性類固醇的微生物降解: 由模式物種延伸至環境樣本
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2017
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Sex steroid hormones (SHs), a major group of endocrine disrupting agents, are often detected in aquatic environments. The most concerned SHs include estrogens (e.g., 17β-estradiol and estrone) and androgens (e.g., testosterone). Among the proposed remediation strategies, bacterial degradation has been considered an efficient and eco-friendly strategy for removing the SHs from the contaminated ecosystems. In this dissertation, I aimed to investigate the metabolic and phylogenetic diversity related to bacterial degradation of SHs from model organisms to the environemnt. By using culturable bacterial strains as model organisms, I demonstrated that strictly aerobic Sphingomonas sp. strain KC8 degrade estrogens through the 4,5-seco pathway; the essential meta-cleavage dioxygenase was isolated and characterized. Furthermore, through the genomic and transcriptomic analyses, I identified the catabolic gene clusters in the 4,5-seco pathway of strain KC8, and in the 2,3-seco pathway for androgen biodegradation of Steroidobacter denitrificans DSM 18526. The omics studies on the model organisms enabled the environmental investigations of steroid biodegradation, for which I used the following approaches: (i) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identification of signature metabolites, (ii) identification of main catabolic players through next-generation sequencing techniques, and (iii) PCR-based identification of functional genes. This study is the first integrated ‘omics’ investigation on the biochemical mechanisms and phylogenetic diversity of steroid biodegradation in the environment. In brief, Introduction provides the background information of SHs, current knowledge on their biodegradation, and my research objectives. The studies of androgen degradation: the genome of the androgen anaerobic decomposer, Steroidobacter denitrificans was completely sequenced and annotated. Transcriptomic data revealed the gene clusters that were distinctly expressed during anaerobic growth on testosterone; besides, I identified the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, suggested as biomarkers to investigate androgen biodegradation. Based on the available biomarkers of androgen degradation, I investigated the biochemical mechanisms and corresponding microorganisms of androgen degradation in the anaerobic and aerobic sewage. Sewage samples collected from the Dihua Sewage Treatment Plant (Taipei, Taiwan) were incubated with testosterone (1 mM) anaerobically or aerobically. Androgen metabolite analysis indicated that denitrifying bacteria in anaerobic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assay showed androgen degradation in anaerobic sewage by Thauera spp. (mainly T. terpenica) through the action of 1-testosterone hydratase/dehydrogenase. Moreover, the 2.3-seco pathway utilized by T. terpenica 58Eu (DSMZ 12139) was also confirmed. By contrast, bacteria in aerobic sewage degraded androgens via the oxygenase-dependent 9,10-seco pathway, and the metagenomic analysis indicated the apparent enrichment of Comamonas spp. (mainly C. testosteroni) and Pseudomonas spp. in sewage incubated with testosterone. I used the degenerate primers derived from the meta-cleavage dioxygenase gene (tesB) of various proteobacteria to track this essential catabolic gene in the sewage. The amplified sequences showed the highest similarity (87–96%) to tesB of C. testosteroni. Using quantitative PCR, I detected a remarkable increase of the 16S rRNA and catabolic genes of C. testosteroni in the testosterone-treated sewage. The studies of estrogen degradation: Using a tiered functional genomics approach, I deciphered the catabolic enzymes and genes involved in estrogen biodegradation by a wastewater isolate, Sphingomonas sp. strain KC8. I identified the initial intermediates of this catabolic pathway, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. Further genomic and transcriptomic analyses revealed that two gene clusters are specifically expressed in strain KC8 cells grown on 17β-estradiol. I also characterized 17β-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A-ring, respectively. The 4-hydroxyestrone 4,5-dioxygenase gene and the characteristic pyridinestrone acid were detected in two wastewater treatment plants and two suburban rivers in Taiwan. In conclusion, the catabolic genes and characteristic metabolites can be used as the biomarkers to investigate fate and biodegradation potential of estrogens in the environment.
