白麴菌Saccharomycopsis fibuligera是一種具有澱粉糖化（saccharolytic）及微弱酒精發酵（fermentative）能力的酵母菌，其高效率分解澱粉的能力，使其常成為發酵工業所用來生產酒精的酵母菌之一。本研究主要的目標在於了解白麴菌在不同的環境狀況下的適應演化過程與其澱粉糖化能力的關係。本研究所使用之白麴菌酵母菌是由全台灣各地製造酒釀的酵母（白穀）中分離出來的，共收集分離了21個不同品系的白麴菌菌株，另外我們也購置一白麴菌菌株(BCRC21511)做為和野外採集之白麴菌菌株的比較。我們發現不同品系的白麴菌菌株有不同的特定澱粉利用能力，所以我藉由測量這些不同品系白麴菌菌株在特定澱粉中的成長速率(growth rate)，來比較這22種不同品系白麴菌菌株間利用特定澱粉能力的差異，實驗結果顯示每一品系菌株利用特定澱粉的能力皆不相同，其中PF32及BCRC21511菌株對不同種類澱粉都有很好的利用能力。由於對澱粉的代謝能力和澱粉酶基因息息相關，所以我分析了這22株白麴菌的兩種澱粉代謝基因ALP1和GLU1的序列，以瞭解不同的白麴菌菌株利用特定澱粉的能力是否和不同族群間澱粉代謝基因的基因多型性(polymorphism)相關。在本研究中我也發現，在白麴菌中除了ALP1和GLU1兩個澱粉代謝基因外尚有一個基因片段，其序列跟GLU1基因有極高的相似性，極有可能是尚未被發現的澱粉代謝基因。為了更進一步以分子生物學的方法研究這些不同澱粉代謝基因對白麴菌的生態適應性影響，我檢驗了白麴菌的有性生殖方式及這些菌株的基因體狀態以方便進行基因研究。我發現這些白麴菌菌株應該都是雙倍體酵母菌，因為少部分的白麴菌菌株會經誘發產生孢子且單一白麴菌孢子萌芽後可藉由細胞融合方式形成雙倍體酵母菌。為了對白麴菌的基因有更進一步的了解，我將白麴菌的三個釀酒酵母菌的同源基因片段(SDH1、CCC2和PDC1)，轉殖到已進行相對基因剔除的釀酒酵母菌細胞中，進行功能性互補試驗(functional complementation assay)，以了解將來是否有可能以已是模式物種且被瞭解比較透徹的釀酒酵母菌作為平台，研究不同白麴菌澱粉代謝基因的特定澱粉代謝能力，另一方面也提供白麴菌的基因資訊資料作為將來進行白麴菌基因鑑識(gene annotation)的參考。

Saccharomycopsis fibuligera is one of the yeast species capable of starch saccharification and alcoholic fermentation. Due to the high starch hydrolysis capability, S. fibuligera is commonly used in fermentation industry for ethanol production. The major goal of my thesis was to understand the influence of starch saccharification capability to the ecological adaptation of S. fibuligera. The 22 S. fibuligera strains used in this thesis were isolated from different breweries in Taiwan including a BCRC21511 strain purchased from BCRC. To test the specific starch utilization capacity of these S. fibuligera strains I grew these 22 S. fibuligera strain in different starch media. My results indicated that different S. fibuligera strains showed different starch utilization capacity; especially the strains PF32 and BCRC21511 showed the best starch utilization capacity for many different starch substrates tested. It is possible that the different starch utilization capacity is due to the genetic variations of amylases genes. It is known that there are two amylase genes in S. fibuligera, ALP1 and GLU1. So, I analyzed the sequence of these two amylase genes of these 22 S. fibuligera strains to check whether the different starch utilization capacity of S. fibuligera strains were affected by the genetic variation of these two amylases genes. In addition to these two amylase genes, I identified an ORF which showed high sequence simularity to GLU1 gene. In order to perform the genetic analysis of these amylase genes in S. fibuligera, I examined whether this species is maintained in a diploid cell stage by placing these 22 S. fibuligera strains under sporulation condition and I found only eight of them could sporulate. I also found that a single spore could form diploid cells. These results suggested that these S. fibuligera strains might be diploid yeasts. To further characterize the function of the S. fibuligera genes, I performed functional complementation assay with Saccharomyces cerevisiae. I cloned and transformed three S. cerevisiae homologous ORFs SDH1, CCC2 and PDC1 of S. fibuligera to relative gene knockout strain of S. cerevisiae to check if the introduction of S. fibuligera ORFs could rescue the gene lost of S. cerevisiae, and the results can also provide information on the gene annotation of S. fibuligera in the furture.

