Production of succinic acid with engineered genetic switches in methylobacterium extorquens

丁二酸是现代化学工业中最重要的12个分子之一。它被用于许多应用，包括生产聚合物，如PBS(聚丁二酸丁酯)。目前，几乎所有的丁二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，使丁二酸行业能够采用“绿色途径”，许多公司已经开始了利用生物原料发酵生产丁二酸的项目，生物原料主要是来自农作物的糖。然而，这种更环保的产品存在争议，因为它将可能用于种植粮食的农田和资源转移到化学工业。在过去的几年里，这场辩论变得越来越重要，因此工业界开始设想使用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是与人类或动物性食品竞争，而是使用从沼泽化(热嵌合转化)废物中提取的生物甲醇。在该项目的初始阶段，已经评估了使用甲基萃取菌生产用于工业过程的琥珀酸的可行性。甲基营养细菌M.extorquens能够消耗甲醇，并具有柠檬酸循环；因此它能够产生琥珀酸。因此，已经进行了SDH基因的敲除，该基因将琥珀酸转化为富马酸。然而，因为这个基因是必不可少的，所以基因敲除的菌株只有在添加苹果酸来挽救柠檬酸循环的情况下才能存活下来。为了绕过这个问题，使丁二酸的产量接近10-20g/L，我们有三个主要目标。1)设计SDH基因敲除株，并辅以可诱导的SDH。这需要设计和实施新的基因开关，其启动子与Extorquens相容。2)类似地，根据琥珀酸生产的需要，设计抑制SDH的可诱导小RNA来调节表达。3)通过基因重组PHB降解途径，将聚羟基丁酸酯(PHB)积累的生物量转化为丁二酸。

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.