臺灣博碩士論文加值系統

頭孢菌素之抗菌機制為抑制細菌的細胞壁合成，因其更具廣效性及較少抗藥株，在臨床上的應用非常廣泛；目前的主要生產方法為先使用工業發酵生產前驅物，再經過數個修飾化學官能基的步驟，而得到具藥效的最終產物。因化學合成反應不若生物反應專一，且又有昂貴、較污染等缺點，故如何更加經濟地生產頭孢菌素類抗生素成為現今重要的課題。
DACS為一種需要2-oxoglutarate及二價鐵催化的氧化酵素，來源為放線菌 Streptomyces clavuligerus，對應基因為 cefF，在頭孢菌素C的生合成途徑中，參與催化 DAOC 生成 DAC 的反應。本篇論文的目的在利用蛋白質工程的方法，提高DACS對非天然受質 DAOG 的產物轉換率，以更有效率的方式生成抗生素前驅物 DAG。
我們先使用單點突變的方法產生F268A、R270L、A185Y、V279I、V307A、T308A、T308K、M309L和H310L 等突變株， 但其中只有T308A的催化活性有些微改進。之後，我們利用任意突變，例如加入化學突變劑、合成不特定引子用於單點突變或擾亂聚合脢連續反應等等方法製造大量突變株，先利用反應物和產物毒性強弱的差別，初步篩選其中約 6600 株菌至490株，再個別做小量活性測試，挑出生成產物量比野生株高的 69株，經進一步定序後，確定最佳的突變株分別是：A80G、S218D及A57V。並針對其中活性最好的 A57V，同一點位置再突變成賴胺酸(Lys)、白胺酸(Leu)、異白胺酸(Ile)、麩胺酸(Glu) 和甘胺酸(Gly)。我們純化野生株及這幾株表現較好的酵素，做進一步更精確的動力學分析，發現其中最好的A57E (KM = 0.89±0.02mM, kcat/ KM = 2.59±0.12/Msec) 酵素活性有野生株 (KM = 3.09±0.60mM, kcat/ KM = 1.39±0.29/Msec) 的百分之一百八十六。這項結果顯示從任意突變挑到的位置結合單點突變的策略之可行性。未來甚至可以結合數個有意義的單點突變，以達到提高催化活性的最終目標。

Medicinally useful cephalosporins are produced by fermentation products with further modifications. Although cephalosporins are superior antibiotics compared with penicillins, their distribution is limited because the processes for further chemical modifications are complicated and costly.
DACS, a nonheme-iron(II) and 2-oxoglutarate-dependent oxygenase, catalyzes the 3’-hydroxylation of DAOC in the biosynthesis of cephalosporins. It can convert its natural substrate analogue DAOG to DAG with relatively low yield. The goal of this study is to raise the accessibility of DACS to DAOG via protein engineering techniques.
We began to design mutations via site-directed mutagenesis at the residues near the binding pocket or C terminal, including F268A, R270L, A185Y, V279I, V307A, T308A, T308K, M309L and H310L. Only T308A showed slightly higher activity. Therefore, we applied random mutagenesis such as chemical mutagenesis, site-directed random mutagenesis, and error-prone PCR. After screening 6600 transformants by several steps, we obtained 69 mutants and sequenced them. The three mutants S218D, A80G and A57V showed the highest activities. Furthermore, the A57 was mutated to K, I, E, L and G. Kinetic parameters of the recombinant DACSs were measured by assays under the optimized condition. The enzyme product of the A57E mutant showed the highest activity (kcat/KM = 2.59±0.12/Msec) which was about 1.86 fold of that of the wild type (kcat/KM = 1.39±0.29/Msec). The data showed that the well-developed screening methods could successfully help us to find the desired mutants derived from random mutagenesis and further manipulations at those residues may improve enzymatic activities.

Abstract............... .................I
Abstract (Chinese)..................... II
Acknowledgement........................III
Acknowledgement (Chinese)...............IV
Abbreviations...........................VI
Introduction...........................1-9
Materials and Methods................10-20
Results and Discussion...............21-51
Conclusion..............................52
Appendix.............................53-54
References...........................55-59

Adrio, J. L., Cho, H., Piret, G. M. and Demain, A. L. (1999) Inactivation of deacetoxycephalosporin C synthase in extracts of Streptomyces clavuligerus during bioconversion of penicillin G to deacetoxycephalosporin G. Enzyme Microb. Technol. 25, 497-501.
