本研究針對160株分離自台灣、中國及俄羅斯等地區所採集之自土壤、水源、植物及菇類之紅色酵母菌，進行親緣關係篩選生產類胡蘿蔔素之潛力菌株。所有菌株利用傳統鑑定及分子鑑定進行菌種確認、分類及親緣關係研究。所有菌株比對大單元核糖體D1/D2區域序列與核醣體內轉錄間隔區片段後，可鑑定者共127株32種，另有33株無法鑑定為現有已知種，這些菌株除了以核醣體D1/D2區域及核醣體外轉錄區間ITS序列進行鑑定外，並與其相近的模式菌株以鄰聚法及最大似然法繪製親緣關係樹研究其親緣關係，同時進行形態觀察與生理生化試驗後，分別歸類為21個未描述種。其中有五株菌兩種無法歸類為任一菌屬，利用RNA聚合酶II基因序列 (RPB1, RPB2)、真核延伸因子序列 (Ef1-α)及粒腺體細胞色素B (CYTB)確認新屬分類，建議此二種歸類為Taiwanozyma。在生產類胡蔔素之潛力菌株分析方面，將所有菌種擇一菌株做為代表共50株進行菌種挑選後，將具紅色素生產潛力之菌種中所有菌株共105株進行篩選。萃取方法試驗後，以酸化法為最佳萃取方法，並擇出7株菌進行最適培養條件探討，根據試驗結果發現: 葡萄糖、蔗糖及果糖為最適生產碳源，並挑出Sprorbolomyces koalae AY3L51、Rhodotorula paludigena NU16L11及Rhodotorula taiwanensis GU1S32等三株菌進行後續實驗。由碳源濃度及碳氮源不同比例測試後得知Sprorbolomyces koalae AY3L51和 Rhodotorula taiwanensis GU1S32在2%碳源及0.1%氮源，Rhodotorula paludigena NU16L11使用4%碳源及0.3%氮源有最佳的色素產量。在透過溫度試驗得知， Sprorbolomyces koalae AY3L51和 Rhodotorula taiwanensis GU1S32於18oC下有較高的類胡蘿蔔素產量，但Rhodotorula paludigena NU16L11則於25oC為最適類胡蘿蔔素生產溫度。另光照試驗發現，紅光可以刺激菌落及色素生長，而藍光及綠光有較弱的表現。前述得知不同波長的光源和溫度對於紅色酵母菌是主要影響生長之因素，故利用紅光可刺激生產與黑暗培養最為對照組進行不同溫度和不同波長光源條件下之酵母菌轉錄體分析，以研究類胡蘿蔔素生產代謝途徑中之基因表現差異。在三株菌中，GO分析以生物合成途徑 (biosynthetic process) 改變影響最多，在Sprorbolomyces koalae AY3L51紅光照射培養後，以NADP合成的調控下降和核醣核蛋白拆解的調控上升為主，在Rhodotorula paludigena NU16L11中紅光照射後表現量皆為下降，而在Rhodotorula taiwanensis GU1S32在胞膜傳輸中有所改變。將所有試驗相互比對後，透過KEGG分析，三株菌的改變途徑在二次代謝，主要影響為脂肪酸代謝途徑之改變。

One hundred and sixty red yeast strains isolated from soil, water, plant and mushroom in Taiwan, China and Russia, respectively, previously in our laboratory were analyzed for phylogenetic relationship among these red yeasts based on sequences of the LSU D1/D2 domain and ITS fragment of rRNA gene, also screened for carotenoid production strains for productivity increasing . In this experiment, showed irradiation and temperature could had effected on color changing. Phylogenetic tree constructed by the sequences of LSU D1/D2 domain 32 recognized and 21 undescribed species, and 5 strains of them not belong to recognized genus. Thirty- three strains representing undescribed species were taxonomical studied based on traditional and molecular characteristics in term of morphology, physiology and phylogenetic analysis by D1/D2 domain and ITS fragment. In addition to 5 strains not belong to recognized genus , sequencing by RNA polymerase (RPB1, RPB2), translation elongation factor 1-alpha (EF1-α) and cytochrome b (CYTB) to confirm phylogenety, proposed as novel genus Taiwanozyma . After pigments and yield analysis, extraction methods’ selection, showed 7 strains had more potential then otherelse. 7 strains over different carbon sources cultivation, demonstrated glucose, sucrose and fructose were better than others, beside, there are three strains showed highest yield, Sprorbolomyces koalae AY3L51, and Rhodotorula paludigena NU16L11, Rhodotorula taiwanensis GU1S32 were chose because the specific peak at HPLC results. These three strains after under carbon and nitrogen sources concentration changing, revealed that Sprorbolomyces koalae AY3L51 and Rhodotorula taiwanensis GU1S32 showed best results at 2% carbon sources with 0.1% nitrogen source, but Rhodotorula paludigena NU16L11 was 4% carbon sources with 0.3% nitrogen source. About temperature, 18oC was better for Sprorbolomyces koalae AY3L51 and Rhodotorula taiwanensis GU1S32, compared to Rhodotorula paludigena NU16L11 was 25 oC. Red light had stimulated cells and pigments production than other color of lights, which were blue and green. Since the condition could effect production, dark cultivation as control compare with red light cultivation to analysis transcriptome. Results showed in GO analysis most of effects were participate in biosynthetic process. As KEGG analysis, most effect are participate in secondary metabolic pathway, which fatty acid biosynthesis pathway was the main difference pathway.

