開發生物質衍生培養基並結合功能導向組學以篩選具對映選擇性神經類固醇合成能力之腸道菌株;Function-Driven Omics for Discovering Gut Bacteria capable of Enantioselective Neurosteroid Synthesis in Biomass-Derived Media

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Please use this identifier to cite or link to this item: https://ir.lib.ncu.edu.tw/handle/987654321/98666

Title:	開發生物質衍生培養基並結合功能導向組學以篩選具對映選擇性神經類固醇合成能力之腸道菌株;Function-Driven Omics for Discovering Gut Bacteria capable of Enantioselective Neurosteroid Synthesis in Biomass-Derived Media
Authors:	黃彥勳;Huang, Yen-Hsun
Contributors:	環境工程研究所
Keywords:	微生物培養;生物質衍生培養基;糖蜜;豆渣;3α-羥基類固醇脫氫酶;神經類固醇;黃體酮;異孕烷醇酮;5β-還原酶;3α-羥基類固醇脫氫酶;microbial cultivation;biomass-derived medium;molasses;okara;Holdemania filiformis;5β-reductase;3α-hydroxysteroid dehydrogenase;neurosteroids;progesterone;isopregnanolone
Date:	2025-06-23
Issue Date:	2025-10-17 13:03:51 (UTC+8)
Publisher:	國立中央大學
Abstract:	微生物掌控著整個生物圈，遠至土壤、海洋，近至人體中的腸道，都能看到微生物的蹤跡，而且他們驅動著整個生態的運作。而且目前幾乎所有產業都仰賴這股微小但強大的力量，例如於環境方面，微生物可以用於淨化廢水；農業方面可以依靠微生物固氮並促進生長；食品工業利用微生物發酵提升風味。這些都凸顯了微生物的重要性。但了解特定菌種的功能性，需要做大量的微生物培養及研究，這個過程勢必會消耗非常多的資源。因此本研究以永續循環為核心，首度利用加工副產品糖蜜及農業廢棄物豆渣構築全植物性 Molasses-Okara Medium（MOM），並比較傳統厭氧培養基 PYG對腸道厭氧菌 Holdemania filiformis 之生長情況。且利用該菌株具有的5β-還原酶(5β-reductase)及 3α-羥基類固醇脫氫酶 (Hydroxysteroid dehydrogenase)，將類固醇激素黃體酮（Progesterone）轉化為一種重要的神經類固醇異孕烷醇酮（Isopregnanolone）。研究發現傳統厭氧培養基PYG對於受質黃體酮的轉化率僅為 57 %。而全植物性培養基MOM於7 天內完全耗盡受質，產出 0.81 mg mL⁻¹ 異孕烷醇酮，整體轉換率達 80 %，顯示此植物性培養基之潛力。後續以開放式管柱層析技術，將目標產物異孕烷醇酮於培養基中分離純化，得到高純度之神經類固醇異孕烷醇酮。本研究顯示，植物性培養基可在無動物來源條件下支援 H. filiformis 快速增殖，並保留其 5β-還原與 3α-脫氫活性，提供放大生產立體專一神經類固醇之綠色流程。此成果不僅降低培養成本與碳足跡，亦呼應 UN SDG 12「責任消費與生產」，為腸道微生物驅動之功能性分子製造奠定重要基礎。 ;Microorganisms govern the entire biosphere, ranging from soil and oceans to the human gut, where their presence is ubiquitous and essential to ecological functioning. Nearly every modern industry relies on these minute yet powerful organisms—for instance, in environmental engineering, microbes are employed for wastewater treatment; in agriculture, they facilitate nitrogen fixation and promote plant growth; and in the food industry, microbial fermentation enhances flavor and nutritional profiles. These examples underscore the critical importance of microbes. However, elucidating the functions of specific strains requires extensive cultivation and experimentation, which in turn consumes significant resources. In response, this study emphasizes sustainable biocycling by pioneering the use of industrial by-product molasses and agricultural waste okara to formulate an entirely plant-based culture medium, termed Molasses-Okara Medium (MOM). The medium’s performance was compared with that of the conventional anaerobic medium PYG in supporting the growth of the gut anaerobe Holdemania filiformis. This strain expresses two key enzymes—5β-reductase and 3α-hydroxysteroid dehydrogenase—which catalyze the bioconversion of the steroid hormone progesterone into an important neurosteroid, isopregnanolone. Experimental results showed that the traditional PYG medium yielded only a 57% conversion rate of progesterone. In contrast, the plant-derived MOM medium achieved complete substrate depletion within 7 days and produced 0.81 mg mL⁻¹ of isopregnanolone, corresponding to an overall conversion rate of 80%, highlighting its promising potential. Subsequent purification using open-column chromatography successfully isolated high-purity isopregnanolone from the culture matrix. This study demonstrates that a fully plant-based culture medium can sustain the rapid growth of H. filiformis under animal-free conditions while preserving its enzymatic activity for stereospecific neurosteroid biosynthesis. These findings offer a green biotechnological route for large-scale neurosteroid production, reducing both cultivation costs and carbon footprint. The work aligns with UN Sustainable Development Goal 12, “Responsible Consumption and Production,” and establishes a foundational model for microbial-driven synthesis of high-value functional molecules.
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