Etude de la voie de biosynthèse des dithiolopyrrolones chez saccharotrix algeriensis NRRL B-24137 : approche génétique et enzymologique

Du fait de l’apparition de microorganismes pathogènes ayant une résistance aux antibiotiques actuels, la recherche de nouvelles molécules bioactives possédant une application médicale est devenue une préoccupation mondiale. Saccharothrix algeriensis, une bactérie filamenteuse de l’ordre des actinomycètes a montré une étonnante capacité à produire des molécules bioactives qui appartiennent aux dithiolopyrrolones, ayant de remarquables propriétés à la fois antibiotiques et anticancéreuses. Lors de ce projet de thèse, l’identification du cluster de gènes de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis est envisagée. Suite au séquençage du génome de Sa. algeriensis, une approche génomique ou « genome mining » est suivie, cette approche a révélé un cluster thi potentiellement responsable de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis. Ce cluster contient 12 gènes, dont 8 gènes de biosynthèse, 3 gènes régulateurs et un gène transporteur. Les analyses in silico des gènes ont montré que la cystéine est le substrat d’une NRPS. Les analyses transcriptionelles ont montré que les trois gènes clés codent pour une NRPS, une thiorédoxine et une thioestérase qui pourraient être impliquées dans la biosynthèse des dithiolopyrrolones. Deux gènes actA et actB codant pour des acyltransférases putatives ont été identifiés. Les analyses transcriptionelles suggèrent qu’actA et actB pourraient être responsables de l’acylation de la pyrrothine. Finalement, la caractérisation de deux activités enzymatiques, acétyltransférase et benzoyltransférase, présentes dans l’extrait brut de Sa. algeriensis, ont permis de déterminer les paramètres optimaux (pH et T °C) de la réaction enzymatique. Enfin, les paramètres cinétiques de ces activités ont des valeurs complètements différentes, ce qui confirme la présence d’au moins deux activités différentes chez Sa. algeriensis. ABSTRACT : Due to the emergence in the last decades of new and old infectious diseases to existing antibiotics, the research for new bioactive molecules which possess medical applications become a global occupation. Saccharothrix algeriensis, filamentous bacteria of actinomycetes order showed a surprising ability to produce bioactive molecules belongs to dithiolopyrrolones with remarkable properties of both antibiotics and anticancer. In this thesis, the identification of dithiolopyrrolones biosynthetic gene cluster in Sa. algeriensis was investigated. Through S. algeriensis genome sequencing, a genomics approach "genome mining" was followed, this approach has revealed a potentially thi cluster responsible for dithiolopyrrolones biosynthesis pathway in Sa algeriensis. This cluster contains 12 genes, including 8 biosynthesis genes, three regulatory genes and one transporter gene. The in silico analysis of this cluster showed that the cysteine is the substrate of the NRPS. The transcriptional analyzes showed that the three key genes which encode for NRPS, thioredoxin and thioesterase could be involved in dithiolopyrrolone biosyntheses. Two genes, actA and actB, encode for two putative acyltransferases were identified, the transcriptional analyzes suggests that these genes may be responsible for the acylation of pyrrothine core. The characterizations of two activities, acetyltransferase and benzoyltransferase, in the crude extract of Sa. algeriensis led the determination of the optimal parameters (pH and T °C) to detect these activities. Moreover, the effect of temperature and pH on these activities was determined. Finally, the kinetic parameters of these activities showed different values, which confirm the presence of, at least, two activities in Sa. algeriensis

Identification of Two Putative Acyltransferase Genes Potentially Implicated in Dithiolopyrrolone Biosyntheses in Saccharothrix algeriensis NRRL B-24137

