1 →3)-β-d-QuipNAc4NAc-(1 →3)-β-d-GalpNAc-(1 →. The 3D model, generated in accordance with 1H,1H-NOE NMR correlations and consisting of ten repeating units, shows a helical structure. In contrast with the other extracellular polysaccharides produced from Colwellia at 4 °C, this molecule displays only a low ice recrystallization inhibition activity

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories : production of human alpha-galactosidase A

This work was supported by ERANET-IB08-007 project from the European Union and its linked national project EUI2008- 03610 to AV. We also appreciate the support from EME2007-08 to NFM from Universitat Autonoma de Barcelona, from Antartide 2010 to MLT and EP, from MIUR Azioni Integrate Italia-Spagna 2010 Prot. IT10LECLM9 to MLT, from MINECO (IT2009-0021) to AV and LT, from AGAUR (2009SGR-108) to AV. AV is also supported by The Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. PS has received predoctoral fellowship from ISCIII, and AV has been distinguished with an ICREA ACADEMIA award (Catalonia, Spain).Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems