希瓦氏菌 MR-1 為一格蘭氏兼性厭氧菌，因具有特殊的電子傳遞通道，能有效進行電子傳遞、金屬還原及偶氮化合物的降解，因此被廣泛應用於生物復育、微生物燃料電池及污水整治中。此菌株亦可於體內生產結構較複雜的細胞色素 c蛋白質，使其可作為特定蛋白質之表達宿主。本研究即基於希瓦氏菌之特殊性質，欲建立有效應的重組蛋白表達系統，藉此提升目標蛋白質的表達量，並改善其催化效率或增加高經濟價值化合物之產量，達成希瓦氏菌於工業應用的可行性。
本研究分別建立自發型與誘導型表達系統：自發型表達系統含有不同啟動子 (PLacI，PJ23100，PJ23105，PJ23109，PTet) 及複製原點 (pBR322及p15A)，並表達綠色螢光蛋白比較其產量，其中以 PLacI 啟動子與高拷貝數之 pBR322 複製原點具有最高螢光強度，達5950 a.u.。結果顯示啟動子與複製原點於希瓦氏菌對蛋白質表達的影響趨勢與大腸桿菌相同，但大腸桿菌常用之複製原點於 MR-1 內的複製量約為四分之一，MR-1中拷貝數分別是 p15A 的 34.9 及 pBR322 的 72.5。本研究首次整合T7 RNA 聚合酶至 MR-1 基因組，並構築 p28m 載體使質體含有 T7 啟動子，經由接合作用轉化至 MR-1達成T7誘導表達系統。由偶氮還原酶、紅色螢光蛋白及碳酸酐酶證實其可行性及蛋白質之功能與活性，其中RFP之螢光強度為原型菌株之 8 倍；含CA之菌株對於二氧化碳轉換活性最高可達 12106 WAU/mg，相較於原型菌株其效率皆有顯著提升。由以上結果可知，T7 表達系統可順利於希瓦氏菌運作，提升了蛋白質表達量及菌株的應用效率。
自發型表達系統後續應用於表達 Mtr 通道蛋白欲提升其電子傳遞效率，然後結果發現表達通道上單一蛋白反而會導致偶氮染料降解能力下降，甚至缺陷，因此對於 Mtr 通道蛋白的調控仍需更深入研究及複雜的調控方式。T7 表達系統則應用提升血基質合成途徑上之 Heme 蛋白，目標提升五胺基酮戊酸 (ALA) 之產量。結果發現於 24 小時 HemD、HemF 及 RsHemA 分別提升 ALA產量至2.16倍、1.13倍及1.33倍，但隨時間增加而下降，經由 qPCR 分析質體並未發現質體丟失，ALA生產不穩定性後續仍須探討及改善。綜合上述二類表達系統，本研究已成功建立了希瓦氏菌重組蛋白表達系統，後續將配合基因調控開拓更廣泛的應用。

In this study, constitutive expression system and inducible T7 system were established and applied to S. oneidensis MR-1. Promoters of PLacI, PJ23100, PJ23105, PJ23109 and PTet were first selected to combine with different replication origins (i.e., pBR322 and p15A) for constitutive expression, thus to find out the optimal orthogonal genetic modules. The highest protein expression was under PLacI promoter and pBR322 origin, reached 6000 a.u. fluorescence intensity. Also, there showed the same trend of promoters and replication origins in MR-1 and E. coli. In inducible T7 system, T7 RNA polymerase was inserted onto MR-1 genome by homologous recombination, and p28m vector was constructed with PT7 promoter and mobilization gene cluster. In this attempt, azo-reductase (AzoRI), red fluorescence protein (RFP) and carbonic anhydrase (CA) were over-expressed. The intensity of RFP in T7 system was almost 2000 a.u., 8 times increased, CO2 transferred in water was dramatically enhanced with CA expressed as 12106 WAU/mg, which showed the feasibility of T7 system in MR-1. The proteins on Mtr pathway were expressed by constitutive system to enhance the electron-transfer efficiency. However, the azo-declorization rate were dramatically decreaced when CymA and MtrA were over-expressed and there were no obvious difference of the ferric reduction activity between the two mutated strain and wild-type MR-1. The T7 system was used to increase the ALA productivity, and its production is 3.96-folds more than that of wild-type MR-1 when HemD is over-expressed.

[1] Ozawa, K., Tsapin, A. I., Nealson, K. H., Cusanovich, M. A., & Akutsu, H. (2000). Expression of a Tetraheme Protein, Desulfovibrio vulgaris Miyazaki F Cytochromec 3, in Shewanella oneidensis MR-1. Applied and Environmental Microbiology, 66(9), 4168-4171.
[2] Sybirna, K., Antoine, T., Lindberg, P., Fourmond, V., Rousset, M., Méjean, V., & Bottin, H. (2008). Shewanella oneidensis: a new and efficient system for expression and maturation of heterologous [Fe-Fe] hydrogenase from Chlamydomonas reinhardtii. BMC Biotechnology, 8(1), 73.
[3] Bretschger, O., Obraztsova, A., Sturm, C. A., Chang, I. S., Gorby, Y. A., Reed, S. B., Zhou, J, et al. (2007). Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants. Applied and Environmental Microbiology, 73(21), 7003-7012.
[4] Heidelberg, J. F., Paulsen, I. T., Nelson, K. E., Gaidos, E. J., Nelson, W. C., Read, T. D., Clayton, R. A., et al. (2002). Genome sequence of the dissimilatory metal ion–reducing bacterium Shewanella oneidensis. Nature Biotechnology, 20(11), 1118.
[5] Studier, F. W., & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology, 189(1), 113-130.
[6] Myers, C. R., & Nealson, K. H. (1988). Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor. Science, 240(4857), 1319-1321.
[7] Derby, H. A., & Hammer, B. W. (1931). Bacteriology of butter IV. Bacteriological studies on surface taint butter. Research Bulletin (Iowa Agriculture and Home Economics Experiment Station), 11(145), 1.
[8] Long, H. F., & Hammer, B. W. (1941). Distribution of Pseudomonas putrefaciens. Journal of Bacteriology, 41, 100-101.
[9] MacDonell, M. T., & Colwell, R. R. (1985). Phylogeny of the Vibrionaceae, and recommendation for two new genera, Listonella and Shewanella. Systematic and Applied Microbiology, 6(2), 171-182.
[10] Pitts, K. E., Dobbin, P. S., Reyes-Ramirez, F., Thomson, A. J., Richardson, D. J., & Seward, H. E. (2003). Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA expression in Escherichia coli confers the ability to reduce soluble Fe (III) chelates. Journal of Biological Chemistry, 278(30), 27758-27765.
[11] Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y., Cohen, B. E., Helms, B. A., Ajo-Franklin, C. M. , et al. (2010). Engineering of a synthetic electron conduit in living cells. Proceedings of the National Academy of Sciences, 107(45), 19213-19218.
[12] Hau, H. H., & Gralnick, J. A. (2007). Ecology and biotechnology of the genus Shewanella. Annual Review of Microbiology., 61, 237-258.
[13] Fredrickson, J. K., Romine, M. F., Beliaev, A. S., Auchtung, J. M., Driscoll, M. E., Gardner, T. S., Rodionov, D. A., et al. (2008). Towards environmental systems biology of Shewanella. Nature Reviews Microbiology, 6(8), 592.
[14] Xu, M., Guo, J., Cen, Y., Zhong, X., Cao, W., & Sun, G. (2005). Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant. International Journal of Systematic and Evolutionary Microbiology, 55(1), 363-368.
[15] Myers, C. R., & Nealson, K. H. (1990). Respiration-linked proton translocation coupled to anaerobic reduction of manganese (IV) and iron (III) in Shewanella putrefaciens MR-1. Journal of Bacteriology, 172(11), 6232-6238.
[16] Venkateswaran, K., Moser, D. P., Dollhopf, M. E., Lies, D. P., Saffarini, D. A., MacGregor, B. J., Burghardt, J. , et al. (1999). Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. International Journal of Systematic and Evolutionary Microbiology, 49(2), 705-724.
[17] Lies, D. P., Hernandez, M. E., Kappler, A., Mielke, R. E., Gralnick, J. A., & Newman, D. K. (2005). Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for biofilms. Applied and Environmental Microbiology, 71(8), 4414-4426.
[18] Marshall, M. J., Beliaev, A. S., Dohnalkova, A. C., Kennedy, D. W., Shi, L., Wang, Z., Reed, S. B., et al. (2006). c-Type cytochrome-dependent formation of U (IV) nanoparticles by Shewanella oneidensis. PLoS Biology, 4(8), e268.
[19] Gorby, Y. A., Yanina, S., McLean, J. S., Rosso, K. M., Moyles, D., Dohnalkova, A., Culley, D. E., et al. (2006). Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proceedings of the National Academy of Sciences, 103(30), 11358-11363.
[20] El-Naggar, M. Y., Wanger, G., Leung, K. M., Yuzvinsky, T. D., Southam, G., Yang, J., Gorby, Y. A., et al. (2010). Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1. Proceedings of the National Academy of Sciences, 107(42), 18127-18131.