The dithiolopyrrolone class of antibiotics has been known to display bacteriostatic activity against both Gram-positive and Gram-negative bacteria and exert other biological activities. Acyltransferase activities are proposed to be responsible for the structural diversity of dithiolopyrrolones produced by Saccharothrix algeriensis NRRL B-24137. Moreover, two activities, pyrrothine N-acetyltransferase and pyrrothine N-benzoyltransferase, are reported to catalyze the formation, respectively, to thiolutin and benzoyl-pyrrothine (BEP) in this bacterium. In this study, two genes encoding two putative acyltransferases were identified in S. algeriensis. The first one, actA, was identified by bioinformatic analysis and by analogy to an acetyltransferase, hlmA, identified in holomycin biosynthetic gene cluster in Streptomyces clavuligerus. The second was identified by purification of both enzymes from the bacterial biomass which provided a semipurified extract. The microsequencing of tryptic peptides from the final protein preparation yielded sequences of eight different fragments, two of them encoded by one gene, actB, in S. algeriensis genome bank. The alignment of actB against the GenBank database revealed significant homology to acyltransferase family. Differential expression of these genes, actA and actB, was then investigated in three different media: (i) semisynthetic medium (SSM), which promotes the production of thiolutin; (ii) SSM supplemented by 1.25 mM benzoic acid (SSM + BA), which promotes the production of both thiolutin and BEP; and (iii) tryptic soy broth (TSB) in which no dithiolopyrrolone derivatives were detected

Etude de la voie de biosynthèse des dithiolopyrrolones chez saccharotrix algeriensis NRRL B-24137 (approche génétique et enzymologique)

Du fait de l apparition de microorganismes pathogènes ayant une résistance aux antibiotiques actuels, la recherche de nouvelles molécules bioactives possédant une application médicale est devenue une préoccupation mondiale. Saccharothrix algeriensis, une bactérie filamenteuse de l ordre des actinomycètes a montré une étonnante capacité à produire des molécules bioactives qui appartiennent aux dithiolopyrrolones, ayant de remarquables propriétés à la fois antibiotiques et anticancéreuses. Lors de ce projet de thèse, l identification du cluster de gènes de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis est envisagée. Suite au séquençage du génome de Sa. algeriensis, une approche génomique ou genome mining est suivie, cette approche a révélé un cluster thi potentiellement responsable de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis. Ce cluster contient 12 gènes, dont 8 gènes de biosynthèse, 3 gènes régulateurs et un gène transporteur. Les analyses in silico des gènes ont montré que la cystéine est le substrat d une NRPS. Les analyses transcriptionelles ont montré que les trois gènes clés codent pour une NRPS, une thiorédoxine et une thioestérase qui pourraient être impliquées dans la biosynthèse des dithiolopyrrolones. Deux gènes actA et actB codant pour des acyltransférases putatives ont été identifiés. Les analyses transcriptionelles suggèrent qu actA et actB pourraient être responsables de l acylation de la pyrrothine. Finalement, la caractérisation de deux activités enzymatiques, acétyltransférase et benzoyltransférase, présentes dans l extrait brut de Sa. algeriensis, ont permis de déterminer les paramètres optimaux (pH et T C) de la réaction enzymatique. Enfin, les paramètres cinétiques de ces activités ont des valeurs complètements différentes, ce qui confirme la présence d au moins deux activités différentes chez Sa. algeriensis.Due to the emergence in the last decades of new and old infectious diseases to existing antibiotics, the research for new bioactive molecules which possess medical applications become a global occupation. Saccharothrix algeriensis, filamentous bacteria of actinomycetes order showed a surprising ability to produce bioactive molecules belongs to dithiolopyrrolones with remarkable properties of both antibiotics and anticancer. In this thesis, the identification of dithiolopyrrolones biosynthetic gene cluster in Sa. algeriensis was investigated. Through S. algeriensis genome sequencing, a genomics approach "genome mining" was followed, this approach has revealed a potentially thi cluster responsible for dithiolopyrrolones biosynthesis pathway in Sa algeriensis. This cluster contains 12 genes, including 8 biosynthesis genes, three regulatory genes and one transporter gene. The in silico analysis of this cluster showed that the cysteine is the substrate of the NRPS. The transcriptional analyzes showed that the three key genes which encode for NRPS, thioredoxin and thioesterase could be involved in dithiolopyrrolone biosyntheses. Two genes, actA and actB, encode for two putative acyltransferases were identified, the transcriptional analyzes suggests that these genes may be responsible for the acylation of pyrrothine core. The characterizations of two activities, acetyltransferase and benzoyltransferase, in the crude extract of Sa. algeriensis led the determination of the optimal parameters (pH and T C) to detect these activities. Moreover, the effect of temperature and pH on these activities was determined. Finally, the kinetic parameters of these activities showed different values, which confirm the presence of, at least, two activities in Sa. algeriensis.TOULOUSE-INP (315552154) / SudocSudocFranceF