1. Alby K, Schaefer D, Bennett RJ. Homothallic and heterothallic mating in the opportunistic pathogen Candida albicans. Nature 460 (7257): 890-3, 2009.
2.Astell CR, Ahlstrom-Jonasson L, Smith M et al. The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae. Cell; 27: 15-23. 1981
3. Bourgeron T, Rustin P, Chretien D, Birch-Machin M, Bourgeois M, Viegas-Pequignot E, Munnich A, Rotig A. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat Genet 11 (2): 144-9, 1995.
4. Brena BM, Pazos C, Franco-Fraguas L, Batista-Viera F. Chromatographic methods for amylases. J Chromatogr B Biomed Appl 684 (1-2): 217-37, 1996.
5. Bullis BL, Lemire BD. Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase. J Biol Chem 269 (9): 6543-9, 1994.
6. Butler G, Kenny C, Fagan A, Kurischko C, Gaillardin C, Wolfe KH. Evolution of the MAT locus and its Ho endonuclease in yeast species. Proc Natl Acad Sci U S A 101 (6): 1632-7, 2004.
7. Chapman KB, Solomon SD, Boeke JD. SDH1, the gene encoding the succinate dehydrogenase flavoprotein subunit from Saccharomyces cerevisiae. Gene 118 (1): 131-6, 1992.
8. Chi Z, Liu G, Wang F, Ju L, Zhang T. Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol Adv 27 (4): 423-31, 2009.
9. Chi Z, Liu J, Ji J, Meng Z. Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu. J Biotechnol 102 (2): 135-41, 2003.
10. Chi Z, Liu J, Zhang W. Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzyme Microb Technol 28 (2-3): 240-245, 2001.
11.De Mot B, De Clercq M, Rousseeuw P. Visco-elastic properties of four currently used tissue conditioners. J Oral Rehabil ;11: 419-427.1984
12. Eksteen JM, Van Rensburg P, Cordero Otero RR, Pretorius IS. Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera. Biotechnol Bioeng 84 (6): 639-46, 2003.
13. Flikweert MT, Kuyper M, van Maris AJ, Kotter P, van Dijken JP, Pronk JT. Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity. Biotechnol Bioeng 66 (1): 42-50, 1999.
14.Frommer DJ. Defective biliary excretion of copper in Wilson's disease. Gut; 15: 125-129. 1974
15. Haber JE. Mating-type gene switching in Saccharomyces cerevisiae. Trends Genet 8 (12): 446-52, 1992.
16.Hesseltine CW. Microbiology of oriental fermented foods. Ann Rec Microbiol 37:575-601. 1983
17. Hicks J, Strathern JN. Interconversion of mating type in S. cerevisiae and the Cassette model for gene transfer. Brookhaven Symp Biol (29): 233-42, 1977.
18. Hohmann S. Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae. J Bacteriol 173 (24): 7963-9, 1991.
19. Hostinova E, Solovicova A, Dvorsky R, Gasperik J. Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain. Arch Biochem Biophys 411 (2): 189-95, 2003.
20. Itoh T, Ohtsuki I, Yamashita I, Fukui S. Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera. J Bacteriol 169 (9): 4171-6, 1987.
21. Kellermann E, Seeboth PG, Hollenberg CP. Analysis of the primary structure and promoter function of a pyruvate decarboxylase gene (PDC1) from Saccharomyces cerevisiae. Nucleic Acids Res 14 (22): 8963-77, 1986.
22. Klar AJ, Fogel S, Radin DN. Switching of a mating-type a mutant allele in budding yeast Saccharomyces cerevisiae. Genetics 92 (3): 759-76, 1979.
23. Knop M. Yeast cell morphology and sexual reproduction--a short overview and some considerations. C R Biol 334 (8-9): 599-606.
24.Knox AM, du Preez JC, Kilian SG. Starch fermentation characteristics of Saccharomyces cerevisiae strains transformed with amylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Enz Microb Technol;34: 453–60. 2004.
25.Korf RP. The terms homothallism and heterothallism. Nature; 170: 534-535. 1952
26. Kostriken R, Strathern JN, Klar AJ, Hicks JB, Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell 35 (1): 167-74, 1983.
27. Kuehne HA, Murphy HA, Francis CA, Sniegowski PD. Allopatric divergence, secondary contact, and genetic isolation in wild yeast populations. Curr Biol 17 (5): 407-11, 2007.
28. Lombardo A, Carine K, Scheffler IE. Cloning and characterization of the iron-sulfur subunit gene of succinate dehydrogenase from Saccharomyces cerevisiae. J Biol Chem 265 (18): 10419-23, 1990.
