豐原素由枯草桿菌 (Bacillus subtilis) F29-3產生，是一種對絲狀真菌具有抑制作用的脂胜肽類抗生素。豐原素是由脂肪酸與胜肽鏈組成，在脂肪酸部分，包含十三種碳數為十五至十七個的脂肪酸，胜肽鏈部分包含十個胺基酸，在第三個胺基酸的L-Tyr與第十個位置的L-Ile形成lactone bond，使豐原素形成環狀結構。豐原素合成酶的基因組大小38 kb，包含五個依序為fenC、fenD、fenE、fenA、fenB的豐原素合成酶基因，在fenC上游有啟動子來調控其表現，已知豐原素是以非核糖體方式合成的。實驗室曾經發現FenC、FenD、FenE、FenA及FenB可以結合成複合體。我利用Ni-NTA agarose對FenC、FenD、FenE、FenA及FenB進行蛋白質結合測試，結果發現FenB只會與FenA結合。另外，進一步縮小FenA與FenB的結合範圍，目前發現對於FenB N端來說，FenA C端為其專一的互動區。對於FenA而言，FenA是以C端500個胺基酸區域與FenB N端538個胺基酸區域結合。此研究顯示豐原素合成酶會以專一性結合的方式形成複合體。

Fengycin is an antifungal antibiotic produced by Bacillus subtilis F29-3, which is composed of a fatty acid and a peptide chain. Fengycin is synthesized nonribosomally by fengycin synthetases, including FenC, FenD, FenE, FenA, and FenB. It is known that fengycin peptide elongates in an orderly fashion during peptide synthesis on FenC, FenD, FenE, FenA, and FenB. Thus, it is hypothesized that specific interactions among fengycin synthetases are critical to the synthesis process. Our earlier studies demonstrated that these five peptide synthetases form a complex in vivo. However, whether these enzymes interact specifically is still unknown. To understand how fengycin is synthesized, this study investigates the interaction between FenA and FenB to elucidate the domains in these two enzymes involved in the interaction. This study demonstrates that the N-terminal region of FenB interacts specifically with the C-terminal region of FenA. Deletion analysis delineates the interaction regions to the N-terminal 538 amino acids of FenB and the C-terminal 500 amino acids of FenA. Because this study finds that FenB interacts only with FenA but not the other fengycin synthesis; the finding suggests that nonribosomal peptide synthesis involves specific interactions among peptide synthetases.

指導教授推薦書………………………………………………………
口試委員會審書………………………………………………………
授權書…………………………………………………………………
致謝…………………………………………………………………….iv
中文摘要……………………………………………………………..…v
英文摘要…………………………………………………………….…vi
緒論…………………………………………………………………..…1
材料與方法…………………………………………………………....10
實驗結果………………………………………………………………14
討論……………………………………………………………………21
圖................................………………………………………………....26
表………………………………………………………………………46
參考文獻………………………………………………………………49

林光慧‧1999‧枯草桿菌F29-3豐原素合成基因fenA及fenB之研究‧國立台灣大學植物學研究所博士論文
林翠品‧1999‧豐原素合成基因fenC及啟動子的研究‧國立陽明大學微生物暨免疫學研究所博士論文
林雅靜‧2003‧豐原素合成酶FenA的功能分析‧長庚大學基礎醫學研究所微生物組碩士論文
陳奇良‧1995‧枯草桿菌F29-3中豐原素合成基因的分析‧國立中興大學植物學研究所博士論文
許鴻猷‧2002‧枯草桿菌F29-3豐原素胜肽合成酶fenE, fenC基因及Phosphopantetheinyl Transferase基因fgp之研究‧國立陽明大學微生物暨免疫學研究所博士論文
黃士豪‧1998‧豐原素合成基因fenD之研究‧國立陽明大學微生物暨免疫學研究所碩士論文
Belshaw, P.J., Walsh, C.T. and Stachelhaus, T. (1999) Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science, 284, 486-489.
Bergendahl, V., Linne, U. and Marahiel, M.A. (2002) Mutational analysis of the C-domain in nonribosomal peptide synthesis. Eur J Biochem, 269, 620-629.
Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res, 7, 1513-1523.
