Metabolism of Styrene by Rhodococcus rhodochrous

1. Eight strains of bacteria able to grow on styrene as a sole source of carbon and energy had been isolated prior to this project. The aim of the project was to discover the pathway for the metabolism of styrene in one of these strains, preferably a novel pathway. 2. Initial characterisation of these eight strains using growth experiments and simultaneous induction led to the selection of three strains for further work, and they were tentatively identified as coryneform bacteria. Simultaneous induction tests on one strain, a possible Rhodococcus sp., suggested that styrene was probably metabolised through phenylacetic acid, a well-established pathway for styrene degradation. The results from another of the three strains were not clear cut. The third strain was clearly using a novel pathway, and so it was selected for further study. 3. The organism which was selected for most of the work described in this thesis, strain 26, was identified as Rhodococcus rhodochrous. A method was developed to grow this strain in a fermenter containing a minimal salts medium, with styrene vapour supplied in the air supply. For larger quantities of cells, a nutrient broth medium was used, with styrene supplied as vapour in order to induce the enzymes involved in styrene degradation. 4. R. rhodochrous 26 is able to grow on styrene, toluene, ethylbenzene and 1-phenylethanol. Simultaneous induction experiments showed that growth on each of these substrates resulted in the ability to oxidise all of the others. 5. Oxidative responses to possible intermediates in known pathways of toluene metabolism showed high activities with toluene cis-glycol and 3-methylcatechol, with lower activities towards other possible substrates. This led to the proposition that a similar pathway was being used for styrene metabolism. 6. Styrene cw-glycol was produced by Pseudomonas putida from styrene, and identified by and n.m.r.. This substrate was oxidised by extracts of R. rhodochrous 26 which had been grown on styrene, in an NAD-dependent reaction. There was also an NAD-dependent oxidation of toluene cw-glycol. Ion exchange chromatography of extracts, coupled with a heat denaturation test, strongly indicated that both toluene c/5-glycol and styrene cz5-glycol were oxidised by the same enzyme. 7. Under anaerobic conditions, in the presence of NAD, extracts transformed styrene cz5-glycol into a compound identified by g.c.-m.s. as vinylcatechol. Toluene cis- glycol was transformed to methylcatechol by the same system. 8. Intact cells incubated with styrene and 3-fluorocatechol (as an inhibitor of catechol oxygenase activity), accumulated a compound identified by g.c.-m.s. and n.m.r. as 3-vinylcatechol. 2-Fluoromuconic acid, identified by and n.m.r., also accumulated under these conditions. 9. When intact cells were incubated with styrene and 3-fluorocatechol in an atmosphere of 50% 18O2 and 50% 18O2, g.c.-m.s. analysis demonstrated the presence of two molecular ions for vinylcatechol, M and M-i-4, showing that initial ring attack is due to a dioxygenase. 10. When grown on styrene, R. rhodochrous 26 accumulated a compound identified by and n.m.r. as 2-vinylmuconic acid. Up to 40% of the styrene substrate may be converted to this product. There was no sign of further metabolism of this product in either growth medium or cell extracts. A similar accumulation of muconic acid was noticed when R. rhodochrous 26 was grown on benzyl alcohol. Cell extracts did not seem to metabolise cis, cis-muconic acid, the expected product of benzyl alcohol breakdown through an ortho cleavage pathway. 11. It was not possible to detect any styrene dioxygenase activity in extracts using an oxygen electrode or spectrophotometric methods. 12. Extracts of R. rhodochrous 26 which had been grown on styrene contained catechol 1,2-oxygenase and catechol 2,3-oxygenase activities. The two enzymes were clearly separated by ion exchange chromatography, each appearing as a single peak. Both enzymes could oxidise catechol, 3-methylcatechol, 4-methylcatechol and vinylcatechol. (Abstract shortened by ProQuest.)