[21] Pirbadian, S., Barchinger, S. E., Leung, K. M., Byun, H. S., Jangir, Y., Bouhenni, R. A., Gorby, Y. A., et al. (2014). Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components. Proceedings of the National Academy of Sciences, 111(35), 12883-12888.
[22] Lovley, D. R. (2012). Electromicrobiology. Annual Review of Microbiology, 66, 391-409.
[23] Harris, H. W., El-Naggar, M. Y., Bretschger, O., Ward, M. J., Romine, M. F., Obraztsova, A. Y., & Nealson, K. H. (2010). Electrokinesis is a microbial behavior that requires extracellular electron transport. Proceedings of the National Academy of Sciences, 107(1), 326-331.
[24] Ma, C., Zhou, S. G., Zhuang, L., & Wu, C. Y. (2011). Electron transfer mechanism of extracellular respiration: a review. Acta Ecologica Sinica, 31(7), 2008-2018.
[25] Marsili, E., Baron, D. B., Shikhare, I. D., Coursolle, D., Gralnick, J. A., & Bond, D. R. (2008). Shewanella secretes flavins that mediate extracellular electron transfer. Proceedings of the National Academy of Sciences, 105(10), 3968-3973.
[26] Von Canstein, H., Ogawa, J., Shimizu, S., & Lloyd, J. R. (2008). Secretion of flavins by Shewanella species and their role in extracellular electron transfer. Applied and Environmental Microbiology, 74(3), 615-623.
[27] Myers, C. R., & Myers, J. M. (1997). Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron (III), fumarate, and nitrate by Shewanella putrefaciens MR-1. Journal of Bacteriology, 179(4), 1143-1152.
[28] Beliaev, A. S., & Saffarini, D. A. (1998). Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe (III) and Mn (IV) reduction. Journal of Bacteriology, 180(23), 6292-6297.
[29] Schuetz, B., Schicklberger, M., Kuermann, J., Spormann, A. M., & Gescher, J. (2009). Periplasmic electron transfer via the c-type cytochromes MtrA and FccA of Shewanella oneidensis MR-1. Applied and Environmental Microbiology, 75(24), 7789-7796.
[30] Shi, L., Squier, T. C., Zachara, J. M., & Fredrickson, J. K. (2007). Respiration of metal (hydr) oxides by Shewanella and Geobacter: a key role for multihaem c‐type cytochromes. Molecular Microbiology, 65(1), 12-20.
[31] Myers, C. R., & Myers, J. M. (2002). MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1. Applied and Environmental Microbiology, 68(11), 5585-5594.
[32] Beliaev, A. S., Saffarini, D. A., McLaughlin, J. L., & Hunnicutt, D. (2001). MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR‐1. Molecular Microbiology, 39(3), 722-730.
[33] Reardon, C. L., Dohnalkova, A. C., Nachimuthu, P., Kennedy, D. W., Saffarini, D. A., Arey, B. W., Moyles, D. (2010)., et al. Role of outer‐membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR‐1. Geobiology, 8(1), 56-68.
[34] Potter, M. C. (1911). Electrical effects accompanying the decomposition of organic compounds. Proceedings of the Royal Society of London B, 84(571), 260-276.
[35] Liu, H., & Logan, B. E. (2004). Electricity generation using an air-cathode single chamber microbial fuel cell in the presence and absence of a proton exchange membrane. Environmental Science & Technology, 38(14), 4040-4046.
[36] Bennetto, H. P., Box, J., Delaney, G. M., Mason, J. R., Roller, S. D., Stirling, J. L., & Thurston, C. F. (1987). Redox-mediated electrochemistry of whole microorganisms: from fuel cells to biosensors. Biosensors, TURNER, APF, KARUBE, I., WILSON, GS (Hrsg.) Oxford University Press, Oxford, 291-314.
[37] Kim, H. J., Park, H. S., Hyun, M. S., Chang, I. S., Kim, M., & Kim, B. H. (2002). A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. Enzyme and Microbial Technology, 30(2), 145-152.
[38] Kim, H. J., Hyun, M. S., Chang, I. S., & Kim, B. H. (1999). A microbial fuel cell type lactate biosensor using a metal-reducing bacterium, Shewanella putrefaciens. Journal of Microbiology and Biotechnology, 9(3), 365-367.
[39] Ringeisen, B. R., Henderson, E., Wu, P. K., Pietron, J., Ray, R., Little, B., Jones-Meehan, J. M., et al. (2006). High power density from a miniature microbial fuel cell using Shewanella oneidensis DSP10. Environmental Science & Technology, 40(8), 2629-2634.
[40] Lovley, D. R. (1991). Dissimilatory Fe (III) and Mn (IV) reduction. Microbiological Reviews, 55(2), 259-287.
