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

目的与假设：该项目的主要目标是在烟草植物中表达细菌酶颗粒甲烷单加氧酶（pMMO）。我们假设，表达这种酶的植物将代谢甲烷，并将这种温室气体转化为效力较低的二氧化碳，同时为下游生物燃料过程生产生物质。这种植物在对甲烷含量高的土壤解毒方面很有价值，例如湿地、前垃圾填埋场或稻田。背景：甲烷是一种强有力的温室气体;它对气候变化的影响比二氧化碳大20倍以上。在全球范围内，超过60%的甲烷排放来自人类活动，包括工业气体和石油系统，牲畜，人工湿地和垃圾填埋场。甲烷营养菌是以甲烷气体为碳源的生物体，是唯一的甲烷生物汇，在全球碳循环中起着关键作用。它们的颗粒甲烷单加氧酶（pMMO）是自然界中主要的甲烷氧化催化剂。它存在于几乎所有的甲烷氧化菌中，将甲烷转化为二氧化碳，产生甲醇作为副产品。模型系统：尽管最近在开发大豆、番茄和生菜等重要植物的转化方案方面取得了进展，但迄今为止的大多数研究都使用烟草作为叶绿体转化的模型系统，因此我们将使用烟草。这将提供一个概念验证，但也是一个可用的植物系统，用于田间试验。工作计划：为了创造这样的植物，我们将在植物叶绿体以及内质网中产生pMMO复合物。叶绿体具有与核基因组分开的基因组。在叶绿体基因组中插入pMMO在技术上更具挑战性，但具有以下优点：叶绿体可以产生和储存大量外源蛋白。由于叶绿体的母系遗传，叶绿体也提供了更好的转基因包容性，从而排除了叶绿体，因此转基因从花粉传播。核转化和靶向内质网（ER）-细胞的蛋白质生产位点-挑战性较低，因此被用作创建pMMO生产植物的替代方法。ER在自然界中具有很强的蛋白质表达和复杂组装能力。我们将测试这些植物对甲烷的解毒能力以及它们的一般健康和生育能力。意义：作为概念验证，我们将在烟草中表达pMMO。这些植物可用作将甲烷转化为二氧化碳的绿色催化剂。这将额外产生用于下游生物燃料工艺的生物质，从而允许“负碳”生物燃料。副产品甲醇已被证明可以刺激植物生长，增加生物燃料生产的生物量。这种植物可以生长在甲烷含量高的土壤上，例如湿地或稻田，用于解毒目的，并最终用于生物质生产。最终，该项目将导致现场测试和行业合作。例如，用pMMO转化水稻可能具有非常宝贵的效益，因为水稻农业的甲烷排放是一个主要的环境问题。稻田约占人类甲烷排放量的20%。总结：我们将在烟草植物中表达细菌酶pMMO。我们假设，表达这种酶的植物将代谢甲烷，并将这种温室气体转化为效力较低的二氧化碳，同时为下游生物燃料过程生产生物质。这种植物在对甲烷含量高的土壤解毒方面很有价值，例如湿地、前垃圾填埋场或稻田。

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.