1. 毕业设计（论文）的内容和要求

在大肠杆菌生产琥珀酸的过程中，随着发酵的进行，发酵液的ph逐渐降低，为了维持最适生长环境，必须添加大量的碱，但也带来了高渗透压的问题。

细胞膜是保护微生物的第一道屏障，改变细胞膜组分可提高细胞对多种胁迫环境的耐受性能。

本课题拟通过过量表达可以改变细胞膜组分的关键性基因，提高菌株对低ph以及高浓度丁二酸的耐受性，从而提高大肠杆菌发酵产琥珀酸的生长性能以及生产强度。

2. 参考文献

（1）Lancaster, J.R., Jr., Steyn, A.J., 2007. Mycobacterium tuberculosis WhiB3 responds to O2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival. Proc. Natl. Acad. Sci. USA 104, 11562#8211;11567. （2）Steyn, A.J., Collins, D.M., Hondalus, M.K., Jacobs, W.R., Jr., Kawakami, R.P., Bloom,B.R., 2002. Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth. Proc. Natl. Acad. Sci. USA 99, 3147#8211;3152. （3）Mycobacterium tuberculosis H37Rv: a [4Fe-4S] cluster co-ordianting protein isulphide reductase. Mol. Microbiol. 63, 1414#8211;1431. （4）Alam, M.S., Garg, S.K., Agrawal, P., 2009. Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37Rv. FEBS J. 276, 76#8211;93. （5）Andersson, C., Helmerius, J., Hodge, D., Berglund, K.A., Rova, U., 2009. Inhibition of succinic acid production in metabolically engineered Escherichia coli by neutralizing agent, organic acids, and osmolarity. Biotechnol. Prog. 25, 116#8211;123. （6）Choi, W.W., Park, S.D., Lee, S.M., Kim, H.B., Kim, Y., Lee, H.S., 2009. The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum. FEMS Microbiol. Lett. 290, 32#8211;38. （7）Glassner, D.A, Datta, R., 1992. Process for the production and purification of succinic acid. US patent 5143834. （8）Gokarn, R.R., Eiteman, M.A., Altman, E., 1998. Expression of pyruvate carboxylase enhances succinate production in Escherichia coli without affecting glucose uptake. Biotechnol. Lett. 20, 795#8211;798. （9）Guettler, M.V., Jain, M.K., Rumler, D., 1996. Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants. US patent 5,573,931. （10）Guettler, M.V., Rumler, D., Jain, M.K., 1999. Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. Int. J. Syst. Bacteriol.49,207#8211;216. （11）Hermann, B.G., Patel, M., 2007. Today's and tomorrow's bio-based bulk chemicals from white tiotechnology: a techno-economic analysis. Appl. Biochem. Biotechnol. 136,361#8211;388. （12）Hong, S.H., Kim, J.S., Lee, S.Y., In, Y.H., Choi, S.S., Rih, J., Kim, C.H., Jeong, H., Hur,C.G., Kim, J.J., 2004. The genome sequ ence of the capnophilic rumen bacterium Mannheimia succiniciproducens. Nat Biotechn ol. 22, 1275#8211;1281. （13）Ikeda, M., Ohnishi, J., Hayashi, M., Mitsuhashi, S., 2006. A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient Llysine production. J. Ind. Microbiol. Biotechnol. 33, 610#8211;615. （14）Inui, M., Murakami, S., Okino, S., Kawaguchi, H., Vert#232;s, A.A., Yukawa, H., 2004.Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7,

3. 毕业设计（论文）进程安排

2017.12-2018.2 文献查阅，完成开题报告，熟悉实验仪器。

2018.3-2018.4 顺反异构酶（cti）关键基因构建以及重组质粒的鉴定 2018.4-2018.5 全局调控蛋白酶（whib）关键基因构建以及重组质粒的鉴定 2018.5-2018.6 双基因改造质粒的表达验证及诱导条件的优化 2018.6-2018.6 重组菌株摇瓶及发酵罐验证，以及代谢过程的优化，完成毕业论文的撰写