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Bacterial isoprene metabolism: a missing link in a key global biogeochemical cycle

Periodic Reporting for period 4 - IsoMet (Bacterial isoprene metabolism: a missing link in a key global biogeochemical cycle)

Reporting period: 2021-05-01 to 2022-12-31

Isoprene is an important biogenic volatile organic compound released into the environment in similar amounts as methane, making it the second most abundant organic trace gas after methane. Although we know a great deal about the biological cycling of methane, we know virtually nothing about how isoprene is recycled in the biosphere before it can escape to the atmosphere. Isoprene is an important building block for the isoprenoid family of compounds which include rubber, cholesterol, vitamin A, carotenoids, monoterpenes. There is a natural biogeochemical cycle for isoprene, with around 600 million tonnes of isoprene (about 1/3 of all BVOCs) being released into the environment by terrestrial plants per year. This represents 1-2% of net primary productivity by land plants re-emitted to the atmosphere as Isoprene. Isoprene has important effects on atmospheric chemistry and can act as both a global warming gas and as a global cooling gas and also affects air quality and so it is really important to know what happens to the isoprene that is emitted by plants and how it is removed from our biosphere. Some plants such as willow and oil palm are being grown in huge amounts for production of biofuels. These are very high emitters of isoprene and so removal of native trees and planting and growing these high isoprene-emitters in their place may have substantial consequences on air quality in these regions. It is important therefore that society needs to able to understand the global isoprene cycle.
The overall aim of the IsoMet project was to obtain a critical, fundamental understanding of the metabolism and ecological importance of biological isoprene degradation and to test the hypothesis that isoprene degrading bacteria play a key role in isoprene cycling in the environment.
Key Objectives were to:
Determine the biological mechanisms by which isoprene is metabolised, both in model bacteria in the laboratory and in the environment.
Use novel methods to study isoprene degradation in the environment.
Elucidate at the mechanistic level how isoprene cycling by microbes is regulated in the environment.

These three broad objectives require a coordinated multidisciplinary approach and use of a wide range of innovative and leading edge techniques and so the specific aims of the project are:

a) Isolate and characterise bacteria that metabolise isoprene.
b) Elucidate the pathways of isoprene metabolism and their regulation.
c) Characterise the enzymes catalysing key steps in isoprene degradation.
d) Identify genes encoding isoprene-degrading enzymes and regulatory mechanisms.
e) Develop functional gene probes for detection of isoprene degraders in the environment.
f) Determine the diversity and activity of isoprene degraders in the environment.
g) Assess the contribution that bacteria make to isoprene cycling in the environment.
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.
We have developed a suite of novel analytical and molecular tools and used them to prove our hypothesis that isoprene-degraders are widespread in the environment and key players in the global isoprene cycle and provide an unprecedented understanding of their metabolism. Our research has produced over 20 high quality peer reviewed benchmark publications which now represents the key body of literature on the microbiology and ecology of these bacteria. The project has provided outstanding training in trace gas microbiology for early career researchers and generated a cohort of scientists that can continue this fascinating area of research. A number of key national and international collaborations have been established with scientists from UK, Denmark, Austria, The Netherlands, South Africa, Thailand, Malaysia.
Microscope image of an isoprene-degrading Rhodococcus
The global isoprene cycle
Genes and enzymes of the isoprene degradation pathway