1. 毕业设计（论文）的内容和要求

本课题通过在大肠杆菌中引入异源四氢嘧啶合成基因簇，构建异源四氢嘧啶生产菌，并对具有较高四氢嘧合成能力的菌株进行进一步的发酵过程调控及产酸途径改造，以减少乙酸的产生及积累，提高碳源的利用率，从而降低生产成本和获得高产量的四氢嘧啶。

要求：学生自主查阅文献，完成文献翻译，制定初步的实验方案；在老师指导下完成实验内容，掌握数据处理和统计的方法；完成毕业论文撰写和答辩PPT的制作。

2. 参考文献

[1] Galinski EA, Pfeiffer HP, Truper HG. 1,4,5,6-tetrahydro-2-methyl-4- pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira [J]. European Journal of Biochemistry, 1985, 149: 135一139. [2]Arahal et al., 2001 D.R. Arahal, M.T. Garc#237;a, C. Vargas, D. C#225;novas, J.J. Nieto, A. Ventosa. Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174 [J]. Int. J. Syst. Evolut. Microbiol., 51 (2001), pp. 1457-1462 [3]C#225;novas et al., 1997 D. C#225;novas, C. Vargas, F. Iglesias-Guerra, L.N. Csonka, D. Rhodes, A. Ventosa, J.J. Nieto. Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes [J]. J. Biol. Chem., 272 (1997), pp. 25794-25801 [4]Peters et al., 1990 P. Peters, E.A. Galinski, H.G. Tr#252;per. The biosynthesis of ectoine [J]. FEMS Microbiol. Lett., 71 (1990), pp. 157-162 [5] Anne UK, Erhard B. Osmotically regulated synthesis of the compatible solute ectoine Bacillus pasteurii and related Bacillus spp [J]. Applied and Environmental Microbiology, 2002 772-783. [6] Mark AM, Raymond SN, Lesley JB. Organic osmoregulatory solutes in cyanobacteria [J]. General. Microbiology, 1984, 130: 2177-2191. [7] Ronit R, Irena P, Hagai G, et al. 13C NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria [J]. Archives of Biochemistry and Biophysics, 1990, 278: 106-112. [8]黄红英，刘爱民，张林普.真细菌的渗透调节机制[J].微生物学通报，1999 26(3): 218-220. [9]朱道辰，刘杏荣.相容性溶质四氢嚓陡及其羚基化衍生物的研究进展 [J].中国生物工程杂志,2011.2: 95-101. [10] Galinski EA,Lippert K.Novel compatible solutes and their potential application aspects as stabilizers in enzyme technology.Rodriguez-Valera F,ed. General and applied of halophilic micro-organisms [J]. New York, NY: Plenum Press, 1991:351-358. [11]Louis P, Truper HG, Galinski EA. Survival of Escherichia coli during drying and storage in the presence of compatible solutes [J]. Applied Microbiology and Biotechnology, 1994, 41:648-688. [12]何建.甜菜碱和四氢嘧啶提高Psezrdomonas putida KT2440耐盐性的分子机理研究[D].南京:南京农业大学，2005. [13]徐蕊.四氢嘧啶合成工程菌株构建[D].大连:大连海事大学，2012. [14] Zhang LH, Wang Y, Zhang C, et al. Supplementation Effect of Ectoine on Thermostability of Phytase [J]. Journal Of Bioscience And Bioengineering, 2006, 102(6): 560-563. [15] Zhang LH, Lang YJ, Wang CX, et al. Promoting effect of compatible solute ectoine on the ethanol fermentation by Zymomonas mobilis CICC10232 [J] Process Biochemistry, 2008, 43: 642-646. [16] Mathumai K, Georg L, Michael S, et al. Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzhermer'sp-amyloid [J]. FEBS Letters, 2005, 579: 4775-4780. [17] Schnoor M, Voss P, Cullen P et al. Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer [J]. Biochemical and Biophysics Research Communications,2004,322:867-872. [18] Antonio V, Joaquin J, Nieto A 0. Biology of moderately halophilic aerobic bacteria [J]. Microbiol Mol Biol Rev, 1998, 62 (2):504-544. [19]Pastor et al., 2010 J.M. Pastor, M. Salvador, M. Argandontilde;a, V. Bernal, M. Reina-Bueno, L.N. Csonka, M. C#225;novas. Ectoines in cell stress protection: uses and biotechnological production [J]. Biotechnol. Adv., 28 (2010), pp. 782-801 [20]闫喜文.四氢嘧啶制备技术研究[D]. .大连:大连海事大学，2011. [21] Shinichi Nagata, Yaoqiang, Akinobu Oshima, et al. Efficient cyclic system to yield ectoine using Brevibacterlum sp. JCM 6894 subjected to osmotic downshock [J]. Bio-technology and bioengineering, 2008, 99(4):941-948. [22] Ning Y, Wu X, Zhang C, et al. Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli [J]. Metab Eng, 2016, 36: 10-18. [23]陈宁.氨基酸发酵工艺学[M].北京:中国轻工业出版社，2007: 346-355. [24]张伟国.氨基酸生产技术及其应用[M].北京:中国轻工业出版社，1997: 23-45. [25]张克旭，陈宁，等.代谢控制发酵[M].北京:中国轻工业出版社，1998: 25-53.

3. 毕业设计（论文）进程安排

2019.3.15-2019.4.15 四氢嘧啶合成基因簇的克隆表达 2019.4.16-2019.4.30 代谢工程大肠杆菌的构建与发酵条件优化 2019.5.1-2019.5.30 重组大肠杆菌产酸途径改造 2019.6.1-2019.06.10 整理数据、撰写论文，准备论文答辩