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

这个小企业创新研究第一阶段项目涉及氯化溶剂降解生物催化剂的开发，这种生物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，启动子在目标化学物质存在、低pH和/或低温下诱导（开启）。该生物催化剂将适用于反应器碱和原位生物处理工艺，用于破坏顽固的氯化溶剂。氯化溶剂是美国地下水和土壤中分布最广、含量最多的污染物，其中许多即使是少量也会直接致癌。目前用于修复被氯化溶剂污染的土壤和/或地下水的技术由于成本高或往往不能破坏污染化学品而受到限制。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大面积扩散的污染物或被困在含水层中的污染物。虽然许多细菌能够破坏氯化溶剂，但这一过程是共同代谢的；即，这些化合物不作为降解生物的生长底物或基因诱导剂。在缺乏有毒诱导底物（如甲苯或苯酚）的情况下，降解往往受到酶合成抑制的限制。Envirogen, Inc.建议使用分子生物学技术开发生物催化剂，不再需要存在有毒诱导底物来破坏氯化溶剂，而是在环境条件（低温或低pH值）或目标化合物本身存在的DNA启动子控制下具有降解基因。克隆的甲苯单加氧酶基因将融合到现有的环境诱导启动子中，以及在拟议项目中分离的启动子中。所得到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破坏氯化溶剂。

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.