臺灣博碩士論文加值系統

靈菌紅素（Prodigiosin）為一種可由Serratia, Pseudomonas, Streptomyces, Vibrio等微生物醱酵合成之二級代謝產物，目前已有文獻證實其具有抗生素特性，亦有抑制癌細胞轉移及免疫抑制等功效。
基於Serratia marcescens之靈菌紅素合成機制為藉由生產前驅物2-methyl-3-n-amyl-pyrrole（MAP）及4-methoxy-2,2’-bipyrrole-5-carbaldehyde（MBC），再以縮合酵素作用所形成，因此本研究先將野生株S. marcescens CH-1利用gene targeting的技術，以single cross homologous recombination的方式剔除pig E（調控MAP合成）與pig M（調控MBC合成）基因所在之DNA片段，成功取得只產MBC或MAP單一前驅物之兩生產菌株（此兩菌株均已失去合成靈菌紅素能力），並分別命名為BMJ816與AMJ817，經電子撞擊式質譜儀確認菌株醱酵純化之產物確實分別為靈菌紅素前驅物MBC與MAP。
再以半合成培養基初步探討共培養兩菌株合成靈菌紅素之成效，於兩菌體生長達指數期前期時進行之，結果顯示靈菌紅素最佳可達到約100 mg / L，證實將兩菌株所產之單一前驅物相互添加可以得到靈菌紅素。進一步檢測前驅物合成後是否釋放於胞內或胞外，發現BMJ816菌株之上清液添加於AMJ817菌株之菌液仍可隨時間增加合成靈菌紅素，最高達37 mg / L，且利用高濃度之前驅物MAP類似物：2,4-dimethyl-3-ethylpyrrole（DEP）添加至BMJ816菌株之上清液亦可生成2,4-dimethyl-3-ethyl-prodiginine，顯示MBC甚至縮合酵素生成後確可釋放至胞外，而將AMJ817菌株之上清液添加至BMJ816菌株之菌液僅能得到8 mg / L之靈菌紅素，結果亦顯示靈菌紅素量不隨時間增長，推測MAP無法釋放至胞外中，或是釋放至胞外後極度不穩定。
添加AMJ817或BMJ816突變株之醱酵培養液至S. marcescens C3之醱酵培養液共培養，研究顯示BMJ816突變株額外合成之MBC可和S. marcescens C3分泌多餘之前驅物MAP合成靈菌紅素，且所需時間較短，共培養40小時後產量即可達4 g / L，較單一S. marcescens C3醱酵液需時60小時為快，惟量不足僅導致僅能加速合成時間而最終產量無異。此外AMJ817突變株額外合成之MAP無法被S. marcescens C3利用，也可能S. marcescens C3並無分泌多餘MBC可供合成靈菌紅素。
此外，利用SDS-PAGE分析由Serratia marcescens SM△R（產靈菌紅素）與BMJ816菌株（不產靈菌紅素）萃取之粗酵素，皆可獲得與縮合酵素分子量一致之片段，進一步經由醱酵結果證實，額外添加由S. marcescens SM△R萃取之粗酵素，確實可於短時間內加速靈菌紅素產生，達110 mg / L ，為對照組之2倍。但由MALDI-TOF結果顯示，萃取之粗酵素無法從資料庫找到高度相似圖譜，需要進一步定序瞭解此萃取酵素為何，是否為一新發現。

Prodigiosin（PG）, a secondary metabolite produced by Serratia marcescens and other gram-negative bacteria, such as Pseudomonas, Streptomyces, and Vibrio. In recently PG is of great interest due to its antibiotic, antifungal, immunosuppressive and antiproliferative properties.
The terminal step in the biosynthesis of PG involves the coupling of a stable bipyrrole, 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde（MBC）, with a volatile monopyrrole, 2-methyl-3-n-amyl-pyrrole（MAP）to form a linear tripyrrole（i.e. PG）. So the single cross homologous recombination to knockout pig E（a regulator of the MAP biosynthesis）and pig M（a regulator of the MBC biosynthesis）were performed to screen for the mutants that produce MBC（named BMJ816）and MAP（named AMJ817） separately, and this study shown that the BMJ816 and AMJ817 can produce MBC and MAP separately and has lost the ability to synthesize PG.
The method of co-synthesis formation of pigment was used in the assay for PG biosynthesis. The experiment was carried out from cultures of two strains grown to early exponential phase in semi-defined medium respectively, and one of the mutant strain was inoculated to another one. It was observed that the PG was formed by the assay and the concentration of PG was 100 mg / L.
On the other hand, this work reveals that co-synthesis of the supernatant of BMJ816 strain and the broth of AMJ817 strain can obtain the concentration of PG to 37 mg / L. With DEP (an analogue of precursor MAP) supplied in the supernatant of BMJ816 strain, it infers that MBC and PCE synthesized can release to extracellular. To co-synthesis with the supernatant of AMJ817 strain and the broth of BMJ816 strain, 8 mg / L of PG was obtained. After incubation, the results shown that MAP maybe released to extracellular, highly unstable and easily to be volatile.
SDS-PAGE analysis of the crude enzyme from Serratia marcescens SM△R （PG producing strain） and BMJ816（non-PG producing strain）was shown that a predicted protein band. In addition to, the fermentation results and its molecular weight is 100 KDa confirmed that feeding with the crude enzyme from S. marcescens SM△R can induce the PG synthesized in seconds, and the concentration of PG was approach to 110 mg / L, which was approximately 2-fold higher than control.

中文摘要 I
英文摘要 IV
誌謝 VII
目錄 IX
表目錄 XI
圖目錄 XII
第一章 前言 1
1.1 Serratia marcescens 1
1.2 靈菌紅素 1
1.3 靈菌紅素之合成機制與參與因子 5
1.4 研究動機與目的 9
第二章 材料與方法 11
2.1 實驗菌株、質體與引子 11
2.2 材料與試劑 12
2.3 實驗步驟 17
第三章 實驗結果與討論 29
3.1 論文研究架構 29
3.2 突變株之選殖 30
3.3 確認突變株部分序列已被刪除並插入可辨識片段 33
3.4 突變株產物之分子量鑑定分析 41
3.5 S. marcescens CH-1與突變株之生長曲線與pH值分析 44
3.6 以LB培養基探討突變株共培養對靈菌紅素合成之影響 46
3.7 以半合成培養基探討突變株共培養對靈菌紅素合成之影響 48
3.8 藉由半合成培養基偵測BMJ816菌株前驅物存在位置 52
3.9 藉由半合成培養基偵測AMJ817菌株前驅物存在位置 54
3.10 添加前驅物MAP之類似物-DEP探討前驅物MBC合成後釋放至胞內或胞外 56
3.11 添加單一突變株之醱酵培養液至S. marcescens C3之醱酵培養液共培養 58
3.12 添加單一突變株之醱酵培養液至S. marcescens SM△R之醱酵培養液共培養 60
3.13 純化縮合酵素並以醱酵方式初步探測其活性 62
第四章 結論與未來展望 69
4.1 結論 69
4.2未來展望 72
參考文獻 73

1. Azambuja, P., D. Feder, E.S. Garcia. Isolation of Serratia marcescens in the midgut of Rhodnius prolixus: impact on the establishment of the parasite Trypanosoma cruzi in the vector. Exp. Parasitol. 107, 89–96 (2004).

2. Castillo, A.W., M. Abal, S. Robine, T.R. Perez. Non-apoptotic concentrations of prodigiosin (H+/Cl- symporter) inhibit the acidification of lysosomes and induce cell cycle blockage in colon cancer cells. Life Sci. 78, 121-127 (2005).

3. Cho, L.K., J.A. Lowe, R.B. Maguire, J.C. Tsang. Relationship of prodigiosin condensing enzyme activity to the biosynthesis of prodigiosin and its precursors in Serratia marcescens. Experientia. 43, 397-399 (1987).

4. Cho, L.K.N., R.B. Maguire, J.C. Tsang. Location and detergent solubilization of prodigiosin condensing enzyme from cell envelopes of Serratia marcescens. Microbios. Letters. 37, 57-63 (1988).

5. Deol, B.S., J.R. Alden, J.L. Still, J. Winkler, A.V. Robertson. The isolation and characterization of monopyrroles from Serratia marcescens. Biochem. Biophys. Res. Commun. 47, 1378-1385 (1972).

