Microbial catabolic pathways for complex aromatics

Abstract

This thesis concerns the microbial degradation of recalcitrant aromatic compounds, and the enzymes responsible for that degradation. The two foci are guaiacylglycerol-β-guaiacyl ether (GGE), which is a model compound representing a key intermediate in the deconstruction of lignin, and 2,4-dinitroanisole (DNAN), which is a widely used component of explosives. The degradation of these compounds is of interest both because of the insights it provides into the evolution of catabolic pathways and because of the potential uses of the microbes and enzymes involved in chemical manufacturing and environmental bioremediation. Chapter 1 reviews the existing literature on the microbial degradation of lignin and nitroaromatic explosives such as DNAN, including consideration of their potential industrial applications. Chapter 2 describes the isolation and characterization of the GGE degrading Erythrobacter sp. SG61-1L strain and compares it with the GGE degradation activity of the previously isolated Sphingomonas sp. SYK-6. SG61-1L was found to degrade GGE significantly more rapidly than SYK-6, although the same pathway appeared to operate in both strains. The first step in GGE degradation by SYK-6 had previously been shown to be catalysed by four stereospecific dehydrogenases and I found seven homologous genes in the SG61-1L genome. These thirteen genes were heterologously expressed in E. coli and the activities of their products were studied with the four stereoisomers of GGE. Two of the SG61-1L enzymes in particular had higher activities for the four GGE isomers than any of the four SYK-6 enzymes. A phylogenetic analysis showed that the GGE dehydrogenase activity of the two strains was mainly confined to a particular subclade of classical short chain dehydrogenases but two enzymes from other clades of these dehydrogenases were also found to have significant GGE activity. Chapter 3 describes the mineralization of DNAN by Nocardioides sp. strain JS1661 and the identification and characterization of a complex demethylase enzyme in this bacterium which catalyses the cleavage of the methyl group on DNAN. The hydrolytic removal of the methyl group forms methanol and 2,4-dinitrophenol, which is mineralized via a well-established catabolic pathway. The demethylase enzyme was purified to more than 90 % purity, revealing two distinctive bands on SDS-PAGE. Tryptic digestion and peptide sequencing of these bands identified two overlapping genes in the genome of this bacterium. The two genes, named dnH1 and dnH2, had 40.5 % nucleotide and 21.3 % amino acid sequence similarity to one another. Their closest known relatives were beta lactamase proteins which had 37 % and 55 % amino acid sequence similarity to dnH1 and dnH2 respectively. Only one set of homologues was found, from Nocardia testacea, which are co-translated via cognate stop-start codons in the same way as dnH1 and dnH2. Chapter 4 is a general discussion of the findings described in the two previous chapters. The consideration of evolutionary issues focuses on the functional and phylogenetic relationships of the two sets of enzymes characterized. While the dehydrogenation of the β-aryl ether linkages in GGE by the GGE dehydrogenases seems to have evolved in three clades of classical short chain dehydrogenases, the O-demethylation of the DNAN by the dnH1/dnH2 enzyme appears to be a new class of activity which is functionally distinct from previous classes of O-demethylase enzymes and only very distantly related to other enzymes, the closest known being beta lactamases. The GGE dehydrogenases may be useful in chemical syntheses requiring various forms of stereospecificity, including but not limited to the use of biomass feedstocks. The DNAN demethylase may prove useful in various bioremediation and biosensors strategies, although challenges will first need to be overcome in respect of its heterologous expression. Show more