STUDIES ON CYTOCHROME P-450 DEPENDENT DETOXIFICATION OF XENOBIOTICS IN BACTERIA

Abstract

Bioremediation has emerged as a potential tool for the conversion of hazardous organic chemicals and wastes into innocuous products. The treatment and disposal of hazardous wastes containing recalcitrant toxic compounds is not feasible based on our current knowledge of conventional waste treatment process engineering. Hence, cost-effective remediation of recalcitrant compounds in the environment requires novel bioremediation alternatives. Since many contaminated sites contain more than one pollutant it is often difficult for natural microorganisms to degrade, efficiently and simultaneously, a mixture of pollutants. Indigenous microorganisms when exposed to a mixture of pollutants may produce toxic intermediates that do not allow an overall reduction of toxicity of biotreated sample to a significant extent. In many cases, natural microorganisms have not evolved the genetic competence to mineralise certain synthetic compounds. The activity of natural pure cultures is often contaminant specific and is less applicable to complex mixtures of pollutants encountered in environmental situations. For this reason the search and isolation of naturally occurring bacterial strains, which rapidly metabolize recalcitrant pollutants, has been only partially successful. Genetically improved single cultures are considered more efficient than mixed cultures in degrading complex chemical wastes. Intense research effort has been directed at construction of bacteria with utility in bioremediation of hazardous wastes. Such genetically engineered organisms can be conferred an enhanced degradative capability for biotreatment of recalcitrant chemicals that confound natural degradation. Improved biocatalysts possessing the desired catabolic or detoxification potential in environmentally robust hosts can be applied for the remediation of contaminated soils and sediments. The use of'microbial inocula' is an attractive proposition provided that it could be cost-effective, with survival oforganisms and rejuvenation ofstarting inocula. The metabolic potential of microorganisms cannot be harnessed universally to develop effective biotechnological processes for remediation of hazardous wastes as the evolution of enzymes with wider substrate range proceeds very slowly in nature. Hence, the most desirable enzymes systems in biodegradation would be those that are involved in transforming and activating a wide variety of compounds rather than being specific. The ability ofcytochrome P-450 enzymes to metabolize an extraordinary spectrum of diverse substrates renders them suitable for detoxification of hazardous wastes. Bacterial cytochrome P-450 cam and human cytochrome P-450 2E1 with their wide substrate range represent ideal catalysts for application in bioremediation. The present study explored the feasibility of expression of native and Nterminus modified human cytochrome P-450 2E1 in E. coli and the environmentally robust host, Pseudomonas putida. Further, the role of luciferase to serve as electron transfer partner for the expressed P-450 2E1 was investigated. The study also evaluated the in vivo catalytic activity ofPseudomonas putida coexpressing A^-terminus modified human cytochrome P-450 2E1 and luciferase. Low yields of the native human cytochrome P-450 2E1 were observed in E. coli and Pseudomonas putida, necessitating N-terminus modification of the hemoprotein to attain enhanced expression in bacterial hosts. The N-terminus modified human cytochrome P-450 2E1 subcloned on broad host range vector, exhibited improved expression in E. coli and Pseudomonas putida co-expressing luciferase. Successful co-expression of N-terminus modified P-450 2E1 and luciferase was obtained in Pseudomonas putida under the optimal growth conditions. The possibility of photo-reduction of modified P-450 2E1 by luciferase was assessed in in vitro assays containing the hemoprotein and luciferase. The generation of the characteristic P-450 peak in the absence of the chemical reductant validated the ability of luciferase to reduce the P-450 2E1 hemoprotein. This reduction was dependent on the concentration of luciferase that was comparable to the chemical reductant, dithionite. Resting cultures of Pseudomonas putida co-expressing cytochrome P-450 2E1 and luciferase incubated with carbon tetrachloride exhibited nearly 30% conversion to chloroform, as determined by GC-MS analysis. The in vivo reductive metabolism of carbon tetrachloride demonstrates that luciferase serves as an alternate electron transfer partner for P-450 2E1. The present study also determined the metabolic competence of engineered Pseudomonas under nutrient stress conditions, utilizing a strain of Pseudomonas putida co-expressing cytochrome P-450 cam and luciferase as a model. This organism provides both the reductive detoxification potential of the hemoprotein and a mechanism of its reduction in the absence of 'normal' P-450 redox partners. The ability of the organism to survive and remain metabolically in competent under nutrient stress was evaluated in soil slurries containing the halogenated substrates, viz. hexachloroethane and y-hexachlorocyclohexane. Genetically engineered Pseudomonas putida co-expressing cytochrome P- 450 cam and luciferase transformed hexachloroethane to tetrachloroethylene 8-10 fold faster than the uninoculated slurries. In addition, the engineerd Pseudomonas also mediated nearly 65% dehalogenation of y-hexachlorocyclohexane to y- 3,4,5,6-tetrachlorocyclohexene in soil slurries under subatmospheric conditions, as confirmed by GC-MS. The present study examined the response of engineered Pseudomonas putida co-expressing cytochrome P-450 cam and luciferase under single and multiple nutrient stress. In addition, the recovery and stability of the dual engineered functions under oligotrophic conditions in soil microcosm and soil slurry was also evaluated. The study also explored the feasibility of utilizing clay minerals as immobilization matrices for the generation of active microbial inocula for bioremediation applications. More than 74% of the cells of the engineered Pseudomonas were culturable after 7 days of multiple nutrient (C,N,P) starvation. Diagnostic COdifference spectra and luminescence exhibited by the cells validated the stability of the dual engineered traits. The engineered organism could be revived after repeated desiccation and starvation using Luria-Bertani medium, benzoate or citrate as nutrients. Significant survival and recovery of viable and culturable cells after slow desiccation observed even after 99 days in soil slurry validate the robust survival attributes of the engineered organism. Prolonged survival with full IV retention of the engineered traits and recovery of 10-15% cells as active catalysts of lyophilized clay-matrix bound Pseudomonas putida even after 600 days establishes the potential of clay-minerals as effective microbial carriers. The results obtained in the present study indicate that GEMs can be designed with broad substrate range detoxification catalysts such as cytochrome P-450 for the development of remediation processes for hazaradous wastes. The results also confirm that alternate electron transfer partners such as luciferase, maybe utilized for cytochrome P-450-dependent bioremediation strategies.