Engineering synthetic pathways to bio-ethylene production in Cupriavidus necator

背景：目前乙烯是通过乙烷裂解生产的，乙烷会产生大量二氧化碳，导致全球变暖。 2000年，蒸汽裂解的一次能源消耗量为30亿吉焦耳，二氧化碳排放量约为2亿吨。乙烯是最常见的塑料聚乙烯的单体，全球年产量约为 8000 万吨。寻找乙烯生产的可持续或碳中性替代品势在必行。 Cupriavidus necator 是一种革兰氏阴性土壤细菌，能够在二氧化碳和氢气的作用下生长，从而能够生产低碳燃料和化学品，同时向环境释放的二氧化碳量最少。该领域的研究处于绿色革命的前沿，从可持续或碳中性来源生产生物乙烯进一步减少了全世界对化石燃料的依赖。 目的：该项目的目的是将 Cupriavidus necator 设计为生产乙烯等碳氢化合物产品的平台。作为概念证明，我们表达了丁香假单胞菌 pv 的 efe 基因（乙烯形成酶）。 paseolicolola，足以在异源宿主和 Ralstonia solanacearum 中产生乙烯。我们已经从基本介质和二氧化碳中产生了乙烯；我们现在正在通过定向进化和代谢工程来提高产量。作为该过程的一部分，我们希望设计一条利用植物的杨途径生产乙烯的合成途径。这为在 C. necator 中实施新途径并将乙烯生产与生长联系起来提供了令人兴奋的机会。乙烯是由 1-氨基环丙烷-1-羧酸 (ACC) 有效生物合成的（ACC）（Zhou 等人，2002），ACC 本身衍生自蛋氨酸，作为杨循环的一个分支（Wang 等人，2002）。该过程非常高效，因为它保留了高能蛋氨酸硫醚键。该途径利用 SAM 合酶、ACC 合酶和 ACC 氧化酶。 1-氨基-环丙烷-1-羧酸（ACC）转化为乙烯会释放氰甲酸，氰甲酸会自发脱羧释放氰化物，氰化物主要通过 CAS 途径解毒（machingura 等，2016）。该途径的实施将为 C. necator 中的氰化物解毒提供机制。培训：该项目将允许在独特的多学科环境中进行培训，包括基因组工程、气体发酵、合成生物学、尖端分子生物学和系统生物学建模。该项目将为学生提供广泛的可转移技能，这些技能在不断发展的生物经济中受到雇主的高度重视。该项目还将提供一些高影响力的出版物。该转化项目将在诺丁汉 BBSRC/EPSRC 合成生物学研究中心 (SBRC) 内进行，该中心由 70 名研究生和博士后研究人员组成 (www.clostron.com/people.php)，当前预算为 2700 万英镑。

Background: Ethylene is currently produced from stream cracking of ethane which produces large quantaties of CO2, contributing to global warming. In 2000, steam cracking had a primary energy use of 3 billion Gigajoules and accounted for approximately 200 millions tons of CO2 emissions. Ethylene is the monomer for the most common plastic, polyethylene, and annual global production is approximately 80 million tons. Finding a sustainable or carbon neutral alternative to ethylene production is imperative. Cupriavidus necator is a gram-negative soil bacterium, capable of growing on CO2 and H2 enabling low carbon fuels and chemicals to be produced with minimal release of CO2 to the environment. Research in this area is at the forefront of the green revolution and the production of bio-ethylene from sustainable or carbon neutral sources further spearheads a diminished reliance on fossils fuels throughout the world.Aim: The aim of this project is to engineer Cupriavidus necator as a platform for the production of hydrocarbon-based products such as ethylene. As proof of concept, we have expressed the efe genes (ethylene forming enzyme) from P. syringae pv. paseolicola, which is sufficient for ethylene production in heterologous hosts and Ralstonia solanacearum. We have generated ethylene from minimal media and from CO2; and we are now in the process of improving production through directed evolution and metabolic engineering. As part of this process we would like to engineer a synthetic pathway for ethylene production utlising the Yang pathway from plants. This provides an exciting opportunity to implement a novel pathway in C. necator and link ethylene production to growth. Ethylene is efficiently biosynthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) (Zhou et al., 2002), which is itself derived from methionine as a branch of the Yang cycle (Wang et al., 2002). This process is energetically efficient as it preserves the high-energy methionine thioether bond. This pathway utilises SAM synthtase, ACC synthase and ACC oxidase. The conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) to ethylene releases cyanoformic acid, which spontaneously decarboxylates to release cyanide, which is principally detoxified by the CAS pathway (machingura et al., 2016). The implementation of this pathway will provide a mechanism for detoxifiying cyanide in C. necator. Training: The project will allow for training in a unique multidisciplinary environment, incorporating genomic engineering, gas fermentation, synthetic biology, cutting edge molecular biology and systems biology modelling. The project will provide the student with a vast array of transferable skills, highly prized by employers in the growing bioeconomy. The project will also provide several high impact publications. This translational project will be carried out within the BBSRC/EPSRC Synthetic Biology Research Centre (SBRC) at Nottingham which comprises 70+ graduate and postdoctoral researchers (www.clostron.com/people.php) and a current budget of £27M.