Production of succinic acid with engineered genetic switches in methylobacterium extorquens

琥珀酸是现代化学工业中最重要的12种分子之一。它被用于许多应用，包括生产聚合物，如PBS（聚丁琥珀酸酯）。目前，几乎所有的琥珀酸生产都来自化石碳。为了寻找其他来源，减少对石油的依赖，使琥珀酸产业能够走“绿色道路”，许多公司已经启动了以生物原料（主要是从作物中提取的糖）为原料，通过发酵生产琥珀酸的项目。然而，这种更环保的产品是有争议的，因为它转移了可能用于种植食品作物的农业用地和资源，转而用于化学工业。在过去的几年里，这一争论变得越来越重要，因此工业界开始设想依赖纤维素或废物的第二代工艺。在这个项目中，用于发酵的原料来源不与人类或动物食品竞争，而是使用通过气化（热化学转化）从废物中提取的生物甲醇。在项目的初始阶段，已经评估了利用敲诈甲基杆菌生产工业用琥珀酸的可行性。嗜甲基化菌具有消耗甲醇和柠檬酸循环的能力；因此，它能够产生琥珀酸。因此，将琥珀酸盐转化为富马酸盐的sdh基因敲除已经完成。然而，由于该基因是必需的，敲除菌株只有在添加苹果酸盐以挽救柠檬酸循环的情况下才能存活。为了避免这个问题，并实现琥珀酸产量在接近10-20g/L的范围内，我们有三个主要目标。1)设计sdh敲除菌株与可诱导的sdh互补。这需要设计和实现新的基因开关，其启动子与敲诈勒索杆菌兼容。2)同样，设计可诱导的小rna，抑制sdh，根据琥珀酸生产的需要调整表达。3)通过基因重组PHB降解途径，将以聚-ß-羟基丁酸（PHB）形式积累的生物质转化为琥珀酸。

Succinic acid is one of the 12 most important molecules in the modern chemical industry. It is used in many applications, including the production of polymers such as PBS (polybutylsuccinate). Currently, almost all of the succinic acid production originates from fossil-carbon. In order to find other sources and rely less on petroleum, so that the succinic industry can embrace a "green approach", many companies have started projects to produce succinic acid by fermentation from biological raw material, mainly sugars derived from crops. However, this greener product is controversial because it diverts agricultural land and ressources that could potentially be used for crops intended for food towards the chemical industry instead. For the past few years, this debate has been gaining in importance and thus industry is starting to envision second generation processes that would rely on cellulose or waste. In this project, the source of raw material used for fermentation does not compete with human or animal food, but rather uses bio-methanol derived from waste by gazeification (thermo-chimio-conversion). In an initial phase of the project, the feasibility of using Methylobacterium extorquens to produce succinic acid for industrial processes has been evaluated. The methylotrophic bacteria M. extorquens is capable of consuming methanol and has the citric acid cycle; it is thus capable of producing succinic acid. Therefore, a knockout of the sdh gene, which converts succinate to fumarate, has already been performed. However, because this gene is essential, the knockout strains survive only with the addition of malate to rescue the citric acid cycle. To circumvent this problem and achieve succinic acid production in a range close to 10-20g/L, we have three main objectives. 1) Engineer a sdh knockout strain complemented with an inducible sdh. This requires the design and implementation of new genetic switches with promoters compatible with M. extorquens. 2) Similarly, engineer inducible small RNAs that repress sdh to tune expression according to succinic acid production needs. 3) Convert the biomass accumulated in the form of poly-ß-hydroxybutyrate (PHB) to succinic acid by genetically rewiring the PHB degradation pathways.