Team:DeNovocastrians/Poster




Engineering benzene-degrading bacteria
Presented by The DeNovocastrians

PI: Prof. Brett Neilan, Dr Karl Hassan, Dr Benjamin Matthews, Dr Bernadette Drabsch.

Advisors: Evan Gibbs, Catherine Dawson and Joachim Steen Larsen.

iGEM team: Aakash Patel, Angela O'Connor, Catherine Mackay, Darcy Kilvert, Dayna Perez, Luke Gerassimou, Ruby Pippen and Zayne Jeffries.

Abstract

Our project looked to eliminate benzene in polluted environments through bioremediation; a process which utilises organisms such as microbes to degrade hazardous substances. Compared to traditional remediation practices, bioremediation is affordable and sustainable. Our project aimed to engineer microorganisms to detect, import and degrade the petrochemical benzene in various environments. Achieving this involved creating a benzene biosensor and cloning the benzene degradation pathway (benABCDE) into Escherichia coli. The biosensor would detect the environmental levels of benzene through a fluorescent protein expression system paired with transcription factors recognising the hydrocarbon. We extracted the benABCDE gene cluster (which encodes an importer and degradation enzymes) from specialized bacteria that naturally break down benzene into energy via the Citric Acid Cycle. Following this, we inserted the benABCDE genes into plasmid vectors to transform the model laboratory species E. coli into a practically useful benzene degrader to clean up polluted sites. We were unable to verify the capability of our cloned benABCD genes to degrade benzene or to fully construct the biosensor. However, we cloned the benE transporter gene and showed it enabled cells to import benzoate from their surroundings, this transportation led to the importation of benzene into our engineered E. coli. We hope our research will lay the groundwork for future research to develop these systems.
Problems leading to inspiration...
One of the greatest threats to the environment, and by extension humankind, is pollution. It is an inherent by-product of human activities and disasters such as oil spills can have devastating impacts on a range of interconnected ecosystems. Most research has focused on the impact of air pollution, but terrestrial and aquatic pollution are also serious threats to both us and the environment.

For example, a crisis unfolded recently in the waters surrounding Mauritius. A ship filled with petrochemicals (chemicals derived from petroleum or natural gas) ran aground and leaked its fuel into the ocean, devastating sea life and causing catastrophic harm to the nearby reef ecosystem (ABC, 2020).

Additionally, contaminated soil accounts for over one-quarter of all hazardous waste. A major source of this contamination comes from the industrial sector, where petrochemical waste, such as the carcinogenic benzene, is spilt into the environment. A common way of dealing with this contamination is to relocate the soil to other areas, rather than remediating the site. This process can be expensive and often ineffective, which is why innovative solutions are necessary for progress.
Introduction
Our city of Newcastle in Australia has a strong industrial history, particularly for its coal mining and export. It has been found the soils in Newcastle are significantly contaminated with heavy metals and hydrocarbons, which have a long-lasting impact on locals (Harvey et al., 2017). However, the city of Newcastle is also committed to protecting and restoring the environment. Numerous environmental management projects and legislations are in place to reduce the impact we have on the environment. Innovation and initiative for solving these important issues is needed for progress. For this reason, our goals for the 2020 iGEM competition were focused around an environmental aspect.


A common pollutant of the industrial sector is the chemically-stable hydrocarbon, benzene. Benzene is a known carcinogen, found inside petrochemicals, industrial intermediates and consumer products (Liu et al., 2020). When oil spills occur, benzene can leach into the environment and contaminate ecosystems. Cars and petrol stations are some of the largest sources of benzene in local environments, with residents living or working close to petrol stations being exposed to higher levels of benzene (Terres et al., 2010). Living in an industrial city, the exposure of benzene and other petrochemicals also increases. Benzene exposure is known to cause diseases such as non-Hodgkin’s lymphoma, leukaemia and multiple myeloma (W.H.O, 2010). As such, the management and removal of pollutants such as benzene is of international importance.

In order to degrade benzene, cells must have the ability to import it into the cell. Our project cloned the benzene transporter encoded by the benE gene, co-localised with the benABCD cluster. This transporter allows bacteria to import extracellular benzene/benzoate SOURCE). The degradation of benzene is encoded by the benABCD genes, involving a multi-step process that degrades benzene into catechol (Ezezika et al., 2006). The benAB genes form a complex encoding a benzoate dioxygenase, together with benC (encoding a reductase) converts the benzoate into benzoate diol. Next, benD (which encodes a dehydrogenase) will use benzoate diol and convert it into catechol (Kitagawa et al., 2001). Catechol can also be degraded into acetyl-CoA and succinyl-CoA, which are fed into the Citric Acid Cycle for ATP energy production and the formation of essential biomolecules (Ezezika et al., 2006).
Methodology
Several species of the genus Rhodococcus and Acinetobacter encode the benABCD cluster (Révész et al., 2019). We cloned the benE gene from a number of bacterial species and introduced them into the laboratory strain of E. coli. Functional assays were performed using gene knockouts of the benE gene as well as the benK gene also thought to be responsible for some benzene/benzoate transportation.

By cloning the benABCD pathway from the Rhodococcus and Acinetobacter species we have identified that the cluster can be shuttled (via a plasmid vector) to transform the commonly used model laboratory species Escherichia coli into a practically useful benzene degrader. We made microbial growth curves to test the growth characteristics of each of our species both before and after insertion of the benABCD and benE genes to quantify the success of our project.