Aharonowitz, Y., Cohen, G. and Martín, J. F. (1992) Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation, and evolution. Annu. Rev. Microbiol. 46, 461-495.
Baldwin, J. E. & Abraham, E. P. (1988) The biosynthesis of penicillins and cephalosporins. Nat. Prod. Rep. 5, 129-145.
Burzlaff, N. I., Rutledge, P. J., Clifton, I. J., Hensgens, C. M., Pickford, M., Adlington, R. M., Roach, P. L. & Baldwin, J. E. (1999) The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. Nature, 401, 721-724.
Cantwell, C. A., Beckmann, R. J., Dortzlaf, J. E., Fisher, D. L., Skatrud, K. L., Yeh, W. -K. and Queener, S. W. (1990) Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum. Curr. Genet. 17, 213-221.
Cedrone, F., Ménez, A. and Quéméneur, E. (2000) Tailoring new enzyme functions by rational redesign. Curr. Opin. Struct. Biol. 10, 405-410.
Chin, H. -S., Sim, J. and Sim, T. -S. (2001) Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion. Biochem. Biophys. Res. Commun. 287, 507-513.
Cho, H., Adrio, J. L., Luengo, G. M., Wolfe, S., Ocran, S., Hintermann, G., Piret, J. M. and Demain, A. L. (1998) Elucidation of the conditions allowing conversion of penicillin G and other penicillins to deacetoxycephalosporins by resting cells and extracts of Streptomyces clavuligerus NP1. Proc. Natl. Acad. Sci. 95, 11544-11548.
Clifton, I. J., Hsueh, L. -C., Baldwin, J. E., Harlos, K. and Schofield, C. J. (2001) Structure of proline 3-hydroxylase: evolution of the family of 2-oxoglutarate dependent oxygenases. Eur. J. Biochem. 268, 6625-6636.
Copeland, R. A. (2000) Enzymes: a practical introduction to structure , mechanism, and data analysis. Wiley-VCH.
Crawford, L., Stepan, A. M., McAda, P. C., Rambosek, J. A., Conder, M. J., Vinci, V. A. and Reeves, C. D. (1995) Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Bio/technology, 13, 58— 62
Dotzlaf, J. E. & Yeh, W. -K. (1989) Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus. J. Biol. Chem. 264, 10219-10227.
Dotzlaf, J. E. & Yeh, W. -K. (1987) Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium. J. Bacteriol. 169, 1611-1618.
Dubus, A., Lloyd, M. D., Lee, H. -J., Schofield, C. J., Baldwin, J. E. and Frère, J. -M. (2001) Probing the penicillin side chain selectivity of recombinant deacetoxycephalosporin C synthase. Cell. Mol. Life Sci. 58, 835-843.
Fersht, A. (1999) Structure and mechanism in protein science: a guide to enzyme
catalysis and protein folding, Freeman, New York.
Ghag, S.K., Brems, D.V., Hassell, T. C. and Yeh, W. -K. (1996) Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli. Biotechnol. Appl. Biochem. 24, 109-119.
Glazer, A. N. & Nikaido, H. (1995) Microbial Biotechnology: Fundamentals of Applied Microbiology, Freeman, New York.
Hegg, E. L. & Que, J. L. (1997) The 2-His-1-carboxylate facial triad: an emerging structural motif in mononuclear non-heme iron(II) enzymes, Eur. J. Biochem. 250, 625—629.
Huang, W., Petrosino, J., Hirsch, M., Shenkin, P. S. and Palzkill, T. (1996) Amino acid sequence determinants of -lactamase structure and activity. J. Mol. Biol. 258, 688-703.
Kimura, H., Izawa, M. and Sumino, Y. (1996) Molecular analysis of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90. Appl. Microbiol. Biotechnol. 44, 589-596.
Kovacevic, S., Weigel, B. L., Tobin, M. B., Ingolia, T. D. and Miller, J. R. (1989) Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol. 171, 754-760.
Lange, S. J. & Que, J. L. (1998) Oxygen activating nonheme iron enzymes. Curr. Opin. Chem. Biol. 2, 159—172.
Lee, H.-J., Lloyd, M. D., Harlos, K., Clifton, I. J., Baldwin, J. E. and Schofield, C. J. (2001) Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS). J. Mol. Biol. 308, 937-948.
