Isolation of DNA Promotors Induced by Environmental Stimuli and Their Use In The Construction of Improved Biocatalysts for Environmental Remediation

这一小型企业创新研究第一阶段项目涉及开发氯化溶剂降解生物催化剂，这些催化剂在环境刺激下表达降解基因。氯化溶剂降解基因将在启动子的控制下克隆，所述启动子在目标化学品存在下、在低pH值下和/或在低温下被诱导（开启）。生物催化剂将适用于反应器基础和现场生物处理工艺，以销毁难降解的氯化溶剂。 氯化溶剂是国家地下水和土壤中最广泛和最丰富的污染物，许多即使少量也是直接致癌的。目前用于修复被氯化溶剂污染的土壤和/或地下水的技术受到其高成本或它们通常不破坏污染化学品的事实的限制。然而，氯化溶剂的生物氧化导致污染物的完全破坏，降解微生物可用于破坏分布在大面积的污染物或截留在含水层中的污染物。虽然许多细菌能够破坏氯化溶剂，但该过程是共代谢的;即化合物不作为降解生物的生长基质或基因诱导剂。在没有毒性诱导底物（如甲苯或苯酚）的情况下，降解通常受到酶合成抑制的限制。Envirogen公司提出使用分子生物学技术来开发生物催化剂，其不再需要存在毒性诱导底物来破坏氯化溶剂，而是具有在DNA启动子控制下的降解基因，所述DNA启动子由环境条件（低温或低pH）或目标化合物本身的存在诱导。克隆的甲苯单加氧酶基因将融合到现有的环境诱导的启动子，并在拟议的项目中分离的启动子。由此产生的生物催化剂将能够在生物反应器中的受控条件下以及在含水层和/或土壤中的特定原位条件下销毁氯化溶剂。

This Small Business Innovative Research Phase I project involves the development of chlorinated solvent-degrading biocatalysts that express degradative genes in response to environmental stimuli. Chlorinated solvent-degrading genes will be cloned under the control of promoters that are induced (turned on) in the presence of target chemicals, under low pH, and/or under low temperature. The biocatalysts will be applicable to both reactorbase( and in situ biotreatment processes for destroying recalcitrant chlorinated solvents. Chlorinated solvents are the most widespread and abundant contaminants of the Nation's groundwater and soils, and many are directly carcinogenic even in small amounts. Current technologies for remediating soils and/or groundwater contaminated with chlorinated solvents are limited by their high costs or the fact that they often do not destroy the contaminating chemicals. Biological oxidation of chlorinated solvents, however, results in the complete destruction of the contaminant, and degradative microorganisms can be used to destroy contaminants spread over large areas or contaminants trapped in aquifers. Although many bacteria are capable of destroying chlorinated solvents, the process is co-metabolic; i.e. the compounds do not act as growth substrates or gene inducers for the degradative organisms. Degradation is often limited by repression of enzyme synthesis in the absence of toxic inducing substrates such as toluene or phenol. Envirogen, Inc. proposes to use molecular biological techniques to develop biocatalysts that no longer require the presence of toxic inducing substrates for destroying chlorinated solvents, but rather have degradative genes under control of DNA promoters that are induced by environmental conditions (low temperature or low pH) or the presence of the target compounds themselves. Cloned toluene monooxygenase genes will be fused to existing environmentally induced promoters, and to promoters isolated during the proposed project. The resulting biocatalysts will be capable of destroying chlorinated solvents under controlled conditions in bioreactors, and also under specific in situ conditions in aquifers and/or soils.