[41] Coursolle, D., Baron, D. B., Bond, D. R., & Gralnick, J. A. (2010). The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. Journal of Bacteriology, 192(2), 467-474.
[42] Myers, J. M., & Myers, C. R. (2001). Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide. Applied and Environmental Microbiology, 67(1), 260-269.
[43] Caccavo, F., Blakemore, R. P., & Lovley, D. R. (1992). A hydrogen-oxidizing, Fe (III)-reducing microorganism from the Great Bay Estuary, New Hampshire. Applied and Environmental Microbiology, 58(10), 3211-3216.
[44] Wielinga, B., Mizuba, M. M., Hansel, C. M., & Fendorf, S. (2001). Iron promoted reduction of chromate by dissimilatory iron-reducing bacteria. Environmental Science & Technology, 35(3), 522-527.
[45] Xu, M., Guo, J., Zeng, G., Zhong, X., & Sun, G. (2006). Decolorization of anthraquinone dye by Shewanella decolorationis S12. Applied Microbiology and Biotechnology, 71(2), 246-251.
[46] Hong, Y., Chen, X., Guo, J., Xu, Z., Xu, M., & Sun, G. (2007). Effects of electron donors and acceptors on anaerobic reduction of azo dyes by Shewanella decolorationis S12. Applied Microbiology and Biotechnology, 74(1), 230-238.
[47] Yang, Y. Y., Du, L. N., Wang, G., Jia, X. M., & Zhao, Y. H. (2011). The decolorisation capacity and mechanism of Shewanella oneidensis MR-1 for methyl orange and acid yellow 199 under microaerophilic conditions. Water Science and Technology, 63(5), 956-963.
[48] Pearce, C. I., Christie, R., Boothman, C., von Canstein, H., Guthrie, J. T., & Lloyd, J. R. (2006). Reactive azo dye reduction by Shewanella strain J18 143. Biotechnology and Bioengineering, 95(4), 692-703.
[49] Khalid, A., Arshad, M., & Crowley, D. E. (2008). Decolorization of azo dyes by Shewanella sp. under saline conditions. Applied Microbiology and Biotechnology, 79(6), 1053-1059.
[50] Sun, J., Bi, Z., Hou, B., Cao, Y. Q., & Hu, Y. Y. (2011). Further treatment of decolorization liquid of azo dye coupled with increased power production using microbial fuel cell equipped with an aerobic biocathode. Water Research, 45(1), 283-291.
[51] Dodson, J. R., Parker, H. L., García, A. M., Hicken, A., Asemave, K., Farmer, T. J., Hunt, A. J., et al. (2015). Bio-derived materials as a green route for precious & critical metal recovery and re-use. Green Chemistry, 17(4), 1951-1965.
[52] Mata, Y. N., Torres, E., Blazquez, M. L., Ballester, A., González, F. M. J. A., & Munoz, J. A. (2009). Gold (III) biosorption and bioreduction with the brown alga Fucus vesiculosus. Journal of Hazardous Materials, 166(2-3), 612-618.
[53] He, Y. R., Cheng, Y. Y., Wang, W. K., & Yu, H. Q. (2015). A green approach to recover Au (III) in aqueous solution using biologically assembled rGO hydrogels. Chemical Engineering Journal, 270, 476-484.
[54] Bai, H., Zhang, Z., Guo, Y., & Jia, W. (2009). Biological Synthesis of Size-Controlled Cadmium Sulfide Nanoparticles Using Immobilized Rhodobacter sphaeroides. Nanoscale Research Letters, 4(7), 717.
[55] Windt, W. D., Aelterman, P., & Verstraete, W. (2005). Bioreductive deposition of palladium (0) nanoparticles on Shewanella oneidensis with catalytic activity towards reductive dechlorination of polychlorinated biphenyls. Environmental Microbiology, 7(3), 314-325.
[56] Konishi, Y., Ohno, K., Saitoh, N., Nomura, T., Nagamine, S., Hishida, H., Uruga, T., et al. (2007). Bioreductive deposition of platinum nanoparticles on the bacterium Shewanella algae. Journal of Biotechnology, 128(3), 648-653.
[57] Marshall, M. J., Beliaev, A. S., Dohnalkova, A. C., Kennedy, D. W., Shi, L., Wang, Z., Reed, S. B., et al. (2006). c-Type cytochrome-dependent formation of U (IV) nanoparticles by Shewanella oneidensis. PLoS Biology, 4(8), e268.
[58] Suresh, A. K., Pelletier, D. A., Wang, W., Broich, M. L., Moon, J. W., Gu, B., Doktycz, M. J., et al. (2011). Biofabrication of discrete spherical gold nanoparticles using the metal-reducing bacterium Shewanella oneidensis. Acta Biomaterialia, 7(5), 2148-2152.