Lee, H. -J., Lloyd, M. D., Harlos, K. and Schofield, C. J. (2000) The effect of cysteine mutations on the activity of recombinant deacetoxycephalosporin C synthase from S. clavuligerus. Biochem. Biophys. Res. Commun. 267, 445-448.
Lloyd, M. D., Lee, H.-J., Harlos, K., Zhang, Z. -H., Baldwin, J. E., Schofield, C. J., Charnock, J. M., Garner, C. D., Hara, T., Terwisscha van Scheltinga, A. C., Valegård, K., Viklund, J. A. C., Hajdu, J., Andersson, I., Danielsson, A. and Bhikhabhai, R. (1999) Studies on the active site of deacetoxycephalosporin C synthase. J. Mol. Biol. 287, 943—960.
Martín, J. F. (1998) New aspects of genes and enzymes for -lactam antibiotic biosynthesis. Appl. Microbiol. Biotechnol. 50, 1-15.
Meyer, A., Schnmid, A., Held, M., Westphal, A. H., Röthlisberger, M., Kohler, H. -P. E., van Berkels, W. J. H. and Witholt, B. (2002) Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution. J. Biol. Chem. 277, 5575-5582.
Morgan, N., Pereira, I. A. C., Andersson, I. A., Adlington, R. M., Baldwin, J. E., Cole, S. C., Crouch, N. P. and Sutherland, J. D. (1994) Substrate specificity of recombinant Streptomyces clavuligerus deacetoxycephalosporin C synthase. Bioorg. Med. Chem. Lett. 4, 1595-1600.
Piret, J., Resendiz, B., Mahro, B., Zhang, J. -Y. Serpe, E., Romero, J., Connors, N. and Demain, A. L. (1990) Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585. Appl. Microbiol. Biotechnol. 32, 560-567.
Roach, P. L., Clifton, I. J., Fulop, V., Harlos, K., Barton, G. J., Hajdu, J., Andersson, I., Schofield, C. J. & Baldwin, J. E. (1995) Structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature, 375, 700—704.
Roach, P. L., Clifton, I. J., Hensgens, C. M. H., Shibata, N., Schofield, C. J., Hajdu, J. & Baldwin, J. E. (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. Nature, 387, 827—830.
Que, J. L. (2000) One motif — many different reactions. Nat. Struct. Biol. 7, 182-184.
Schofield, C. J., Baldwin, J. E., Byford, M. F., Clifton, I., Hajdu, J., Hensgens, C. and Roach, P. (1997) Proteins of the penicillin biosynthesis pathway. Curr. Opin. Struct. Biol. 7, 857-864.
Schofield, C. J. & Zhang, Z. -H. (1999) Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr. Opin. Struct. Biol. 9, 722—731.
Shen, Y. -Q., Wolfe, S., and Demain, A. L. (1984) Desacetoxycephalosporin C synthetase: Importance of order of cofactor/reactant addition. Enzyme Microb. Technol. 6, 402—404.
Shibata, N., Lloyd, M.D., Baldwin, J. E. and Schofield, C. J. (1996) Adipoyl-6-aminopenicillanic acid is a substrate for deacetoxycephalosporin C synthase (DAOCS). Bioorg. Med. Chem. Lett. 6, 1579-1584.
Sim J., and Sim, T.-S. (2001) In vitro conversion of penicillin and ampicillin by recombinant Streptomyces clavuligerus NRRL3585 deacetoxycephalosporin C synthase. Enzyme Microbial. Technol. 29, 240-245.
Sim J., and Sim, T.-S. (2000) Mutational evidence supporting the involvement of triparitie residues His183, Asp185, and His 243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis. Biosci. Biotechnol. Biochem. 64, 828-832.
Valegård K., Terwisscha van Scheltinga, A. C., Lloyd, M. D., Hara, T., Ramaswamy, S., Perrakis, A., Thompson, A., Lee, H. -J., Baldwin, J. E., Schofield, C. J., Hajdu, J. & Andersson, I. (1998) Structure of a cephalosporin synthase. Nature, 394, 805—809.
Velasco, J., Adrio, J. L., Angel Moreno, M., Diez, B., Soler, G. and Barredo, J. L.(2000) Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum. Nat. Biotechnol. 18, 857-861.
Zhang, Z. -H., Ren, J.-S., Stammers, D. K., Baldwin, J. E., Harlos, K. & Schofield, C. J. (2000) Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase. Nat. Struct. Biol. 7, 127—133.