The final acylation step in aromatic dithiolopyrrolone biosyntheses: Identification and characterization of the first bacterium N-benzoyltransferase from Saccharothrix algeriensis NRRL B-24137

The last step in the biosynthesis of dithiolopyrrolone antibiotics was thought to involve the transfer of acyl group from acyl-CoA to pyrrothine/holothin core. In Saccharothrix algeriensis NRRL B-24137, two acyltransferases, an acetyltransferase and a benzoyltransferase were proposed to catalyze this step. We have previously identified, in Sa. algeriensis genome, two open read frames, actA and actB patiently encoded these enzymes. This study focuses primarily on the characterization of the protein encoded by actA. After cloning and expressing of actA in Escherichia coli BL21, the recombinant protein encoded by actA was purified. Selectivity of ActA for pyrrothine/holothin as substrate and different acyl-CoA as co-substrate was evaluated using two acyls-groups, linear and aromatic. The enzyme was shown to prefer aromatic groups over linear groups as donor group; further neither product nor transfer was observed for linear groups. Therefore ActA has been determined to be a pyrrothine/holothin N-benzoyltransferase which can either pyrrothine (Km of 72 μM) or holothin (Km of 129.5 μM) as substrates and benzoyl-CoA (Km of 348.65 and 395.28 μM) as co-substrates for pyrrothine and holothin, respectively. The optimum pH and temperature has been shown to be 8, 40 °C, respectively. ActA is the first enzyme characterized as N-benzoyltransferase in bacteria

Coronary arteries bypass grafting in dialysis dependent end stage renal disease, results and outcome

Background: The objective is to assess the results and outcome of patients with dialysis dependent end stage renal failure who underwent coronary artery bypass grafting.Methods: In this retrospective study we analyzed the medical records of dialysis dependent end stage renal failure patients who underwent isolated coronary artery bypass grafting from January 2008 till December 2012 at queen alia heart institute. The total number of patients were 62 out of 6522 (0.95%). 42 of them were male (68%), the average age was 48±12 year, the duration of dialysis was 17±7 months. The medical profile of the patients, renal condition and duration of dialysis were analyzed. Preoperative cardiac condition and risk factors were also analyzed. Operative variables and postoperative results including mortality and post-operative renal and cardiac related complications were also analyzed.Results: Total in hospital mortality was 5 (8%), the cause of mortality was renal failure related causes in 2 patients, sepsis in 2 patients, cardiac causes in one patient. Average time for extubation, ICU stay and hospital stay was 9±2.3 hours, 27±7 hours and 7 days respectively. The average amount of blood loss postoperatively was 450±55 ml; five patients (8%) were resent to the operating theatre for exploration resternotomy because of bleeding. The average creatinine level 6.5±1.5 mg/dl. Six patients need dialysis in the first 24 hours of the operation (9.7%), most patients were returned to the usual protocol of dialysis, which was in average of 3 times per week.Conclusions: Coronary artery bypass grafting is the standard treatment in patient of end stage renal disease with coronary artery disease. Surgery is the ideal treatment in three vessel and left main disease according to SYNTAX trial. Those patients are high risk candidate for surgery, but proper preoperative, intraoperative and postoperative management decrease the risk and make the procedure safe.

Enzymatic Synthesis of Dithiolopyrrolone Antibiotics Using Cell-Free Extract of Saccharothrix algeriensis NRRL B-24137 and Biochemical Characterization of Two Pyrrothine N-Acyltransferases in This Extract.