29. Machida M, Ohtsuki I, Fukui S, Yamashita I. Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae. Appl Environ Microbiol 54 (12): 3147-55, 1988.
30. McGill C, Shafer B, Strathern J. Coconversion of flanking sequences with homothallic switching. Cell 57 (3): 459-67, 1989.
31.Mercer JF. The molecular basis of copper-transport diseases. Trends Mol Med; 7: 64-69. 2001
32. Mortimer RK, Romano P, Suzzi G, Polsinelli M. Genome renewal: a new phenomenon revealed from a genetic study of 43 strains of Saccharomyces cerevisiae derived from natural fermentation of grape musts. Yeast 10 (12): 1543-52, 1994.
33.Nasmyth K, Stillman D, Kipling D. Both positive and negative regulators of HO transcription are required for mother-cell-specific mating-type switching in yeast. Cell; 48: 579-587. 1987
34. Oyedotun KS, Sit CS, Lemire BD. The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction. Biochim Biophys Acta 1767 (12): 1436-45, 2007.
35. Pasari AB, Korus RA, Heimsch RC. A model for continuous fermentations with amylolytic yeasts. Biotechnol Bioeng 33 (3): 338-43, 1989.
36.Pena MM, Lee J, Thiele DJ. A delicate balance: homeostatic control of copper uptake and distribution. J Nutr; 129: 1251-1260. 1999
37. Robinson KM, von Kieckebusch-Guck A, Lemire BD. Isolation and characterization of a Saccharomyces cerevisiae mutant disrupted for the succinate dehydrogenase flavoprotein subunit. J Biol Chem 266 (32): 21347-50, 1991.
38. Rusche LN, Rine J. Switching the mechanism of mating type switching: a domesticated transposase supplants a domesticated homing endonuclease. Genes Dev 24 (1): 10-4.
39. Sambongi Y, Wakabayashi T, Yoshimizu T, Omote H, Oka T, Futai M. Caenorhabditis elegans cDNA for a Menkes/Wilson disease gene homologue and its function in a yeast CCC2 gene deletion mutant. J Biochem 121 (6): 1169-75, 1997.
40. Schaaff I, Green JB, Gozalbo D, Hohmann S. A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations. Curr Genet 15 (2): 75-81, 1989.
41. Schmidt DM, Saghbini M, Scheffler IE. The C-terminus of the succinate dehydrogenase IP peptide of Saccharomyces cerevisiae is significant for assembly of complex II. Biochemistry 31 (36): 8442-8, 1992.
42. Strathern JN, Klar AJ, Hicks JB, Abraham JA, Ivy JM, Nasmyth KA, McGill C. Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell 31 (1): 183-92, 1982.
43. Tamai Y, Tanaka K, Kaneko Y, Harashima S. HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette. Appl Microbiol Biotechnol 55 (3): 333-40, 2001.
44. Thornton RJ, Eschenbruch R. Homothallism in wine yeasts. Antonie Van Leeuwenhoek 42 (4): 503-9, 1976.
45. Tsuyoshi N, Fudou R, Yamanaka S, Kozaki M, Tamang N, Thapa S, Tamang JP. Identification of yeast strains isolated from marcha in Sikkim, a microbial starter for amylolytic fermentation. Int J Food Microbiol 99 (2): 135-46, 2005.
46.Ueda M, Okada H, Tanaka A et al. Induction and subcellular localization of enzymes participating in propionate metabolism in Candida tropicalis. Arch Microbiol; 136: 169-176. 1983
47. Valachova K, Horvathova V. Starch degradation by glucoamylase Glm from Saccharomycopsis fibuligera IFO 0111 in the presence and absence of a commercial pullulanase. Chem Biodivers 4 (5): 874-80, 2007.
48.Wickerham LJ, Lockwood LB, Pettijohn OG, Ward GE. Starch Hydrolysis and Fermentation by the Yeast Endomycopsis Fibuliger. J Bacteriol; 48: 413-427. 1944
49. Wu X, Moore JK, Haber JE. Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae. Mol Cell Biol 16 (2): 657-68, 1996.
50. Yoshimoto H, Fukushige T, Yonezawa T, Sakai Y, Okawa K, Iwamatsu A, Sone H, Tamai Y. Pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of alpha-ketoisocaproate for isoamyl alcohol formation in Saccharomyces cerevisiae. J Biosci Bioeng 92 (1): 83-5, 2001.
51. Yuan DS, Stearman R, Dancis A, Dunn T, Beeler T, Klausner RD. The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake. Proc Natl Acad Sci U S A 92 (7): 2632-6, 1995.