ABSTRACT
Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15µM) and D3 for MhpB (IC50 110µM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the ß-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.
Subject(s)
Dioxygenases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Lignin/metabolism , Protocatechuate-3,4-Dioxygenase/antagonists & inhibitors , Pseudomonas fluorescens/enzymology , Rhodococcus/enzymology , Acetaldehyde Dehydrogenase Inhibitors/pharmacology , Dioxygenases/metabolism , Disulfiram/pharmacology , Enzyme Inhibitors/chemistry , Fermentation/drug effects , Hydroxamic Acids/chemistry , Hydroxybenzoates/metabolism , Protocatechuate-3,4-Dioxygenase/metabolism , Pseudomonas fluorescens/drug effects , Pseudomonas fluorescens/metabolism , Rhodococcus/drug effects , Rhodococcus/metabolism , Tricarboxylic Acids/metabolismABSTRACT
The aromatic polymer lignin represents a possible renewable source of aromatic chemicals, if biocatalytic routes for lignin breakdown can be developed. The availability of a genome sequence for Rhodococcus jostii RHA1, a bacterium that breaks down lignin, has allowed the application of a targeted pathway engineering strategy to lignin breakdown to produce vanillin, a valuable food/flavor chemical. A gene deletion strain of R. jostii RHA1 in which the vanillin dehydrogenase gene had been deleted, when grown on minimal medium containing 2.5% wheat straw lignocellulose and 0.05% glucose, was found to accumulate vanillin with yields of up to 96 mg/L after 144 h, together with smaller amounts of ferulic acid and 4-hydroxybenzaldehyde.