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate

Study of the biosynthesis pathway of dithiolopyrrolones in Saccharothrix algeriensis NRRL B-24137 : Genetic and enzymological approaches

Du fait de l’apparition de microorganismes pathogènes ayant une résistance aux antibiotiques actuels, la recherche de nouvelles molécules bioactives possédant une application médicale est devenue une préoccupation mondiale. Saccharothrix algeriensis, une bactérie filamenteuse de l’ordre des actinomycètes a montré une étonnante capacité à produire des molécules bioactives qui appartiennent aux dithiolopyrrolones, ayant de remarquables propriétés à la fois antibiotiques et anticancéreuses. Lors de ce projet de thèse, l’identification du cluster de gènes de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis est envisagée. Suite au séquençage du génome de Sa. algeriensis, une approche génomique ou « genome mining » est suivie, cette approche a révélé un cluster thi potentiellement responsable de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis. Ce cluster contient 12 gènes, dont 8 gènes de biosynthèse, 3 gènes régulateurs et un gène transporteur. Les analyses in silico des gènes ont montré que la cystéine est le substrat d’une NRPS. Les analyses transcriptionelles ont montré que les trois gènes clés codent pour une NRPS, une thiorédoxine et une thioestérase qui pourraient être impliquées dans la biosynthèse des dithiolopyrrolones. Deux gènes actA et actB codant pour des acyltransférases putatives ont été identifiés. Les analyses transcriptionelles suggèrent qu’actA et actB pourraient être responsables de l’acylation de la pyrrothine. Finalement, la caractérisation de deux activités enzymatiques, acétyltransférase et benzoyltransférase, présentes dans l’extrait brut de Sa. algeriensis, ont permis de déterminer les paramètres optimaux (pH et T °C) de la réaction enzymatique. Enfin, les paramètres cinétiques de ces activités ont des valeurs complètements différentes, ce qui confirme la présence d’au moins deux activités différentes chez Sa. algeriensis.Due to the emergence in the last decades of new and old infectious diseases to existing antibiotics, the research for new bioactive molecules which possess medical applications become a global occupation. Saccharothrix algeriensis, filamentous bacteria of actinomycetes order showed a surprising ability to produce bioactive molecules belongs to dithiolopyrrolones with remarkable properties of both antibiotics and anticancer. In this thesis, the identification of dithiolopyrrolones biosynthetic gene cluster in Sa. algeriensis was investigated. Through S. algeriensis genome sequencing, a genomics approach "genome mining" was followed, this approach has revealed a potentially thi cluster responsible for dithiolopyrrolones biosynthesis pathway in Sa algeriensis. This cluster contains 12 genes, including 8 biosynthesis genes, three regulatory genes and one transporter gene. The in silico analysis of this cluster showed that the cysteine is the substrate of the NRPS. The transcriptional analyzes showed that the three key genes which encode for NRPS, thioredoxin and thioesterase could be involved in dithiolopyrrolone biosyntheses. Two genes, actA and actB, encode for two putative acyltransferases were identified, the transcriptional analyzes suggests that these genes may be responsible for the acylation of pyrrothine core. The characterizations of two activities, acetyltransferase and benzoyltransferase, in the crude extract of Sa. algeriensis led the determination of the optimal parameters (pH and T °C) to detect these activities. Moreover, the effect of temperature and pH on these activities was determined. Finally, the kinetic parameters of these activities showed different values, which confirm the presence of, at least, two activities in Sa. algeriensis

Coronary arteries bypass grafting in dialysis dependent end stage renal disease, results and outcome