摘要.....II
Abstract III
致謝辭 IV
目錄 V
表目錄 VII
圖目錄 VIII
壹、前言 1
一、 類胡蘿蔔素之化學結構與功效 1
(一) 結構 1
(二) 功能 1
(三) 來源 2
二、 紅色酵母菌 3
(一) 紅色酵母菌定義、分類 3
(二) 分布 3
(三) 應用 3
三、 類胡蘿蔔素於Rhodotorula之合成途徑 4
四、 微生物性類胡蘿蔔素之生產 4
(一) 菌種多樣性 4
(二) 碳源利用 6
(三) 溫度 6
(四) 光照 6
五、 紅色酵母菌基因研究 6
六、 本研究之目的 6
貳、材料與方法 8
一、 藥品: 8
二、 培養基 8
(一) 葡萄糖酵母麥芽抽取物洋菜膠培養基 (Dextrose-Yeast extract and Malt extract Agar, YMA) 8
(二) 酵母菌氮源基礎洋菜培養基 (Yeast nitrogen base, YNB) 8
(三) 碳源試驗培養基 8
(四) 酵母碳源基礎洋菜膠培養基 (Yeast carbon base, YCB) 9
(五) 氮源試驗培養基 9
(六) 酵母抽取物葡萄糖培養基 (Yeast extract peptone dextrose, YPD) 9
(七) 酵母抽取物硫酸銨培養基 (Yeast extract ammonium sulfate, YA) 9
三、 器材 9
四、 儀器 10
(一) 恆溫箱 10
(二) 電泳儀器 10
(三) 分析儀器 10
(四) 顯微鏡 10
(五) 其他儀器 10
五、 研究方法 10
(一) 菌種活化 10
(二) 酵母菌傳統鑑定 10
(三) 酵母菌分子鑑定法 17
(四) 親緣關係分析 20
(五) 類胡蘿蔔素生產菌株篩選試驗 20
(六) 類胡蘿蔔素生產試驗 20
(七) 色素萃取 21
(八) 分析方法 21
(九) 轉錄體分析 22
參、結果與討論 24
一、 菌種系統分類研究 24
(一) 分子鑑定 24
(二) 酵母菌菌種生理形態特徵 29
(三) 親緣關係 30
二、 菌種描述 45
三、 類胡蘿蔔素生產菌株篩選 50
(一) 萃取法試驗 50
(二) 菌種初步篩選 50
(三) 菌株確認篩選 52
四、 色素生產條件最佳化探討 59
(一) 碳源種類及濃度 59
(二) 氮源濃度 60
(三) 溫度 60
(四) 光照 63
(五) 最佳條件 63
五、 GO酵素途徑分析 69
(一) Sporobolomyces koalae AY3L51 69
(二) Rhodotorula paludigena NU16L11 70
(三) Rhodotorula taiwanensis GU1S32 71
(四) GO 資料庫比對分析 72
(五) KEGG之分析 74
肆、結論 76
伍、參考文獻 78
附錄一…………...………………………………………………………………………………76-97
附錄二……….…………………………………………………………………………………98-102
附錄三………………………………..….…………………………………………………..……103
附錄四…………………………….……………….…………………………………………104-113
附錄五…………………………….……………….…………………………………………114-119

杜似鵑，梅艷珍，胡耀輝，齊斌。2008。紅法夫酵母生產蝦青素的培養條件研究。食品科學。Vol. 29，No. 08 pp.441-444。
Bernstein PS, Li B, Vachali P P, Gorusupudi A, Shyam R, Henriksen B S, Nolan, J M (2016). Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease. Progress in retinal and eye research, 50, 34-66.
Bhosale P, Bernstein PS (2004). β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea. Journal of Industrial Microbiology and Biotechnology, 31(12), 565-571.
Boussiba S, Vonshak A (1991). Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis1. Plant and Cell Physiology, 32(7), 1077-1082.
