We isolated and characterised many isoprene degrading bacteria from a variety of soils and leaves proving the widespread nature of these types of bacteria in the environment. We showed that they are abundant in environments where there are high isoprene-emitting trees such as polar, willow and oil palm. In depth analysis of several of these isoprene degraders, including a gram positive Rhodococcus and a gram negative Variovorax has identified the pathways of isoprene degradation. They all use a soluble diiron centre monooxygenase called isoprene monooxygenase and a glutathione transferase enzyme, followed by two dehydrogenases to metabolise isoprene. We have shown that this is a common pathway found in all isolates. The genes encoding these enzymes are all clustered on the genome of isoprene degraders and are found on large plasmids in several of them. These isoprene gene clusters are evolutionarily related and are regulated by isoprene and its oxidation products. Genetic systems have been developed for Rhodococcus and Variovorax and we have shown that iso genes are switched on in response to isoprene.
Detailed proteomics, transcriptomics and molecular genetics analyses enabled us to elucidate the pathways and regulation of isoprene degradation in Rhodococcus and Variovorax. We have purified the isoprene monooxygenase from Rhodococcus and it consists of a three component oxygenase, a reductase, a Rieske-like ferredoxin and a coupling protein. All are essential for activity in vitro. This novel and complex multicomponent enzyme that oxygenates isoprene to an epoxide. This metabolite is toxic but the bacteria are smart and use another enzyme, a glutathione transferase, to detoxify the epoxide and then assimilate its carbon into cellular material before it kills the bacterium. The isoprene monooxygenase is a versatile enzyme with a relatively high affinity for isoprene; it can also cooxidise a range of alkenes. We have discovered that octyne is a potent inhibitor which allows us to validate isoprene oxidation by this system in vivo with environmental samples.
We have developed functional gene probes to capture isoprene monooxygenase genes directly from the environment. This enables us to carry out cultivation-independent studies on these bacteria directly in the environment without the need to cultivate them in the laboratory. Using functional gene probes we have discovered that isoprene degrading bacteria can be detected in many different soil environments and especially on the leaves of isoprene-emitting trees such as willow, poplar and oil palm. A quantitative PCR assay we developed has allowed us to show that they are abundant in soils and on leaves of isoprene emitting trees, thus proving that these bacteria are key players in the global isoprene cycle.
DNA stable isotope probing and metagenomics has been used to determine the distribution and diversity of active isoprene degrading bacteria in willow and poplar (from the UK) and oil palm from Malaysia. These are hotspots of isoprene production, especially oil palm which is the highest known producer of isoprene and is grown in huge areas of the tropics. Our work has shown that isoprene degraders thrive on the leaves of these trees. We have rescued the genomes of key players in isoprene degradation from plants, including Variovorax, Rhodococcus, Gordonia, Ramlibacter, Sphingopyxsis, Nocardioides. They all contain the isoprene monooxygenase and isoprene pathway genes. Using this DNA sequence information and targeted enrichment strategies, we have now isolated these new isoprene degraders, investigation mechanisms of regulation of isoprene metabolism and demonstrated their biotechnological potential for the oxidation of alkanes and alkenes and production of chiral epoxides.
