21ENGBIO_pMMO in plants for methane detoxification and as a carbon negative biofuel

Objective and Hypothesis:
The main objective of the project is to express the bacterial enzyme, particulate methane monooxygenase (pMMO) in tobacco plants. We hypothesise that plants expressing this enzyme will metabolise methane and turn this greenhouse gas into the less potent carbon dioxide whilst producing biomass for downstream biofuel processes. Such plants will be valuable in detoxifying soil high in methane, for example wetlands, ex-landfill sites or paddy fields.

Background:
Methane is a potent greenhouse gas; its impact on climate change is over 20 times greater than carbon dioxide. Globally, over 60% of methane emissions come from human activities including industrial gas and petroleum systems, livestock, artificial wetlands, and landfills. Methanotrophic bacteria, organisms that live on methane gas as their carbon source, function as the only biological methane sink and perform a critical role in the global carbon cycle. Their particulate methane monooxygenase (pMMO) is the predominant methane oxidation catalyst in nature. Present in nearly all methanotrophs it converts methane into carbon dioxide producing methanol as a by-product.

Model system: Although recent progress has been made in developing transformation protocols for important plants such as soybean, tomato, and lettuce, most studies to date use tobacco as a model system for chloroplast transformation, and hence we will use tobacco. This will provide a proof-of concept but also a usable plant system for field trials.

Work plan:
To create such plants, we will produce the pMMO complex in plant chloroplasts as well as on the endoplasmic reticulum. Chloroplast have a separate genome to the nuclear genome. pMMO insertion in the chloroplast genome is technically more challenging but has the following advantages: Chloroplast can produce and store large amounts of foreign proteins. Chloroplasts also provide better transgene containment due to the maternal inheritance of chloroplasts, which excludes chloroplasts and therefore the transgenes from pollen transmission.
Nuclear transformation and targeting to the endoplasmic reticulum (ER) -the cell's protein production site- is less challenging and therefore is used as an alternative approach to create pMMO-producing plants. The ER by nature has great capacity for protein expression and complex assembly.
We will test these plants for their capability of detoxifying methane as well as for their general health and fertility.

Significance:
As a proof of concept we will express pMMO in tobacco. These plants can be used as green catalysts to convert methane to carbon dioxide. This will additionally produce biomass for downstream biofuel processes allowing for a 'carbon-negative' biofuel. The by-product methanol has been shown to stimulate plant growth increasing the resulting biomass for biofuel production. Such plants can be grown on soil high in methane, for example wetlands or rice paddy fields for detoxification purposes and ultimately for biomass production. Eventually this project should lead to field testing and industry collaborations. For example, transforming rice with pMMO could be of invaluable benefit as methane emissions from rice agriculture are a major environmental problem. Paddy fields account for around 20% of human-related methane emissions.

Summary:
We will express the bacterial enzyme pMMO in tobacco plants. We hypothesise that plants expressing this enzyme will metabolise methane and turn this greenhouse gas into the less potent carbon dioxide whilst producing biomass for downstream biofuel processes. Such plants will be valuable in detoxifying soil high in methane, for example wetlands, ex-landfill sites or rice paddy fields.

Methane is a potent greenhouse gas with a 20 times higher impact on global warming than carbon dioxide. Methanotrophic bacteria feed on methane and convert it to carbon dioxide, producing methanol as a by-product. For this, bacteria utilise a specific enzyme, the particulate methane monooxygenase (pMMO). pMMO is the predominant methane oxidation catalyst in nature and has the potential to enable conversion of the potent greenhouse gas methane into the less potent carbon dioxide. We will modify tobacco plants to express pMMO, enabling them to detoxify methane. The by-product methanol will enhance plant growth and produce biomass for downstream biofuel processes. Such plants can be grown for detoxification purposes on soil high in methane, e.g. wetlands, ex-landfill sites or rice paddy fields.

To achieve this, we will apply two plant transformation approaches: a) chloroplast transformation using gene bombardment and b) Agrobacterium-mediated nuclear transformation.

a) pMMO is composed of three subunits coded for by a single bacterial operon. The expression of bacterial operons and production of active enzymes consisting of various subunits is possible in plant chloroplasts due to the prokaryotic nature of this organelle.

b) As a parallel approach we will use nuclear transformation using constructs with self-cleaving peptides to produce the three subunits simultaneously in one plasmids under one promoter. Self-cleaving peptides are coded for between the individual pMMO subunits. This results in the production of one polypeptide which is then cleaved at the self-cleaving peptides by inducing ribosomal skipping during translation resulting in individual subunits with the correct stoichiometry.

The plants created will be tested for their pMMO enzymatic capacity in vitro and in vivo as well as for health and fertility.