[59] Konishi, Y., Tsukiyama, T., Ohno, K., Saitoh, N., Nomura, T., & Nagamine, S. (2006). Intracellular recovery of gold by microbial reduction of AuCl4− ions using the anaerobic bacterium Shewanella algae. Hydrometallurgy, 81(1), 24-29.
[60] Wang, H., Law, N., Pearson, G., Van Dongen, B. E., Jarvis, R. M., Goodacre, R., & Lloyd, J. R. (2010). Impact of silver (I) on the metabolism of Shewanella oneidensis. Journal of Bacteriology, 192(4), 1143-1150.
[61] Wu, R., Cui, L., Chen, L., Wang, C., Cao, C., Sheng, G., Zhao, F., et al. (2013). Effects of bio-Au nanoparticles on electrochemical activity of Shewanella oneidensis wild type and ΔomcA/mtrC mutant. Scientific Reports, 3, 3307.
[62] Ng, C. K., Sivakumar, K., Liu, X., Madhaiyan, M., Ji, L., Yang, L., Cao, B., et al. (2013). Influence of outer membrane c‐type cytochromes on particle size and activity of extracellular nanoparticles produced by Shewanella oneidensis. Biotechnology and Bioengineering, 110(7), 1831-1837.
[63] Venkateswaran, K., Moser, D. P., Dollhopf, M. E., Lies, D. P., Saffarini, D. A., MacGregor, B. J., Burghardt, J., et al. (1999). Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. International Journal of Systematic and Evolutionary Microbiology, 49(2), 705-724.
[64] Konstantinidis, K. T., Serres, M. H., Romine, M. F., Rodrigues, J. L., Auchtung, J., McCue, L. A., Fredrickson, J. K., et al. (2009). Comparative systems biology across an evolutionary gradient within the Shewanella genus. Proceedings of the National Academy of Sciences, 106(37), 15909-15914.
[65] Saffarini, D. A., & Nealson, K. H. (1993). Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1. Journal of Bacteriology, 175(24), 7938-7944.
[66] Murray, A. E., Lies, D., Li, G., Nealson, K., Zhou, J., & Tiedje, J. M. (2001). DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes. Proceedings of the National Academy of Sciences, 98(17), 9853-9858.
[67] Beliaev, A. S., Klingeman, D. M., Klappenbach, J. A., Wu, L., Romine, M. F., Tiedje, J. M., ... & Zhou, J. (2005). Global transcriptome analysis of Shewanella oneidensis MR-1 exposed to different terminal electron acceptors. Journal of Bacteriology, 187(20), 7138-7145.
[68] Song, W., Juhn, F. S., Naiman, D. Q., Konstantinidis, K. T., Gardner, T. S., & Ward, M. J. (2008). Predicting σ28 promoters in eleven Shewanella genomes. FEMS Microbiology Letters, 283(2), 223-230.
[69] Gao, H., Wang, X., Yang, Z. K., Palzkill, T., & Zhou, J. (2008). Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses. BMC Genomics, 9(1), 42.
[70] Rodionov, D. A., Novichkov, P. S., Stavrovskaya, E. D., Rodionova, I. A., Li, X., Kazanov, M. D., Permina, E. A., et al. (2011). Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus. BMC Genomics, 12(1), S3.
[71] Kasai, T., Kouzuma, A., Nojiri, H., & Watanabe, K. (2015). Transcriptional mechanisms for differential expression of outer membrane cytochrome genes omcA and mtrC in Shewanella oneidensis MR-1. BMC Microbiology, 15(1), 68.
[72] Kim, J. S. (2016). Genome editing comes of age. Nature Protocols, 11(9), 1573.
[73] Nakashima, N., & Miyazaki, K. (2014). Bacterial cellular engineering by genome editing and gene silencing. International Journal of Molecular Sciences, 15(2), 2773-2793.
[74] Zimmermann, A., Reimmann, C., Galimand, M., & Haas, D. (1991). Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli. Molecular Microbiology, 5(6), 1483-1490.
[75] Myers, J. M., & Myers, C. R. (2000). Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone. Journal of Bacteriology, 182(1), 67-75.
[76] Gao, W., Liu, Y., Giometti, C. S., Tollaksen, S. L., Khare, T., Wu, L., & Zhou, J., et al. (2006). Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1. BMC Genomics, 7(1), 76.
[77] Burns, J. L., & DiChristina, T. J. (2009). Anaerobic respiration of elemental sulfur and thiosulfate by Shewanella oneidensis MR-1 requires psrA, a homolog of the phsA gene of Salmonella enterica serovar typhimurium LT2. Applied and Environmental Microbiology, 75(16), 5209-5217.
