Sphingomonas wittichii RW1 is one out of three strains capable of metabolizing dibenzo-p-dioxin as a sole source for carbon and energy, accordingly, has received extensive research from many scientists worldwide [3, 16]. Dioxins are a group of chemically related aromatics that include polychlorinated dibenzo-p-dioxins and dibenzofurans. These compounds are chemically inert, recalcitrant, lipid soluble, and toxic to humans and animals [17]. Dioxins are released to the environment as by products from the manufactory of pesticides and herbicides, incineration of wastes, or can be produced naturally from forest fires and volcanoes eruption; therefore, dioxins have become ubiquitous pollutants in the environment [21]. Reports have stated that more than 50% of the dioxins excluded to the environment is from incineration of municipal solid waste. These persistent compounds remain in the sediments for decades which is why it is necessary measure dioxin concentration if waste water effluent [19].
Degradation rates of S. wittichii RW1 for dibenzo-p-dioxin (DD) and dibenzofuran (DBF) under laboratory condition are relatively high, 5 and 8 h for DD and DBF, respectively, when grown on 5 mM of each substrate [24]. However, their degradation capabilities in the environment are restricted to nutrient availability, generation of dead end products, trace concentrations of dioxins, water activity, and low adaptation to soil environment [4, 5].
Attempts to engineer S. wittichii RW1 to improve its degradation capabilities have succeeded by introducing membrane superchannels from Sphingomonas sp. A1. These superchannels facilitated the incorporation of polyaromatic hydrocarbons into S. wittichii RW1 cells thus enhanced the degradation rate of hazardous compounds in contaminated sites [1]. Interestingly, genome analysis of S. wittichii RW1 revealed that this strain contained genes that carried the ability to perform all the steps included in the biphenyl degradation pathway except the dehydrogenation of cis 2,3-dihydro-2,3-dihydroxybiphenyl and this was the only missing gene that prevented it from growing on biphenyl. Engineering this strain by introducing an appropriate dehydrogenase from a biphenyl degrader enabled S. wittichii RW1 to grow on biphenyl at relatively high rates [8].
Acetate is an important carbon source and easy to use compound. It is both aerobically and anaerobically metabolized by bacteria. As a result, inhibiting such microbial activities leads to acetate accumulation in soil. Acetate concentrations varies according to type of soil being the highest in salt-marsh sediments that ranges between 0.1 and 1.0 mM [10]. Acetate is produced in soils by several pathways, it can be produced anaerobically by bacteria via acetogenesis or through anaerobic degradation of cellobiose [12]. Evidence for release of acetate from fine roots and associated mycorrhiza have also been detected [11]. Acetate is a preferable carbon source by S. wittichii RW1 and its presence in the environment would have a negative impact on DD and DBF degradation. In addition, expression of most of the genes for DD and DBF degradation is down regulated when grown on acetate compared to their growth on DD and DBF [9]. Oxygen uptake by S. wittichii RW1 resting cells grown on acetate and subjected to DD and DBF was relatively less compared to resting cells on DD and DBF [24].In addition, acetate has been reported to inhibit the degradation rate of some polyaromatic hydrocarbons (PAH) in the environment probably due to its preference by PAH degrading microorganisms [20]. The presence of more than one carbon source in the environment where one is more favorable than the other leads to diauxic growth where the organism cannot metabolize the unfavorable compound until the favorable one is totally consumed [13]. Therefore, we hypothesized that blocking the acetate utilization pathway in S. wittichii RW1 would prevent it from using acetate when present along with DD and DBF in contaminated sites. Engineered strains of S. wittichii RW1 will obtain all their carbon source from contaminated aromatics instead of utilizing acetate. Few studies have been conducted on improving biodegradation capabilities in S. wittichii RW1, however, improving biodegradation capabilities by engineering metabolic pathways has not been reported elsewhere.