Chang, L.K., Chen, C.L., Chang, Y.S., Tschen, J.S., Chen, Y.M. and Liu, S.T. (1994) Construction of Tn917ac1, a transposon useful for mutagenesis and cloning of Bacillus subtilis genes. Gene, 150, 129-134.
Chen, C.L., Chang, L.K., Chang, Y.S., Liu, S.T. and Tschen, J.S. (1995) Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis in Bacillus subtilis. Mol Gen Genet, 248, 121-125.
Chmara, H. (1985) Inhibition of glucosamine synthase by bacilysin and anticapsin. J Gen Microbiol, 131 ( Pt 2), 265-271.
De Crecy-Lagard, V., Marliere, P. and Saurin, W. (1995) Multienzymatic non ribosomal peptide biosynthesis: identification of the functional domains catalysing peptide elongation and epimerisation. C R Acad Sci III, 318, 927-936.
Donadio, S., Staver, M.J., McAlpine, J.B., Swanson, S.J. and Katz, L. (1991) Modular organization of genes required for complex polyketide biosynthesis. Science, 252, 675-679.
Ehmann, D.E., Trauger, J.W., Stachelhaus, T. and Walsh, C.T. (2000) Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases. Chem Biol, 7, 765-772.
Eppelmann, K., Stachelhaus, T. and Marahiel, M.A. (2002) Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics. Biochemistry, 41, 9718-9726.
Gocht, M. and Marahiel, M.A. (1994) Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis. J Bacteriol, 176, 2654-2662.
Gokhale, R.S., Tsuji, S.Y., Cane, D.E. and Khosla, C. (1999) Dissecting and exploiting intermodular communication in polyketide synthases. Science, 284, 482-485.
Gutierrez, S., Diez, B., Montenegro, E. and Martin, J.F. (1991) Characterization of the Cephalosporium acremonium pcbAB gene encoding alpha-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains. J Bacteriol, 173, 2354-2365.
Hahn, M. and Stachelhaus, T. (2004) Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains. Proc Natl Acad Sci U S A, 101, 15585-15590.
Hopwood, D.A. and Sherman, D.H. (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet, 24, 37-66.
Kado, C.I. and Liu, S.T. (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol, 145, 1365-1373.
Kleinkauf, H. and Von Dohren, H. (1996) A nonribosomal system of peptide biosynthesis. Eur J Biochem, 236, 335-351.
Koch, U. (1988) Fengycin: Strukturäufklarung eines mikroheterogenen Lipopeptolidantibiotikums. Dissertation, zur Erlangung des Grades eines Doktors, der Naturwissenschaften. Eberhard-Karls-Universität zu Tübingen, Tübingen, Germany.
Kohli, R.M., Trauger, J.W., Schwarzer, D., Marahiel, M.A. and Walsh, C.T. (2001) Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases. Biochemistry, 40, 7099-7108.
Lambalot, R.H., Gehring, A.M., Flugel, R.S., Zuber, P., LaCelle, M., Marahiel, M.A., Reid, R., Khosla, C. and Walsh, C.T. (1996) A new enzyme superfamily - the phosphopantetheinyl transferases. Chem Biol, 3, 923-936.
Lin, G.H., Chen, C.L., Tschen, J.S., Tsay, S.S., Chang, Y.S. and Liu, S.T. (1998) Molecular cloning and characterization of fengycin synthetase gene fenB from Bacillus subtilis. J Bacteriol, 180, 1338-1341.
Lin, T.P., Chen, C.L., Chang, L.K., Tschen, J.S. and Liu, S.T. (1999) Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis. J Bacteriol, 181, 5060-5067.
Linne, U., Doekel, S. and Marahiel, M.A. (2001) Portability of epimerization domain and role of peptidyl carrier protein on epimerization activity in nonribosomal peptide synthetases. Biochemistry, 40, 15824-15834.
Linne, U., Stein, D.B., Mootz, H.D. and Marahiel, M.A. (2003) Systematic and quantitative analysis of protein-protein recognition between nonribosomal peptide synthetases investigated in the tyrocidine biosynthetic template. Biochemistry, 42, 5114-5124.
Lipmann, F. (1980) Bacterial production of antibiotic polypeptides by thiol-linked synthesis on protein templates. Adv Microb Physiol, 21, 227-266.