Boutigny A-L, Gautier A, Basler R, Dauthieux F, Leite S, Valade R, Aguayo J, Ioos R, Laval V (2019). Metabarcoding targeting the EF1 alpha region to assess Fusarium diversity on cereals. PloS one, 14(1), e0207988-e0207988.
Braunwald T, Schwemmlein L, Graeff-Hönninger S, French WT, Hernandez R, Holmes WE, Claupein W (2013). Effect of different C/N ratios on carotenoid and lipid production by Rhodotorula glutinis. Applied Microbiology and Biotechnology, 97(14), 6581-6588.
El-Banna AaA, El-Razek AaA, El-Mahdy AR (2012). Some Factors Affecting the Production of Carotenoids by Rhodotorula glutinis var. glutinis. Food and Nutrition Sciences, 3, 64-71.
Elfeky N, Elmahmoudy M, Zhang Y, Guo J, Bao Y (2019). Lipid and Carotenoid Production by Rhodotorula glutinis with a Combined Cultivation Mode of Nitrogen, Sulfur, and Aluminium Stress. Applied Sciences, 9(12), 2444.
Ellison SL (2016). Carotenoids: Physiology. In CABALLERO B, FINGLAS P M, & TOLDRÁ F eds. Encyclopedia of Food and Health. Oxford: Academic Press, 670-675.
Ferdes M, Ungureanu C, Mihalcea A, Ana C, Mocanu E (2011). The Influence of the Carbon Source on Torularhodin Pigment Biosynthesis. Revista de Chimie, 62.
Gong G, Liu L, Zhang X, Tan T (2019). Multi-omics metabolism analysis on irradiation-induced oxidative stress to Rhodotorula glutinis. Appl Microbiol Biotechnol, 103(1), 361-374.
Gong G, Zhang X, Tan T (2020a). Simultaneously enhanced intracellular lipogenesis and beta-carotene biosynthesis of Rhodotorula glutinis by light exposure with sodium acetate as the substrate. Bioresour Technol, 295, 122274.
Goodwin TW (1993). [29] Biosynthesis of carotenoids: An overview. In Methods in Enzymology. Academic Press, 330-340.
Gusakov AV, Kondratyeva EG, Sinitsyn AP (2011). Comparison of two methods for assaying reducing sugars in the determination of carbohydrase activities. International journal of analytical chemistry, 2011, 283658-283658.
Hall BG (2013). Building Phylogenetic Trees from Molecular Data with MEGA. Molecular Biology and Evolution, 30(5), 1229-1235.
Head SR, Komori HK, Lamere SA, Whisenant T, Van Nieuwerburgh F, Salomon DR, Ordoukhanian P (2014). Library construction for next-generation sequencing: overviews and challenges. BioTechniques, 56(2), 61-passim.
Hernández-Almanza A, Cesar Montanez J, Aguilar-González MA, Martínez-Ñvila C, Rodríguez-Herrera R, Aguilar CN (2014). Rhodotorula glutinis as source of pigments and metabolites for food industry. Food Bioscience, 5, 64-72.
Hu K, Zhu XL, Mu H, Ma Y, Ullah N, Tao YS (2016). A novel extracellular glycosidase activity from Rhodotorula mucilaginosa: its application potential in wine aroma enhancement. Letters in Applied Microbiology, 62(2), 169-176.
Kong W, Yang S, Agboyibor C, Chen D, Zhang A, Niu S (2019). Light irradiation can regulate the growth characteristics and metabolites compositions of Rhodotorula mucilaginosa. J Food Sci Technol, 56(12), 5509-5517.
Kot AM, Błażejak S, Gientka I, Kieliszek M, Bryś J (2018). Torulene and torularhodin: “new” fungal carotenoids for industry Microbial Cell Factories, 17(1), 49.
Kot AM, Błażejak S, Kieliszek M, Gientka I, Bryś J (2019a). Simultaneous Production of Lipids and Carotenoids by the Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater. Appl Biochem Biotechnol, 189(2), 589-607.
Kot AM, Błażejak S, Kieliszek M, Gientka I, Bryś J, Reczek L, Pobiega K (2019b). Effect of exogenous stress factors on the biosynthesis of carotenoids and lipids by Rhodotorula yeast strains in media containing agro-industrial waste. World journal of microbiology & biotechnology, 35(10), 157-157.
Kot AM, Błażejak S, Kurcz A, Gientka I, Kieliszek M (2016). Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 100(14), 6103-6117.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35(6), 1547-1549.
Kurtzman CP, Robnett CJ (1998). Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek, 73(4), 331-371.
Lin X, Gao N, Liu S, Zhang S, Song S, Ji C, Dong X, Su Y, Zhao ZK, Zhu B (2017). Characterization the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation. Yeast, 34(8), 335-342.