6. Ding, M.J., R.P. Williams. Biosynthesis of prodigiosin by white strains of Serratia marcescens isolated from patients. J. Clin. Microbiol. 17, 476-480 (1983).

7. Dauenhauer, S.A., R.A. Hull, R.P. Williams. Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis. J. Bacteriol. 158, 1128–1132 (1984).

8. Demain, A. L. in Fifty Years of Antimicrobials: Past Perspectives and Future Trends (eds Hunter, P. A., Darby, G. K. & Russell, N. J.) 205–228 (Society for General Microbiology, Cambridge, 1995).

9. D''Alessio, R., A. Bargiotti, O. Carlini, F. Colotta, M. Ferrari, P. Gnocchi , A. Isetta, N. Mongelli, P. Motta, A. Rossi, M. Rossi, M. Tibolla, E. Vanotti. Synthesis and immunosuppressive activity of novel prodigiosin derivatives. J. Med. Chem. 43, 2557–2565 (2000).

10. Eaves, G.N., C.D. Jeffries. Isolation and properties of an exocellular nuclease of Serratia marcescens. J. Bacteriol. 85, 273–278 (1963).

11. Frstner, A. Chemistry and Biology of Roseophilin and the Prodigiosin Alkaloids: A Survey of the Last 2500 Years. Angew. Chem. Int. Ed. 42, 3582-3603 (2003).

12. Goldschmidt, M.C., R.P. Williams. Thiamine-induced formation of the monopyrrole moiety of prodigiosin. J. Bacteriol. 96, 609–616 (1968).

13. Gerber, N.N. Prodigiosin-like pigments. CRC Crit. Rev. Microbiol. 3, 469–485 (1975).

14. Grimont, P.A.D., F. Grimont. The genus Serratia. Annu. Rev. Microbiol. 32, 221-248 (1978).

15. Heinemann, B., A.J. Howard, H.J. Palocz. Influence of dissolved oxygen levels on production of L-asparaginase and prodigiosin by Serratia marcescens. Appl. Microbiol. 19, 5, 800-804 (1970).

16. Hejazi, A., F.R. Falkiner. Serratia marcescens. J. Med. Microbiol. 46, 903-12 (1997).

17. Harris, A.K., N.R. Williamson, H. Slater, A. Cox, S. Abbasi, I. Foulds, H.T. Simonsen, F.J. Leeper, G.P. Salmond. The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation. Microbiology. 150, 3547-3560 (2004).

18. Katz, D.S., R.J. Sobieski. Detection of Pigment Precursors in White Clinical Strains of Serratia marcescens. J. Clin. Microbiol. 9, 301-303 (1979).

19. Kobayashi, N., Y. Ichikawa. A protein associated with prodigiosin formation in Serratia marcescens. Microbiol. Immunol. 33, 257–263 (1989).

20. Mortellaro, A., S. Songia, P. Gnocchi, M. Ferrari, C. Fornasiero, R. D''Alessio, Isetta A, Colotta F, Golay J. New immunosuppressive drug PNU156804 blocks IL-2-dependent proliferation and NF-kappa B and AP-1 activation. J. Immunol. 162, 7102–7109 (1999).

21. Montaner, B., S. Navarro, M. Pique, M. Vilaseca, M. Martinell, E. Giralt, J. Gil, R. Perez - Tomas. Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Br. J. Pharmacol. 131, 585-593 (2000).

22. Manderville, R.A. Synthesis, Proton-Affinity and Anti-Cancer Properties of the Prodigiosin-Group Natural Products. Curr. Med. Chem. - Anti-Cancer Agents. 1, 195-218 (2001).

23. Purkayastha, M., R.P. Williams. Association of pigment with the cell envelope of Serratia marcescens (Chromobacterium prodigiosum). Nature. 187, 349–350 (1960).

24. Reid, J.D., D.M. Ogrydziak. Chitinase-overproducing mutant of Serratia marcescens. Appl. Environ. Microbiol. 41, 664-9 (1981).

25. Rosenberg, M. Isolation of Pigmented and Nonpigmented Mutants of Serratia marcescens with Reduced Cell Surface Hydrophobicity. J. Bacteriol. 160, 480-482 (1984).