[78] Gao, H., Yang, Z. K., Barua, S., Reed, S. B., Romine, M. F., Nealson, K. H., Zhou, J. et al. (2009). Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA. The ISME Journal, 3(8), 966.
[79] Chen, X., Sun, G., & Xu, M. (2011). Role of iron in azoreduction by resting cells of Shewanella decolorationis S12. Journal of Applied Microbiology, 110(2), 580-586.
[80] Qiu, D. (2010). Comparative genomics analysis and phenotypic characterization of Shewanella putrefaciens W3-18-1: Anaerobic respiration, bacterial microcompartments, and lateral flagella.
[81] Pyne, M. E., Moo-Young, M., Chung, D. A., & Chou, C. P. (2015). Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli. Applied and Environmental Microbiology, 81(15), 5103-5114.
[82] Myers, C. R., & Myers, J. M. (1997). Replication of plasmids with the p15A origin in Shewanella putrefaciens MR‐1. Letters in Applied Microbiology, 24(3), 221-225.
[83] Ozawa, K., Yasukawa, F., Fujiwara, Y., & AKUTSU, H. (2001). A simple, rapid, and highly efficient gene expression system for multiheme cytochromes c. Bioscience, Biotechnology, and Biochemistry, 65(1), 185-189.
[84] Rachkevych, N., Sybirna, K., Boyko, S., Boretsky, Y., & Sibirny, A. (2014). Improving the efficiency of plasmid transformation in Shewanella oneidensis MR-1 by removing ClaI restriction site. Journal of Microbiological Methods, 99, 35-37.
[85] Hajimorad, M., & Gralnick, J. A. (2016). Towards enabling engineered microbial-electronic systems: RK2-based conjugal transfer system for Shewanella synthetic biology. Electronics Letters, 52(6), 426-428.
[86] Guiney, D. G., & Yakobson, E. (1983). Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2. Proceedings of the National Academy of Sciences, 80(12), 3595-3598.
[87] Szpirer, C. Y., Faelen, M., & Couturier, M. (2001). Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1. Journal of Bacteriology, 183(6), 2101-2110.
[88] VerBerkmoes, N. C., Bundy, J. L., Hauser, L., Asano, K. G., Razumovskaya, J., Larimer, F., Stephenson, J. L., et al. (2002). Integrating “top-down” and “bottom-up” mass spectrometric approaches for proteomic analysis of Shewanella oneidensis. Journal of Proteome Research, 1(3), 239-252.
[89] Romine, M. F., Elias, D. A., Monroe, M. E., Auberry, K., Fang, R., Fredrickson, J. K., Lipton, M. S., et al. (2004). Validation of Shewanella oneidensis MR-1 small proteins by AMT tag-based proteome analysis. Omics: a Journal of Integrative Biology, 8(3), 239-254.
[90] Gao, H., Pattison, D., Yan, T., Klingeman, D. M., Wang, X., Petrosino, J., Palzkill, T., et al. (2008). Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display. PLoS One, 3(8), e2983.
[91] Schicklberger, M., Bücking, C., Schuetz, B., Heide, H., & Gescher, J. (2011). Involvement of the Shewanella oneidensis decaheme cytochrome MtrA in the periplasmic stability of the β-barrel protein MtrB. Applied and Environmental Microbiology, 77(4), 1520-1523.
[92] Ross, D. E., Ruebush, S. S., Brantley, S. L., Hartshorne, R. S., Clarke, T. A., Richardson, D. J., & Tien, M. (2007). Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. Applied and Environmental Microbiology, 73(18), 5797-5808.
[93] Beliaev, A. S., Thompson, D. K., Khare, T., Lim, H., Brandt, C. C., Li, G., Nealson, K. H., et al. (2002). Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors. Omics: a Journal of Integrative Biology, 6(1), 39-60.
[94] Cao, Y., Li, X., Li, F., & Song, H. (2017). CRISPRi–sRNA: Transcriptional–Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis. ACS Synthetic Biology, 6(9), 1679-1690.
[95] Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172.
[96] Choi, D., Lee, S. B., Kim, S., Min, B., Choi, I. G., & Chang, I. S. (2014). Metabolically engineered glucose-utilizing Shewanella strains under anaerobic conditions. Bioresource Technology, 154, 59-66.
[97] Liu, T., Yu, Y. Y., Deng, X. P., Ng, C. K., Cao, B., Wang, J. Y., Song, H., et al. (2015). Enhanced Shewanella biofilm promotes bioelectricity generation. Biotechnology and Bioengineering, 112(10), 2051-2059.