Liu Y, Hodson M, Hall B (2006). Loss of the flagellum happened only once in the fungal lineage: Phylogenetic structure of Kingdom Fungi inferred from RNA polymerase II subunit genes. BMC evolutionary biology, 6, 74.
Maoka T (2011). Carotenoids in marine animals. Marine drugs, 9(2), 278-293.
Marsden WL, Gray PP, Nippard GJ, Quinlan MR (1982). Evaluation of the DNS method for analysing lignocellulosic hydrolysates. Journal of Chemical Technology and Biotechnology, 32(7-12), 1016-1022.
Mathew L, Salustra U, Cheryl S, Bonnee R (2019). The Oleaginous Red Yeast Rhodotorula/Rhodosporidium: A Factory for Industrial Bioproducts.
Pi HW, Anandharaj M, Kao YY, Lin YJ, Chang JJ, Li WH (2018). Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous β-carotene and cellulase production. Scientific Reports, 8(1), 10850.
Rapoport A, Guzhova I, Bernetti L, Buzzini P, Kieliszek M, Kot AM (2021). Carotenoids and Some Other Pigments from Fungi and Yeasts. Metabolites, 11(2), 92.
Razavi SH, Marc I (2006). Effect of Temperature and pH on the Growth Kinetics and Carotenoid Production by Sporobolomyces ruberrimus H110 Using Technical Glycerol as Carbon Source. Iranian Journal of Chemistry and Chemical Engineering (IJCCE), 25(3), 59-64.
Sakaki H, Nochide H, Komemushi S, Miki W (2002). Effect of active oxygen species on the productivity of torularhodin by Rhodotorula glutinis No. 21. J Biosci Bioeng, 93(3), 338-340.
Sies H, Stahl W (1998). Lycopene: Antioxidant and Biological Effects and its Bioavailability in the Human. Proceedings of the Society for Experimental Biology and Medicine, 218(2), 121-124.
Singh P, Tsuji M, Singh S, Roy U, Hoshino T (2013). Taxonomic characterization, adaptation strategies and biotechnological potential of cryophilic yeasts from ice cores of Midre Lovénbreen glacier, Svalbard, Arctic. Cryobiology, 66.
Supek F, Bošnjak M, Škunca N, Šmuc T (2011). REVIGO summarizes and visualizes long lists of gene ontology terms. PloS one, 6(7), e21800.
Takaichi S (2011). Carotenoids in algae: distributions, biosyntheses and functions. Marine drugs, 9(6), 1101-1118.
Tang W, Wang Y, Zhang J, Cai Y, He Z (2019). Biosynthetic Pathway of Carotenoids in Rhodotorula and Strategies for Enhanced Their Production. J Microbiol Biotechnol, 29(4), 507-517.
Tkáčová J, Furdíková K, Klempová T, Ďurčanská K, Čertík M (2015). Screening of carotenoid-producing strains isolated from natural sources. Acta Chimica Slovaca, 8(1), 34-38.
Voaides C & Dima R (2011). Effect of carbon source on carotenoid production by Rhodotorula sp. Archiva Zootechnica, 14(3), 75-83.
Wang QM, Bai FY (2008). Molecular phylogeny of basidiomycetous yeasts in the Cryptococcus luteolus lineage (Tremellales) based on nuclear rRNA and mitochondrial cytochrome b gene sequence analyses: proposal of Derxomyces gen. nov. and Hannaella gen. nov., and description of eight novel Derxomyces species. FEMS Yeast Res, 8(5), 799-814.
Wang Q M, Groenewald M, Takashima M, Theelen B, Han PJ, Liu XZ, Boekhout T, Bai FY (2015). Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses. Stud Mycol, 81, 27-53.
Wang QM, Theelen B, Groenewald M, Bai FY, Boekhout T (2014). Moniliellomycetes and Malasseziomycetes, two new classes in Ustilaginomycotina. Persoonia, 33, 41-47.
White T, Bruns T, Lee S, Taylor,J, Innis M, Gelfand D, Sninsky J (1990). Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In, 315-322.
Wirth F, Goldani LZ (2012a). Epidemiology of Rhodotorula: an emerging pathogen. Interdisciplinary perspectives on infectious diseases, 2012, 465717-465717.
Wirth F, & Goldani LZ (2012b). Experimental Rhodotorulosis infection in rats. Apmis, 120(3), 231-235.
Xie W, Liu M, Lv X, Lu W, Gu J, Yu H (2014). Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnol Bioeng, 111(1), 125-133.
Yen H-W, Yang YC (2012a). The effects of irradiation and microfiltration on the cells growing and total lipids production in the cultivation of Rhodotorula glutinis. Bioresource Technology, 107, 539-541.