Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23651
標題: 極端高鹽太古生物之分類鑑定、比較基因體分析及其生物科技應用潛力之研究
The identification, characterization, comparative genomics of Haloterrigena species and their biotechnological potentials
作者: 丁俊彥
Ding, Jiun-Yan
關鍵字: 極端高鹽太古生物
haloarchaea
分類鑑定
比較基因體
聚羥基烷酯
解脂酶解酯酶錳觸酶鹵烷降解
脂肪酸降解
生物科技
identification and characterization
comparative genomics
pohyhydroxyalkanoate
lipase
esterase
manganese catalase
haloalkane degradation
fatty acid degradation
biotechnology
出版社: 生命科學系所
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摘要: 自美國聖地牙哥曬鹽場中所分離純化出的 11 株兼性厭氧嗜鹽性微生物 H 系列分離株,經細胞膜核心脂質分析顯示,其脂質鍵結模式為甘油二醚鍵,證實 H 系列屬於極端高鹽太古生物,同時這些分離株均具有生成生物可降解性聚酯 – 聚羥基烷酯的能力,因此對 H 系列進行詳細的研究以便提升其應用價值。在本研究中,經 16S 核糖體 RNA 基因的系統演化分析顯示,H 系列的分離株應屬於 Haloterrigena 這屬的成員,且至少分屬於兩個新種。在膜脂質的種類分析顯示,分離株具有 PG、PGP-Me 與 S2-DGD,亦證實了 16S 核糖體 RNA 基因的分析結果。在特性分析上,H 系列均具有觸酶活性、可還原硝酸鹽、能利用檸檬酸,但不具有氧化酶活性,顯示這些分離株可能屬於兼性厭氧性微生物。其中除了分離株 strain H1之外,多數分離株具有分解酪蛋白、明膠、聚山梨醇 20/80、卵磷脂與色胺酸的能力。基於分生與生化特性的差異,將此 11 個分離株進一步分成三個新種:Htg. mahii、Htg. proteolyticus 與 Htg. esterolyticus。其中 strain H13 是 H 系列中唯一用於大量生產聚羥基烷酯的物種,因此使用了低通量散彈槍定序法,針對此物種進行比較基因體的分析,並試圖獲取聚羥基烷酯生成相關途徑的資料以及開發更多有潛力的生物應用。經脈衝式電泳分析與計算後,strain H13 的基因體大小約為 3.96 Mb。在經定序占全基因體總長 15.45 % 的序列後,可推算出完整基因體的 GC 組成率應接近 64.98 %,同時在此部分基因體中可鑑定出 1,207 個編碼基因,其基因體編碼比率為 73.64 %,推算之蛋白質等電點偏低,其中位數為 4.22。這些結果顯示,strain H13 具有與其它極端高鹽太古生物相同的適應模式。在功能性基因分析中,參與能量、脂肪酸與無機離子代謝之基因出現率較其它物種為高,但在醣類與核苷酸代謝方面則較低,顯示 strain H13 在碳源利用上可能偏好使用脂肪酸而非糖類。在代謝途徑的重建中,可以重建出參與糖新生、丙酮酸代謝、檸檬酸-甲基天門冬胺酸循環、支鏈胺基酸生成與降解、脂肪酸降解、聚羥基烷酯生成與鹵烷降解等途徑,並由此推論胺基酸與脂肪酸是可被 strain H13 利用之碳源,用於合成糖類與聚羥基烷酯。鹵烷降解途徑負責了鹵烷類的脫鹵反應,可供做環境復育的研究之用。在部分基因體中也發現到了參與甜菜鹼生成酵素與肉鹼運輸蛋白等基因,推論 strain H13 可能具有能使用有機相容質的能力,同時也發現額外的過氧化氫代謝酵素 – 錳觸酶,且初步證實其蛋白活性。在錳觸酶的蛋白結構預測上,其結構特徵介於中溫性與高溫性生物之間,推論可能屬於耐高溫酵素,可廣泛應用在不同溫度條件的工業或食品加工製程中。在單一碳源利用上,strain H13 可以利用三丁酸甘油酯,顯示其具有解脂酶/解酯酶活性,雖未能從部分基因體中比對到可能的基因,但仍突顯出 strain H13 在應用於生質柴油生產的可能性。
H series stands for 11 extreme halophilic microorganisms which were isolated from solar saltern in Chulta Vista, California. The presence of glycerol diether moieties indicates these isolates are members of haloarchaea. All of them confirmed to be able to accumulate the biodegradable polyesters, poly-beta-hydroxyalkanoates (PHAs), which makes them more potentially in biotechnological applications. The 16S rRNA gene phylogeny and presence of major glycolipid, S2-DGD, indicate those isolates are the member of the genus Haloterrigena. All isolates are catalase-positive but oxidase-negative strains and capable to reduce nitrate and utilize citrate suggesting they are facultative anaerobes. Most strains can hydrolyze casein, gelatin, Tween 20/80, lecithin and typtophan, except for strain H1. Based on the phylogenetic analysis and biochemical tests, three new species, Htg. mahii, Htg. proteolyticus and Htg. esterolyticus are proposed. Htg. esterolyticus strain H13 of the isolates was choosen for the genomic investigation with the strategy named “low-pass shotgun sequencing”. The genome size of strain H13 was estimated about 3.96 Mb by PFGE. After library construction, clones were randomly sequenced until the total contig length was covered ~15 % of the genome. From the partial genome sequence, 1,207 CDSs could be identified and the coding ratio was estimated about 73.64 %. The GC content of the genome and median pI of proteins were 64.98 % and 4.22, respectively. Both characters indicated that strain H13 possesses the similar strategy in adaption under hash conditions like other haloarchaea. The functional genome snapshot shows strain H13 possesses higher gene content involved in energy, fatty acid and inorganic ion metabolism but less in carbohydrate and nucleotide metabolism. This suggests strain H13 may prefer to used fatty acids rather than carbohydrates. Gluconeogenesis, pyruvate metabolism, TCA-methylasparte cycle, BCAA biosynthesis and degradation, fatty acid oxidation, PHA biosynthesis and haloalkane degradation could be reconstructed from the partial genome. Those findings suggest amino acids and fatty acids could server good carbon sources for the biosynthesis of sugars and PHAs. The presence of full set of genes of haloalkane degradation suggests that strain H13 could be applied in bioremediation. Genes required for organic compatible solutes utilizing could be identified suggesting strain H13 may have a relative delicate stress adaptation strategy. The manganese catalase from strain H13 shares some structure features with the mesophilic and thermophilic counterparts which suggests it could be a thermotolerant enzyme with broadly usage at different temperatures. The abilities of utilizing tributyrylglyceride and olive oil indicate strain H13 contains esterase/lipase which can be applied in biodiesel production; however, it is not yet confirmed in the partial genome of strain H13.
URI: http://hdl.handle.net/11455/23651
其他識別: U0005-0607201121300300
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