[98] Yang, Y., Ding, Y., Hu, Y., Cao, B., Rice, S. A., Kjelleberg, S., & Song, H. (2015). Enhancing bidirectional electron transfer of Shewanella oneidensis by a synthetic flavin pathway. ACS Synthetic Biology, 4(7), 815-823.
[99] Min, D., Cheng, L., Zhang, F., Huang, X. N., Li, D. B., Liu, D. F., Yu, H. Q., et al. (2017). Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing Conduit for Pollutant Degradation. Environmental Science & Technology, 51(9), 5082-5089.
[100] Gardner, L. C., & Cox, T. M. (1988). Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron. Journal of Biological Chemistry, 263(14), 6676-6682.
[101] Von Wettstein, D., Gough, S., & Kannangara, C. G. (1995). Chlorophyll biosynthesis. The Plant Cell, 7(7), 1039.
[102] Liu, et al. (2014) "Microbial production and applications of 5-aminolevulinic acid." Applied Microbiology and Biotechnology, 98.17, 7349-7357.
[103] Sasaki, K., Watanabe, M., & Tanaka, T. (2002). Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid. Applied Microbiology and Biotechnology, 58(1), 23-29.
[104] Beck, T. J., Kreth, F. W., Beyer, W., Mehrkens, J. H., Obermeier, A., Stepp, H., Baumgartner, R., et al. (2007). Interstitial photodynamic therapy of nonresectable malignant glioma recurrences using 5‐aminolevulinic acid induced protoporphyrin IX. Lasers in Surgery and Medicine, 39(5), 386-393.
[105] Bhowmick, R., & Girotti, A. W. (2010). Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy. Free Radical Biology and Medicine, 48(10), 1296-1301.
[106] Rebeiz, C. A., Montazer-Zouhoor, A., Hopen, H. J., & Wu, S. M. (1984). Photodynamic herbicides: 1. Concept and phenomenology. Enzyme and microbial technology, 6(9), 390-396.
[107] Tanaka, T. (1992). Promotive effects of 5-aminolevulinic acid on yield of several crops. Proc. 19th Ann. Meet. Plant Growth Regulator soc. of America, 237-242.
[108] Kuramochi, H., Konnai, M., Tanaka, T., & Hotta, Y. (1997). U.S. Patent No. 5,661,111. Washington, DC: U.S. Patent and Trademark Office.
[109] Sasaki, K., Tanaka, T., & Nagai, S. (1998). Use of photosynthetic bacteria for the production of SCP and chemicals from organic wastes. In Bioconversion of Waste Materials to Industrial Products (pp. 247-292). Springer, Boston, MA.
[110] Choorit, W., Saikeur, A., Chodok, P., Prasertsan, P., & Kantachote, D. (2011). Production of biomass and extracellular 5-aminolevulinic acid by Rhodopseudomonas palustris KG31 under light and dark conditions using volatile fatty acid. Journal of Bioscience and Bioengineering, 111(6), 658-664.
[111] Saikeur, A., Choorit, W., Prasertsan, P., Kantachote, D., & Sasaki, K. (2009). Influence of precursors and inhibitor on the production of extracellular 5-aminolevulinic acid and biomass by Rhodopseudomonas palustris KG31. Bioscience, Biotechnology, and Biochemistry, 73(5), 987-992.
[112] Tangprasittipap, A., Prasertsan, P., Choorit, W., & Sasaki, K. (2007). Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant Rhodobacter sphaeroides. Biotechnology Letters, 29(5), 773-778.
[113] Weinstein, J. D., & Beale, S. I. (1983). Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis. Journal of Biological Chemistry, 258(11), 6799-6807.
[114] Yang, P., Liu, W., Cheng, X., Wang, J., Wang, Q., & Qi, Q. (2016). A new strategy for production of 5-aminolevulinic acid in recombinant Corynebacterium glutamicum with high yield. Applied and Environmental Microbiology, 82(9), 2709-2717.
[115] Gibson K, Laver W, Neuberger A (1958) Initial stages in the biosynthesis of porphyrins. 2. The formation of δ-aminolaevulic acid from glycine and succinyl-coenzyme A by particles from chicken erythrocytes. Biochem J 70:71–81
[116] Sasikala C, Ramana CV, Rao PR (1994) 5-Aminolevulinic acid: a potential herbicide/insecticide from microorganisms. Biotechnol Progr 10:451–459
[117] Hornberger, U., Liebetanz, R., Tichy, H. V., & Drews, G. (1990). Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain. Molecular and General Genetics MGG, 221(3), 371-378.
[118] Drolet, M., & Sasarman, A. (1991). Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter. Molecular and General Genetics MGG, 226(1-2), 250-256.
