Lee Jintae, Cao Li, Ow Saw Yen, Barrios-Llerena Martin E, Chen Wilfred, Wood Thomas K, Wright Phillip C
Artie McFerrin Department of Chemical Engineering and Department of Biology, 220 Jack E. Brown Building, Texas A & M University, College Station, Texas 77843-3122, USA.
J Proteome Res. 2006 Jun;5(6):1388-97. doi: 10.1021/pr060008t.
Metabolically engineered Escherichia coli has previously been used to degrade cis-1,2-dichloroethylene (cis-DCE). The strains express the six genes of an evolved toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green, which formed a reactive epoxide) with either (1) gamma-glutamylcysteine synthetase (GSHI, which forms glutathione) and the glutathione S-transferase IsoILR1 from Rhodococcus AD45 (which adds glutathione to the reactive cis-DCE epoxide) or (2) with an evolved epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA F108L/I219L/C248I which converts the reactive cis-DCE epoxide to a diol). Here, the impact of this metabolic engineering for bioremediation was assessed by investigating the changes in the proteome through a quantitative shotgun proteomics technique (iTRAQ) by tracking the changes due to the sequential addition of TOM-Green, IsoILR1, and GSHI and due to adding the evolved EchA versus the wild-type enzyme to TOM-Green. For the TOM-Green/EchA system, 8 proteins out of 268 identified proteins were differentially expressed in the strain expressing EchA F108L/I219L/C248I relative to wild-type EchA (e.g., EchA, protein chain elongation factor EF-Ts, 50S ribosomal subunits L7/L12/L32/L29, cysteine synthase A, glycerophosphodiester phosphodiesterase, iron superoxide dismutase). For the TOM-Green/IsoILR1/GSHI system, the expression level of 49 proteins was changed out of 364 identified proteins. The induced proteins due to the addition of TOM-Green, IsoILR1, and GSHI were involved in the oxidative defense mechanism, pyruvate metabolism, and glutathione synthesis (e.g., 30S ribosomal subunit proteins S3 and S16, 50S ribosomal subunit protein L20, alkyl hydroperoxide reductase, lactate dehydrogenase, acetate kinase, cysteine synthase A). Enzymes involved in indole synthesis, fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle were repressed (e.g., tryptophanase, acetyl-CoA carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase). Hence, the metabolic engineering that leads to enhanced aerobic degradation of 1 mM cis-DCE (2.4-4-fold more chloride ions released) and reduced toxicity from cis-DCE epoxide results in enhanced synthesis of glutathione coupled with an induced stress response as well as repression of fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle.
代谢工程改造的大肠杆菌此前已被用于降解顺式-1,2-二氯乙烯（顺式-DCE）。这些菌株表达了来自洋葱伯克霍尔德菌G4的进化甲苯邻单加氧酶的六个基因（TOM-Green，其形成一种反应性环氧化物），同时表达（1）γ-谷氨酰半胱氨酸合成酶（GSHI，其形成谷胱甘肽）和来自红球菌AD45的谷胱甘肽S-转移酶IsoILR1（其将谷胱甘肽添加到反应性顺式-DCE环氧化物上），或者（2）来自放射形土壤杆菌AD1的进化环氧化物水解酶（EchA F108L/I219L/C248I，其将反应性顺式-DCE环氧化物转化为二醇）。在此，通过定量鸟枪法蛋白质组学技术（iTRAQ）研究蛋白质组的变化，追踪由于依次添加TOM-Green、IsoILR1和GSHI以及将进化的EchA与野生型酶添加到TOM-Green中所导致的变化，从而评估这种代谢工程对生物修复的影响。对于TOM-Green/EchA系统，在表达EchA F108L/I219L/C248I的菌株中，相对于野生型EchA，在268个鉴定出的蛋白质中有8种蛋白质差异表达（例如，EchA、蛋白质链延伸因子EF-Ts、50S核糖体亚基L7/L12/L32/L29、半胱氨酸合成酶A、甘油磷酸二酯磷酸二酯酶、铁超氧化物歧化酶）。对于TOM-Green/IsoILR1/GSHI系统，在364个鉴定出的蛋白质中有49种蛋白质的表达水平发生了变化。由于添加TOM-Green、IsoILR1和GSHI而诱导的蛋白质参与氧化防御机制、丙酮酸代谢和谷胱甘肽合成（例如，30S核糖体亚基蛋白质S3和S16、50S核糖体亚基蛋白质L20、烷基过氧化氢还原酶、乳酸脱氢酶、乙酸激酶、半胱氨酸合成酶A）。参与吲哚合成、脂肪酸合成、糖异生和三羧酸循环的酶受到抑制（例如，色氨酸酶、乙酰辅酶A羧化酶、磷酸烯醇丙酮酸羧激酶、苹果酸脱氢酶）。因此，导致1 mM顺式-DCE有氧降解增强（释放的氯离子多2.4 - 4倍）和顺式-DCE环氧化物毒性降低的代谢工程，导致谷胱甘肽合成增强，同时诱导应激反应以及脂肪酸合成、糖异生和三羧酸循环受到抑制。