26. Silverman, M.P., E.F. Munoz. Effect of iron and salt on prodigiosin synthesis in Serratia marcescens. J Bacteriol. 114, 999-1006 (1973).

27. Sole, M., N. Rius, A. Francia, J.G. Loren. The effect of pH on prodigiosin production by non-proliferating cells of Serratia marcescens. Lett. Appl. Microbiol. 19, 341-4 (1994).

28. Slater, H., M. Crow, L. Everson, G.P. Salmond. Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-dependent and -independent pathways. Mol. Microbiol. 47, 303–320 (2003).

29. Sunaga, S., H. Li, Y. Sato, Y. Nakagawa, T. Matsuyama. Identification and characterization of the pswP gene required for the parallel production of prodigiosin and serrawettin W1 in Serratia marcescens. Microbiol. Immunol. 48, 723–728 (2004).

30. Song, M.J., J. Bae, D.S. Lee, C.H. Kim, J.S. Kim, S.W. Kim, S.I. Hong .Purification and Characterization of Prodigiosin Produced by Integrated Bioreactor from Serratia sp. KH-95. J. Biosci. Bioeng. 101, 157-161 (2006).

31. Tsang, J.C., J.S. Feng. Effect of polymyxin B on the synthesis of prodigiosin and its precursors in Serratia marcescens. J. Antibiot. 36, 1564-1566 (1983).

32. Tsuji, R.F., M. Yamamoto, A. Nakamura, T. Kataoka, J. Magae, K. Nagai, M. Yamasaki. Selective immunosuppression of prodigiosin 25-C and FK506 in the murine immune system. J. Antibiot. 43, 1293-1301 (1990).

33. Williams, R,P,, C.L. Gott, S.M. Qadri. Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids. J. Bacteriol. 106, 444-8 (1971).

34. Williams, R.P. Biosynthesis of Prodigiosin, a Secondary Metabolite of Serratia marcescens. Appl. Microbiol. 25, 396-402 (1973).

35. Williams, R. P., S.M. Quadri. in The Genus Serratia (eds Von Graevenitz, A. & Rubin, S. J.) 31–75 (Boca Raron: CRC Press Inc, 1980).

36. Williamson, N.R., H.T. Simonsen, R.A. Ahmed, G. Goldet, H. Slater, L. Woodley, F.J. Leeper, G.P. Salmond. Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces. Mol. Microbiol. 56, 971–989 (2005).

37. Wei, Y.H., W.C. Chen. Enhanced production of prodigiosin-like pigment from Serratia marcescens SMΔR by medium improvement and oil-supplement strategies. J. Biosci. Bioeng. Vol. 99, No. 6, 616-622 (2005).

38. Wei, Y.H., W.J. Yu, W.C. Chen. Enhanced undecylprodigiosin production from Serratia marcescens SS-1 by medium formulation and amino-acid supplementation. J. Biosci. Bioeng. Vol. 100, No. 4, 466-471 ( 2005).

39. Williamson, N.R., H.T. Simonsen, A.K. Harris, F.J. Leeper, G.P. Salmond . Disruption of the copper efflux pump (CopA) of Serratia marcescens ATCC 274 pleiotropically affects copper sensitivity and production of the tripyrrole secondary metabolite, prodigiosin. J Ind Microbiol. Biotechnol. 33, 151-158 (2006).

40. Williamson, N.R., P.C. Fineran, F.J. Leeper, G.P. Salmond. The biosynthesis and regulation of bacterial prodiginines. Nat. Rev. Microbiol. 4, 887-899 (2006).

41. Yamashita, M., Y. Nakagawa, H. Li, T. Matsuyama. Silica gel- dependent production of prodigiosin and serrawettins by Serratia marcescens in a liquid culture. Microb. Environ. 16, 250-254 (2001).

42. 姚蕙芳：由Serratia marcescens FC-R1 生產紅色色素之研究，國立台灣大學農業化學研究所，碩士論文，(2000)。

43. 余宛儒：以Serratia marcescens C3生產天然抗癌藥物-靈菌紅素之醱酵製程開發，私立元智大學生物科技暨生物資訊研究所，碩士論文，(2006)。