[119] Leong, S. A., Williams, P. H., & Ditta, G. S. (1985). Analysis of the 5’-regulatory region of the gene for δ-aminolevulinic acid synthetase of Rhizobium meliloti. Nucleic Acids Research, 13(16), 5965-5976.
[120] Alexander, F. W., Sandmeier, E., Mehta, P. K., & Christen, P. (1994). Evolutionary relationships among pyridoxal‐5’‐phosphate‐dependent enzymes. The FEBS Journal, 219(3), 953-960.
[121] Astner, I., Schulze, J. O., Van den Heuvel, J., Jahn, D., Schubert, W. D., & Heinz, D. W. (2005). Crystal structure of 5‐aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans. The EMBO Journal, 24(18), 3166-3177.
[122] Schauer, S., Chaturvedi, S., Randau, L., Moser, J., Kitabatake, M., Lorenz, S., Wall, K., et al. (2002). Escherichia coli glutamyl-tRNA reductase trapping the thioester intermediate. Journal of Biological Chemistry, 277(50), 48657-48663.
[123] Wang, L., Wilson, S., & Elliott, T. (1999). A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium. Journal of Bacteriology, 181(19), 6033-6041.
[124] Kang, Z., Wang, Y., Gu, P., Wang, Q., & Qi, Q. (2011). Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose. Metabolic Engineering, 13(5), 492-498.
[125] Chung, S. Y., Seo, K. H., & Rhee, J. I. (2005). Influence of culture conditions on the production of extra-cellular 5-aminolevulinic acid (ALA) by recombinant E. coli. Process Biochemistry, 40(1), 385-394.
[126] Zhang, J., Kang, Z., Chen, J., & Du, G. (2015). Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli. Scientific Reports, 5, 8584.
[127] Feng, L., Zhang, Y., Fu, J., Mao, Y., Chen, T., Zhao, X., & Wang, Z. (2016). Metabolic engineering of Corynebacterium glutamicum for efficient production of 5‐aminolevulinic acid. Biotechnology and Bioengineering, 113(6), 1284-1293.
[128] Shemin, D., & Russell, C. S. (1953). δ-AMINOLEVULINIC ACID, ITS ROLE IN THE BIOSYNTHESIS OF PORPHYRINS AND PURINES1. Journal of the American Chemical Society, 75(19), 4873-4874.
[129] Beale, S. I. (1971). Studies on the biosynthesis and metabolism of δ-aminolevulinic acid in Chlorella. Plant Physiology, 48(3), 316-319.
[130] Van Der Werf, M. J., & Zeikus, J. G. (1996). 5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene. Applied and Environmental Microbiology, 62(10), 3560-3566.
[131] Van Der Werf, M. J., & Zeikus, J. G. (1996). 5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene. Applied and Environmental Microbiology, 62(10), 3560-3566.
[132] Lin, J., Fu, W., & Cen, P. (2009). Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production. Bioresource Technology, 100(7), 2293-2297.
[133] Noh, M. H., Lim, H. G., Park, S., Seo, S. W., & Jung, G. Y. (2017). Precise flux redistribution to glyoxylate cycle for 5-aminolevulinic acid production in Escherichia coli. Metabolic Engineering, 43, 1-8.
[134] Malik, Z., & Djaldetti, M. (1979). 5-aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced friend erythroleukemic cells. Cell Differentiation, 8(3), 223-233.
[135] Wagnières, G., Jichlinski, P., Lange, N., Kucera, P., & van den Bergh, H. (2014). Detection of bladder cancer by fluorescence cystoscopy: from bench to bedside-the hexvix story (No. EPFL-BOOK-196444). Taylor & Francis.
[136] Wilbur, K. M., & Anderson, N. G. (1948). Electrometric and colorimetric determination of carbonic anhydrase. Journal of Biological Chemistry, 176(1), 147-154.
[137] Segall-Shapiro, T. H., Sontag, E. D., & Voigt, C. A. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. Nature Biotechnology, 36(4), 352.
[138] Mauzerall, D., & Granick, S. (1956). The occurrence and determination of δ-aminolevulinic acid and porphobilinogen in urine. Journal of Biological Chemistry, 219(1), 435-446.
[139] Sanders, C., Turkarslan, S., Lee, D. W., & Daldal, F. (2010). Cytochrome c biogenesis: the Ccm system. Trends in Microbiology, 18(6), 266-274.
[140] Milewska, K., Węgrzyn, G., & Szalewska-Pałasz, A. (2015). Transformation of Shewanella baltica with ColE1-like and P1 plasmids and their maintenance during bacterial growth in cultures. Plasmid, 81, 42-49.