Evolution of efficient pathways for degradation of anthropogenic chemicals

Abstract

Anthropogenic compounds used as pesticides, solvents and explosives often persist in the environment and can cause toxicity to humans and wildlife. The persistence of anthropogenic compounds is due to their recent introduction into the environment; microbes in soil and water have had relatively little time to evolve efficient mechanisms for degradation of these new compounds. Some anthropogenic compounds are easily degraded, whereas others are degraded very slowly or only partially, leading to accumulation of toxic products. This review examines the factors that affect the ability of microbes to degrade anthropogenic compounds and the mechanisms by which new pathways emerge in nature. New approaches for engineering microbes with enhanced degradative abilities include assembly of pathways using enzymes from multiple organisms, directed evolution of inefficient enzymes, and genome shuffling to improve microbial fitness under the challenging conditions posed by contaminated environments.

Figure 1

Pathways for a) degradation of atrazine in Pseudomonas sp. strain ADP and b) degradation of pentachlorophenol in Sphingobium chlorophenolicum sp. Strain ATCC 39723,. Highlighted in blue is the part of each pathway that converts the anthropogenic compound into an intermediate in a standard metabolic pathway.

Figure 2

Examples of promiscuous activities that a) resemble the normal transformation and are due to substrate ambiguity; and b) result in transformations that are quite different from the normal transformation, yet involve common elementary chemical steps. Values for k_cat/K_M in units of M^-1s^-1 are shown in red for normal activities and in blue for promiscuous activities. Kinetic parameters are from references 70 (homoserine kinase), 71 (alkaline phosphatase), 10 (o-succinylbenzoate synthase), and 72 (tetrachlorohydroquinone dehalogenase). (*) A value for k_cat/K_M for tetrachlorohydroquinone dehalogenase cannot be determined due to severe substrate inhibition.

Figure 3

Binding of a) o-succinylbenzoate and b) a promiscuous substrate (N-acetyl methionine) to the active site of o-succinylbenzoate synthase. Reprinted from Reference 11 with permission from Elsevier.

Figure 4

Pathways for degradation of 4-nitrotoluene in Pseudomonas sp. Strain 4-NT and Mycobacterium Strain HL 4-NT-1.

Figure 5

The consequences of recruiting a flavin monooxygenase to hydroxylate PCP include uncoupling of formation of C4a-hydroperoxyflavin and hydroxylation of substrate, which wastes NADPH and generates H₂O₂, and formation of toxic tetrachlorobenzoquinone.

Figure 6

An engineered pathway for degradation of paraoxon that utilizes enzymes from four different microbes.

Figure 7

An engineered pathway for degradation of cis-1,2-dichloroethylene that uses enzymes with promiscuous activities that have been enhanced by DNA shuffling (toluene o-monooxygenase (TOM) from) or site-saturation mutagenesis of active site residues (epoxide hydrolase from Agrobacterium radiobacteri AD1 (EchA)).