Sourced fragments of biosensor construct from Acinetobacter. Amplified BenM binding region/BenM promoter, CatM promoter and CatM binding region. Ordered mCherry fluorscence, GFP, BenM and CatM transcription factor sequences. Attempted to join fragments together to form construct using overlap extension PCR(Hilgarth and Lanigan, 2020) and confirmed by checking size of newly formed DNA via gel electrophoresis.
Results
The benABCD genes were extracted from Rhodococcus strain 9 and Acinetobacter baylyi ADP1. The genes were then amplified and cloned into an over expression vector pTTQ18. This was then transformed into Escherichia coli and growth analysis was performed. The results of the growth curve showed that our benABCDtransformants were unable to tolerate benzoate and grow.



A growth curve analysis was undertaken in order to assess the ability of the transformants to survive in a benzoate containing solution. The strains transformed with benE were able to grow in a concentration of 15.625mM benzoate. The benE equivalent knockout strains (E. Coli ΔydcO) were unable to grow at the 15.625mM benzoate concentration. This suggests that the benE transporter was being expressed in the transformants and that it enabled these cells to import benzoate and utilise it as a carbon source.

In order to determine whether our benE transformants were able to import benzoate we undertook a high performance liquid chromatography (HPLC) analysis. Cells were grown in 15mM benzoate solution for 14 hours, following this, cells were removed and HPLC was undertaken on the solution.

The levels of benzoate remaining in the benE equivalent knockout (E. Coli ΔydcO) were similar to the levels in the 15mM benzoate solution (which did not have cells grown within it), suggesting the knockout cells did not have the ability to import benzoate.

A slight reduction can be seen in the benzoate levels of the solution which contained the Rhodococcus strain 9 benE transformants and a large reduction is seen in the levels of the solution which contained E. fergusonii benE transformants when compared to the knock out. This suggests that the benE gene was being expressed, and that the BenE protein enabled the cells to import benzene from their environments.



Our initial attempts to join the DNA fragments together to construct the biosensor were unsuccessful. However, by ordering new primers, increasing annealing temperature during PCR and using optimisation protocol (Hilgarth and Lanigan, 2020), we were able to join some fragments together. We were able to join CatM binding to mCherry, CatM gene to CatM promoter to CatM gene and BenM gene to BenM binding to GFP.

However, We were unsuccessful in transforming the constructed fragments into an organism for testing.

All the parts/DNA we were looking at.

benABCD from Rhodococcus strain 9(BBa_K3694023) and Acinetobacter baylyi ADP1(BBa_K3694024).
The benABCD gene clusters isolated from Rhodococcus strain 9, is the benzene degradation pathway degrading benzene into catechol. The cluster encodes for a benzoate 1,2-dioxygenase (benABC) which converts benzene into Cis-1,2-dihydroxycyclohexadiene. This is then converted to catechol by the benD encoded dehydrogenase

Theoritical Biosensor, sourced fragmetns from ADP1(BBa_K3694026)
Biosensor gene construct that would work in E.coli. The construct contains transcription factor CaTM and its binding site connected to a downstream fluorescent mCherry gene. The transcription factor, in the presence of its ligand catechol or cis,cis-Muconate, would bind its binding region to recruit RNAP and transcribe the downstream fluorescent protein gene. The construct also contains benM transcription factor and its binding region which is connected to a downstream green fluorescence gene. In the presence of benzene and cis,cis-Muconate, the transcription factor binds to the binding region to recruit RNAP and transcribe the downstream fluorescent protein gene.

BenE from Rhodococcus strain 9(BBa_K3694029), Acinetobacter baylyi ADP1(BBa_K3694027) and E.fergusonii(BBa_K3694028)
benE gene obtained from ADP1 genomic DNA - encodes for a transmembrane symporter which enables cells to import benzene/benzoate from their environment.
Conclusion/Future directions
Making a cell-line capable of degrading benzene and other petrochemicals will be of huge benefit to society. Currently, cleaning up oil spills requires significant manpower and is very costly (Jernelöv, 2010). However, with our system, it will be possible to use the resources provided by nature. Petrochemicals could be degraded by releasing specially engineered microbes into polluted sites. However, before engineered organisms can be released, ethical considerations need to be taken into account.



Data collected from construction of biosensor can be used by future teams to develop the full biosensor construct. This biosensor will have the ability for rapid detection of benzene, which is useful for preliminary examination of pollution in an environment.

The data we have collected from the benzoate degradation and transporter assays can be used to optimise transformation and degradation for bioremediation of local environments. These experiments help lay the foundations for the creation of a highly developed organism with many genes for degradation of complex poly-aromatic hydrocarbons. The work done here can be further built upon to contain a “suicide switch”, whereby the microbes will die off when all the hydrocarbons in the system has been degraded.
Acknowledgements
Thank you to Professor Brett Neilan, Dr Karl Hassan, Dr Benjamin Matthews, Dr Bernadette Drabsch, Joachim Steen Larsen, Evan Gibbs and Catherine Dawson.

Thank you to iGEM for creating a wonderful platform for teams around the world to showcase their skills in synthetic biology and work towards solving real-world problems.

Thank you to the following external supporters:





References

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