Enhanced expression of a bacterial gene for pesticide degradation in a common soil fungus

Abstract

The wide dispersion of toxic chemicals throughout the varied soil and water structures of our earth has stimulated the development of bioremediation technologies which may be applied to many different environmental situations. One of the new developing technologies involves the genetic engineering of natural microorganisms with new degradative capabilities for bioaugmentation of select, contaminated environments. In these studies, a hybrid genetic system formed by the combination of a bacterial structural gene in a fungal expression system is developed and productively introduced into the common soil fungus, Gliocladium virens. The hybrid gene was formed by combining the constitutive promoter and translational signals from a fungal gpd gene (encoding glyceraldehyde-3-phosphate-dehydrogenase) with the structural region of the bacterial opd gene (organophosphate degrading gene). The fungal expression region of gpd included 1071 bp of the 5′-flanking region (the promoter) and 735 bp of the 3′-flanking region (the terminator). A set of opd expression vectors was constructed containing different length of promoter and terminator regions and used to transform a native strain of G. virens, a hardy soil-inhabiting fungus. Organophosphate hydrolase (OPH, encoded by the opd gene) activity was detected in several of the genetic transformants, and the levels of expression were greatly enhanced (over twenty-fold) as compared to transformants with nonhomologous fungal genetic sequences. Furthermore, it was possible to increase the enzymatic activity another fifteen to thirty-five fold by adding 0.1 mM concentrations of Co +2 in the growth medium. Thus, these studies have demonstrated the ability to genetically augment the metabolic capabilities of a naturally-occurring fungus, rendering them capable of ex situ environmental remediation of a variety of widely used neurotoxic, OP pesticides.