Background: The objective is to assess the results and outcome of patients with dialysis dependent end stage renal failure who underwent coronary artery bypass grafting.Methods: In this retrospective study we analyzed the medical records of dialysis dependent end stage renal failure patients who underwent isolated coronary artery bypass grafting from January 2008 till December 2012 at queen alia heart institute. The total number of patients were 62 out of 6522 (0.95%). 42 of them were male (68%), the average age was 48±12 year, the duration of dialysis was 17±7 months. The medical profile of the patients, renal condition and duration of dialysis were analyzed. Preoperative cardiac condition and risk factors were also analyzed. Operative variables and postoperative results including mortality and post-operative renal and cardiac related complications were also analyzed.Results: Total in hospital mortality was 5 (8%), the cause of mortality was renal failure related causes in 2 patients, sepsis in 2 patients, cardiac causes in one patient. Average time for extubation, ICU stay and hospital stay was 9±2.3 hours, 27±7 hours and 7 days respectively. The average amount of blood loss postoperatively was 450±55 ml; five patients (8%) were resent to the operating theatre for exploration resternotomy because of bleeding. The average creatinine level 6.5±1.5 mg/dl. Six patients need dialysis in the first 24 hours of the operation (9.7%), most patients were returned to the usual protocol of dialysis, which was in average of 3 times per week.Conclusions: Coronary artery bypass grafting is the standard treatment in patient of end stage renal disease with coronary artery disease. Surgery is the ideal treatment in three vessel and left main disease according to SYNTAX trial. Those patients are high risk candidate for surgery, but proper preoperative, intraoperative and postoperative management decrease the risk and make the procedure safe.

Proteomic characterization of secretomes from selected fungal strains with high biotechnological potential

Introduction: Filamentous fungi are among the most potent degraders of lignocellulosic biomass due to their ability to thrive in lignocellulose-rich environments; they produce a high number and a broad variety of enzymes that have different and complementary catalytic activities. A large screening of the fungal CIRM-CF collection (http://cirm.esil.univ-mrs.fr/) using robotic methods specifically developed in-house led to the identification of filamentous fungi that deconstruct efficiently polysaccharides. These investigations enabled the unbiased identification of fungal strains issued from biodiversity with high biotechnological potential. To understand their mechanism of action, some of these fungal strains were further investigated using state-of-the-art genomic, transcriptomic and proteomic techniques.Methods: In this context, collaboration between BBF and PAPPSO aims at characterizing the fungal extracellular enzyme production. Fungal strains are cultured on different inducers to favour the secretion of enzymes active on ligncellulosic biomass and the corresponding secretomes (the proteins present in the culture supernatant) are further analyzed using a bottom-up proteomics approach coupling nano liquid chromatography to high resolution mass spectrometry (nano LC-HRMS). X!Tandem Pipeline, a software developed by PAPPSO was used to perform proteins identification from peptide MS/MS spectra.Results: Over the last ten years, hundreds of fungal secretomes have been analyzed. Each secretome contains an average number of 100 specific enzymes for a given growth condition, which means that several thousands of lignocellulose-acting enzymes have been identified. The analysis of Fusarium verticillioides secretomes is given as an example. Secreted proteins of F. verticillioides from induced or non-induced culture condition at 3 different kinetic times were analyzed in one injection on a Q-Exactive+ mass spectrometer (Thermo Scientific) summing up to 735 proteins identified in all samples. The comparative approach revealed the abundant secretion of copper-containing enzymes acting on polysaccharides in the culture condition containing metals.Conclusions: The comparative analysis of fungal secretomes using advanced proteomics (PAPPSO) highlighted the cooperation between fungal enzymes for enhanced degradation of complex lignocellulosic substrates and some discrepancies in carbohydrate-active enzymes (CAZymes) sets dedicated to different types of biomass.Novel Aspect: A robust proteomic approach was established and validated for large-scale proteomic analysis of fungal secretomes from biodiversity to understand the enzymatic mechanisms of biomass degradation in perspective of biotechnological applications

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5\_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5\_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5\_2 enzymes towards galactose-containing saccharides.We are grateful to Pom Geslin-Vinck for her assistance in constructing some of the CgrAlcOx loop variants. This study was supported by (a) the ‘Centre National de la Recherche Scientifique’ (CNRS), (b) the French ‘Agence Nationale de la Recherche’ (ANR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) through the joint ‘Projet de Recherche Collaboratif International’/‘Strategic Partnership Grants for Projects’ program, supporting the project entitled ‘FUNTASTIC—Fungal copper radical oxidases as new biocatalysts for the valorization of biomass carbohydrates and alcohols’ (ANR-17-CE07-0047, NSERC STPGP 493781–16) and (c) the Spanish Ministry of Innovation and Sciences (PID2019-106370RB-I00).Peer ReviewedPostprint (published version