A metabolomics approach for targeted isolation and production of bioactive secondary metabolites in microbial isolates from extreme environments

This thesis was previously held under moratorium from 22/08/2019 to 22/08/2021.Marine microorganisms produce unique secondary metabolites, which are responsible for a variety of biologically active molecules with a wide range of pharmaceutical properties. There is a lack of novel effective drugs for metabolic diseases, cancer and parasite infections as well as TNF-alpha inhibitors hence; underexplored marine bacteria could be an important source for new bioactive molecules. Two strains of Muricauda ruestringensis (SBT531 and SBT587) were isolated from geothermal intertidal pools in Iceland and two strains of Micromonospora sp. N17 and N74 (SBT 687 and SBT692) were isolated from the Mediterranean sponge Phorbas tenacior from the Santorini volcanic complex of Crete. Bioactive metabolites production in thermophile strains of M. ruestringensis (SBT531 and SBT587) and Micromonospora sp. (SBT687 and SBT692) were identified and isolated, by using a metabolomics approach to analyse the liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) data sets. The LC HRMS data was processed by using the modified version of Mzmine 2.10 software, dereplicated with an in-house EXCEL macro coupled to the AntiMarin and Dictionaryof Natural Products (DNP) database to be statistically evaluated by multivariate analysis in SIMCA v15.02. Orthogonal partial least squares discriminant analysis (OPLS-DA) in SIMCA was used to predict and pinpoint the biologically active secondary metabolites. Up-scaling was optimised to increase the production yield of the target metabolites. Additionally, specific bioassay screening determined the activity, if any, from the crude extracts, fractions and isolated compounds. The total ethyl acetate organic extracts of SBT531 and SBT587 were active inhibitors in target-based functional assays: alpha-glucosidase and protein-tyrosine phosphatase 1B (PTP1B) that are a therapeutic target for the treatment of diabetes and other metabolic syndromes. The fractionation of SBT531 afforded a series of bioactive alpha and beta-hydroxy acid derivatives and allowed the definition of a preliminary structure-activity relationship based on their relative potency. Aseanostatin P6 (13-methyltetradecanoic acid) was the major compound identified, followed by two derivatives, 3-hydroxy-13-methyltetradecanoic acid and 2-hydroxy-14-methylhexadecanoic acid. On the other hand, the initial organic crude extracts of SBT687 and SBT692 showed inhibition activity against sea lice and inhibition of TNF alpha respectively, however after further fermentation scale-up the fractions were missing the inhibition activity.Marine microorganisms produce unique secondary metabolites, which are responsible for a variety of biologically active molecules with a wide range of pharmaceutical properties. There is a lack of novel effective drugs for metabolic diseases, cancer and parasite infections as well as TNF-alpha inhibitors hence; underexplored marine bacteria could be an important source for new bioactive molecules. Two strains of Muricauda ruestringensis (SBT531 and SBT587) were isolated from geothermal intertidal pools in Iceland and two strains of Micromonospora sp. N17 and N74 (SBT 687 and SBT692) were isolated from the Mediterranean sponge Phorbas tenacior from the Santorini volcanic complex of Crete. Bioactive metabolites production in thermophile strains of M. ruestringensis (SBT531 and SBT587) and Micromonospora sp. (SBT687 and SBT692) were identified and isolated, by using a metabolomics approach to analyse the liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) data sets. The LC HRMS data was processed by using the modified version of Mzmine 2.10 software, dereplicated with an in-house EXCEL macro coupled to the AntiMarin and Dictionaryof Natural Products (DNP) database to be statistically evaluated by multivariate analysis in SIMCA v15.02. Orthogonal partial least squares discriminant analysis (OPLS-DA) in SIMCA was used to predict and pinpoint the biologically active secondary metabolites. Up-scaling was optimised to increase the production yield of the target metabolites. Additionally, specific bioassay screening determined the activity, if any, from the crude extracts, fractions and isolated compounds. The total ethyl acetate organic extracts of SBT531 and SBT587 were active inhibitors in target-based functional assays: alpha-glucosidase and protein-tyrosine phosphatase 1B (PTP1B) that are a therapeutic target for the treatment of diabetes and other metabolic syndromes. The fractionation of SBT531 afforded a series of bioactive alpha and beta-hydroxy acid derivatives and allowed the definition of a preliminary structure-activity relationship based on their relative potency. Aseanostatin P6 (13-methyltetradecanoic acid) was the major compound identified, followed by two derivatives, 3-hydroxy-13-methyltetradecanoic acid and 2-hydroxy-14-methylhexadecanoic acid. On the other hand, the initial organic crude extracts of SBT687 and SBT692 showed inhibition activity against sea lice and inhibition of TNF alpha respectively, however after further fermentation scale-up the fractions were missing the inhibition activity

Secondary Analysis of Electronic Health Records

Health Informatics; Ethics; Data Mining and Knowledge Discovery; Statistics for Life Sciences, Medicine, Health Science