Engineering synthetic pathways to bio-ethylene production in Cupriavidus necator

背景：乙烯目前是由乙烷的裂化过程产生的，这一过程会产生大量的二氧化碳，导致全球变暖。2000年，蒸汽裂解的一次能源使用量为30亿千兆焦耳，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。寻找一种可持续的或碳中性的替代乙烯生产是势在必行的。necator Cupriavidus necator是一种革兰氏阴性土壤细菌，能够在CO2和H2上生长，从而生产低碳燃料和化学品，同时向环境释放最少的CO2。这一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：该项目的目的是设计Cupriavidus necator作为生产碳氢化合物产品（如乙烯）的平台。作为概念的证明，我们从丁香P. pv中表达了efe基因（乙烯形成酶）。在异源寄主和龙葵中产生乙烯是足够的。我们用最少的介质和二氧化碳生产乙烯；我们现在正处于通过定向进化和代谢工程提高产量的过程中。作为这个过程的一部分，我们想要设计一个合成途径，利用植物的杨途径来生产乙烯。这提供了一个令人兴奋的机会，在C. necator中实现一种新的途径，并将乙烯生产与生长联系起来。乙烯是由1-氨基环丙烷-1-羧酸（ACC）高效生物合成的（Zhou et al., 2002），它本身来源于蛋氨酸，是Yang循环的一个分支（Wang et al., 2002）。这个过程的能量效率很高，因为它保留了高能量的蛋氨酸硫醚键。该途径利用SAM合成酶、ACC合成酶和ACC氧化酶。1-氨基-环丙烷-1-羧酸（ACC）转化为乙烯释放氰甲酸，氰甲酸自发脱羧释放氰化物，主要通过CAS途径解毒（machingura et al., 2016）。该途径的实现将提供一种解毒氰化物的机制。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、前沿分子生物学和系统生物学建模。该项目将为学生提供大量可转移的技能，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。这个转化项目将在诺丁汉的BBSRC/EPSRC合成生物学研究中心（SBRC）内进行，该中心由70多名研究生和博士后研究人员组成（www.clostron.com/people.php），目前的预算为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.