Lipmann, F., Gevers, W., Kleinkauf, H. and Roskoski, R., Jr. (1971) Polypeptide synthesis on protein templates: the enzymatic synthesis of gramicidin S and tyrocidine. Adv Enzymol Relat Areas Mol Biol, 35, 1-34.
Liu, F., Garneau, S. and Walsh, C.T. (2004) Hybrid nonribosomal peptide-polyketide interfaces in epothilone biosynthesis: minimal requirements at N and C termini of EpoB for elongation. Chem Biol, 11, 1533-1542.
Loeffler, W., Tschen, J.S., Vanittanakom, N., Kulger, M., Konorpp, E. and Wu, T.G. (1986) Antifungal effects of bacilysin and fengycin from Bacillus subtilis F29-3. A comparison with activities of other Bacillus antibiotics. J Phytopathol, 115, 204-213.
MacCabe, A.P., Riach, M.B., Unkles, S.E. and Kinghorn, J.R. (1990) The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis. Embo J, 9, 279-287.
Marahiel, M.A., Stachelhaus, T. and Mootz, H.D. (1997) Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis. Chem Rev, 97, 2651-2674.
May, J.J., Kessler, N., Marahiel, M.A. and Stubbs, M.T. (2002) Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases. Proc Natl Acad Sci U S A, 99, 12120-12125.
O'Connor, S.E., Walsh, C.T. and Liu, F. (2003) Biosynthesis of epothilone intermediates with alternate starter units: engineering polyketide-nonribosomal interfaces. Angew Chem Int Ed Engl, 42, 3917-3921.
Quadri, L.E., Weinreb, P.H., Lei, M., Nakano, M.M., Zuber, P. and Walsh, C.T. (1998) Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry, 37, 1585-1595.
Rieder, H., Heinrich, G., Breuker, E., Simlot, M.M. and Pfaender, P. (1975) Bacitracin synthetase. Methods Enzymol, 43, 548-559.
Rogers, H.J. and Garrett, A.J. (1965) The Interrelationship between Mucopeptide and Ribitol Teichoic Acid Formation as Shown by the Effect of Inhibitors. Biochem J, 96, 231-243.
Shu, H.Y., Lin, G.H., Wu, Y.C., Tschen, J.S. and Liu, S.T. (2002) Amino acids activated by fengycin synthetase FenE. Biochem Biophys Res Commun, 292, 789-793.
Sieber, S.A. and Marahiel, M.A. (2003) Learning from nature's drug factories: nonribosomal synthesis of macrocyclic peptides. J Bacteriol, 185, 7036-7043.
Smith, D.J., Burnham, M.K., Bull, J.H., Hodgson, J.E., Ward, J.M., Browne, P., Brown, J., Barton, B., Earl, A.J. and Turner, G. (1990) Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. Embo J, 9, 741-747.
Stachelhaus, T., Huser, A. and Marahiel, M.A. (1996a) Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases. Chem Biol, 3, 913-921.
Stachelhaus, T., Mootz, H.D., Bergendahl, V. and Marahiel, M.A. (1998) Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J Biol Chem, 273, 22773-22781.
Stachelhaus, T., Mootz, H.D. and Marahiel, M.A. (1999) The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol, 6, 493-505.
Stachelhaus, T., Schneider, A. and Marahiel, M.A. (1996b) Engineered biosynthesis of peptide antibiotics. Biochem Pharmacol, 52, 177-186.
Stachelhaus, T. and Walsh, C.T. (2000) Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase. Biochemistry, 39, 5775-5787.
Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R. and Morris, H.R. (1996) The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J Biol Chem, 271, 15428-15435.
Trauger, J.W., Kohli, R.M., Mootz, H.D., Marahiel, M.A. and Walsh, C.T. (2000) Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature, 407, 215-218.
Tschen, J.S.-M. (1987) Control of Rizoctonia soanli by Bacillus subtilis. Transactions of the Mycological Society of Japan., 28, 483-493.
Turgay, K., Krause, M. and Marahiel, M.A. (1992) Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes. Mol Microbiol, 6, 2743-2744.
Vanittanakom, N., Loeffler, W., Koch, U. and Jung, G. (1986) Fengycin--a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot (Tokyo), 39, 888-901.