Sex steroid hormones (SHs), a major group of endocrine disrupting agents, are often detected in aquatic environments. The most concerned SHs include estrogens (e.g., 17β-estradiol and estrone) and androgens (e.g., testosterone). Among the proposed remediation strategies, bacterial degradation has been considered an efficient and eco-friendly strategy for removing the SHs from the contaminated ecosystems. In this dissertation, I aimed to investigate the metabolic and phylogenetic diversity related to bacterial degradation of SHs from model organisms to the environemnt. By using culturable bacterial strains as model organisms, I demonstrated that strictly aerobic Sphingomonas sp. strain KC8 degrade estrogens through the 4,5-seco pathway; the essential meta-cleavage dioxygenase was isolated and characterized. Furthermore, through the genomic and transcriptomic analyses, I identified the catabolic gene clusters in the 4,5-seco pathway of strain KC8, and in the 2,3-seco pathway for androgen biodegradation of Steroidobacter denitrificans DSM 18526. The omics studies on the model organisms enabled the environmental investigations of steroid biodegradation, for which I used the following approaches: (i) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identification of signature metabolites, (ii) identification of main catabolic players through next-generation sequencing techniques, and (iii) PCR-based identification of functional genes. This study is the first integrated ‘omics’ investigation on the biochemical mechanisms and phylogenetic diversity of steroid biodegradation in the environment. In brief, Introduction provides the background information of SHs, current knowledge on their biodegradation, and my research objectives. The studies of androgen degradation: the genome of the androgen anaerobic decomposer, Steroidobacter denitrificans was completely sequenced and annotated. Transcriptomic data revealed the gene clusters that were distinctly expressed during anaerobic growth on testosterone; besides, I identified the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, suggested as biomarkers to investigate androgen biodegradation. Based on the available biomarkers of androgen degradation, I investigated the biochemical mechanisms and corresponding microorganisms of androgen degradation in the anaerobic and aerobic sewage. Sewage samples collected from the Dihua Sewage Treatment Plant (Taipei, Taiwan) were incubated with testosterone (1 mM) anaerobically or aerobically. Androgen metabolite analysis indicated that denitrifying bacteria in anaerobic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assay showed androgen degradation in anaerobic sewage by Thauera spp. (mainly T. terpenica) through the action of 1-testosterone hydratase/dehydrogenase. Moreover, the 2.3-seco pathway utilized by T. terpenica 58Eu (DSMZ 12139) was also confirmed. By contrast, bacteria in aerobic sewage degraded androgens via the oxygenase-dependent 9,10-seco pathway, and the metagenomic analysis indicated the apparent enrichment of Comamonas spp. (mainly C. testosteroni) and Pseudomonas spp. in sewage incubated with testosterone. I used the degenerate primers derived from the meta-cleavage dioxygenase gene (tesB) of various proteobacteria to track this essential catabolic gene in the sewage. The amplified sequences showed the highest similarity (87–96%) to tesB of C. testosteroni. Using quantitative PCR, I detected a remarkable increase of the 16S rRNA and catabolic genes of C. testosteroni in the testosterone-treated sewage. The studies of estrogen degradation: Using a tiered functional genomics approach, I deciphered the catabolic enzymes and genes involved in estrogen biodegradation by a wastewater isolate, Sphingomonas sp. strain KC8. I identified the initial intermediates of this catabolic pathway, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. Further genomic and transcriptomic analyses revealed that two gene clusters are specifically expressed in strain KC8 cells grown on 17β-estradiol. I also characterized 17β-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A-ring, respectively. The 4-hydroxyestrone 4,5-dioxygenase gene and the characteristic pyridinestrone acid were detected in two wastewater treatment plants and two suburban rivers in Taiwan. In conclusion, the catabolic genes and characteristic metabolites can be used as the biomarkers to investigate fate and biodegradation potential of estrogens in the environment.
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Keywords
biodegradation, ecophysiology, estrogen, extradiol dioxygenase, functional genomics, 13C-metabolomics, RNA-Seq, Sphingomonas, steroid hormones, androgen, Comamonas, sewage treatment plant, Illumina MiSeq, denitrifying bacteria, molybdoenzyme, Steroidobacter, Thauera, biodegradation, ecophysiology, estrogen, extradiol dioxygenase, functional genomics, 13C-metabolomics, RNA-Seq, Sphingomonas, steroid hormones, androgen, Comamonas, sewage treatment plant, Illumina MiSeq, denitrifying bacteria, molybdoenzyme, Steroidobacter, Thauera