Rui Lingyun, Kwon Young Man, Reardon Kenneth F, Wood Thomas K
Department of Chemical Engineering, University of Connecticut, Storrs, CT 06269-3222, USA.
Environ Microbiol. 2004 May;6(5):491-500. doi: 10.1111/j.1462-2920.2004.00586.x.
Aerobic, co-metabolic bioremediation of trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-DCE) and other chlorinated ethenes with monooxygenase-expressing microorganisms is limited by the toxic epoxides produced as intermediates. A recombinant Escherichia coli strain less sensitive to the toxic effects of cis-DCE, TCE and trans-1,2-dichloroethylene (trans-DCE) degradation has been created by engineering a novel pathway consisting of eight genes including a DNA-shuffled toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green), a newly discovered glutathione S-transferase (GST) from RhodococcusAD45 (IsoILR1), found to have activity towards epoxypropane and cis-DCE epoxide, and an overexpressed E. coli mutant gamma-glutamylcysteine synthetase (GSHI*). Along with IsoILR1, another new RhodococcusAD45 GST, IsoILR2, was cloned that lacks activity towards cis-DCE epoxide and differs from IsoILR1 by nine amino acids. The recombinant strain in which TOM-Green and IsoILR1 were co-expressed on separate plasmids degraded 1.9-fold more cis-DCE compared with a strain that lacked IsoILR1. In the presence of IsoILR1 and TOM-Green, the addition of GSH1* resulted in a sevenfold increase in the intracellular GSH concentration and a 3.5-fold improvement in the cis-DCE degradation rate based on chloride released (2.1 +/- 0.1 versus 0.6 +/- 0.1 nmol min(-1) mg(-1) protein at 540 microM), a 1.8-fold improvement in the trans-DCE degradation rate (1.29 +/- 0.03 versus 0.71 +/- 0.04 nmol x min(-1) mg(-1) protein at 345 microM) and a 1.7-fold improvement in the TCE degradation rate (6.8 +/- 0.24 versus 4.1 +/- 0.16 nmol x min(-1) mg(-1) protein at 339 microM). For cis-DCE degradation with TOM-Green (based on substrate depletion), V(max) was 27 nmol x min(-1) mg(-1) protein with both IsoILR1 and GSHI* expressed compared with V(max) = 10 nmol x min(-1) mg(-1) protein for the GST(-)GSHI*(-) strain. In addition, cells expressing IsoILR1 and GSHI* grew 78% faster in rich medium than a strain lacking these two heterologous genes.
利用表达单加氧酶的微生物对三氯乙烯（TCE）、顺式-1,2-二氯乙烯（cis-DCE）及其他氯化乙烯进行需氧共代谢生物修复，受到作为中间产物产生的有毒环氧化物的限制。通过构建一条由八个基因组成的新途径，已培育出一种对cis-DCE、TCE和反式-1,2-二氯乙烯（trans-DCE）降解的毒性作用不太敏感的重组大肠杆菌菌株。该新途径包括来自洋葱伯克霍尔德菌G4的经DNA改组的甲苯邻位单加氧酶（TOM-Green）、新发现的来自红球菌AD45的谷胱甘肽S-转移酶（GST）（IsoILR1），发现其对环氧丙烷和顺式-DCE环氧化物具有活性，以及一个过表达的大肠杆菌突变体γ-谷氨酰半胱氨酸合成酶（GSHI*）。除了IsoILR1，还克隆了另一种新的红球菌AD45 GST，IsoILR2，它对顺式-DCE环氧化物没有活性，与IsoILR1有九个氨基酸的差异。将TOM-Green和IsoILR1共表达在不同质粒上的重组菌株，与缺乏IsoILR1的菌株相比，对cis-DCE的降解能力提高了1.9倍。在存在IsoILR1和TOM-Green的情况下，添加GSH1使细胞内谷胱甘肽浓度增加了7倍，基于氯离子释放的cis-DCE降解率提高了3.5倍（在540 microM时为2.1±0.1对0.6±0.1 nmol min⁻¹ mg⁻¹蛋白质），反式-DCE降解率提高了1.8倍（在345 microM时为1.29±0.03对0.71±0.04 nmol·min⁻¹ mg⁻¹蛋白质），TCE降解率提高了1.7倍（在339 microM时为6.8±0.24对4.1±0.16 nmol·min⁻¹ mg⁻¹蛋白质）。对于用TOM-Green进行的cis-DCE降解（基于底物消耗），在同时表达IsoILR1和GSHI时，V(max)为27 nmol·min⁻¹ mg⁻¹蛋白质，而对于GST(-)GSHI*(-)菌株，V(max)=10 nmol·min⁻¹ mg⁻¹蛋白质。此外，表达IsoILR1和GSHI*的细胞在丰富培养基中的生长速度比缺乏这两个异源基因的菌株快78%。
