Development of a Commercially Viable Itaconic Acid Fermentation Process

甲基丙烯酸酯目前是从石化原料中生产的，用于制造一系列散装和特种聚合物。例如，甲基丙烯酸甲酯(2-甲基丙烯酸甲酯)被用作聚甲基丙烯酸甲酯的单体，聚甲基丙烯酸甲酯是一种透明、耐紫外线、易于回收的生物相容性聚合物。该聚合物及其混合物被用于多种应用，如建筑、油漆、涂料、汽车零部件、生物医学材料以及有机玻璃和有机玻璃，从而支持了一个庞大、多样化的供应链。丙烯酸酯行业每年需要2000千吨甲基丙烯酸甲酯，市场规模为30亿美元。Lucite International在全球甲基丙烯酸酯单体、甲基丙烯酸甲酯和甲基丙烯酸的市场占有率超过35%，其中28%的产量在英国。因此，甲基丙烯酸酯的制造和使用为英国经济做出了重要贡献。对石化原料的依赖对丙烯酸酯工业未来的可持续性来说是一个越来越大的风险因素。石油储备正在迅速耗尽，这已经导致原料成本上升。从长远来看，人们对原料供应的可得性感到担忧。因此，需要向可再生原料过渡，以“防患于未然”供应甲基丙烯酸酯单体。Lucite正在探索引入一种新的混合生物和化学催化工艺的潜力，以可再生原料生产甲基丙烯酸。拟议的工艺包括通过发酵生产有机酸，然后在近临界或超临界水中碱催化脱酸以生产甲基丙烯酸。最大的优势是这两个过程都在水中运行。这避免了将有机酸从发酵液中分离出来的需要，否则将需要昂贵的结晶过程。化学催化阶段已经在EPSRC资助的诺丁汉大学CASE项目中成功开发，使用衣康酸和柠檬酸作为底物。该项目的目的是开发一种改进的发酵生产衣康酸的方法，并证明在去除细胞后，原始发酵液可以直接进入热水过程。传统的衣康酸发酵依赖于丝状真菌的使用，而且效率相当高。然而，在发酵过程中(因为真菌以颗粒形式生长)和细胞内水平都存在传质问题，因为乌头必须从线粒体转移到细胞质。这两个问题都导致了生产率的有限。此外，真菌工艺需要酸性pH，而碱催化的杂交工艺需要中性盐。因此，我们将开发工程菌来生产衣康酸。该项目将包括在大肠杆菌中过度表达柠檬酸合成酶、乌头酸水合酶和乌头酸脱羧酶，作为概念的初步验证。随后，将开发一个两阶段发酵过程，首先在有氧条件下快速生长以生产生物催化剂，然后切换到厌氧条件下使用非生长细胞生产衣康酸。虽然这将自动抑制TCA循环的下游反应，但还需要进一步的代谢工程来开发强大的制造工艺。因此，代谢模型将被用来设计高效地产生衣康酸前体的菌株，并且不会将前体和产物转移到非生产性代谢中。一些初步设计将在甲基丙烯酸生产的混合工艺中进行建造和测试。这将为未来的后续项目提供一个平台，以基于设计构建代谢工程生物催化剂，并开发完全集成的杂交过程

Methacrylates are currently produced from petrochemical feedstocks and are used to manufacture a range of bulk and specialist polymers. For example, methylmethacrylate (2-methylpropenoic acid methyl ester) is used as the monomer for polymethylmethacrylate, a transparent, UV-resistant, biocompatible polymer which can easily be recycled. The polymer and its blends are used for numerous applications, such as construction, paints, coatings, automotive components, biomedical materials and as Perspex and Plexiglas, thus supporting a large, diverse supply chain. The acrylics industry requires 2000 kilotonnes of methylmethacrylate annually and the market size is 3 Billion USD. Lucite International has over 35% of the global market share for methacrylate monomers, methyl methacrylate and methacrylic acid, with 28% of their production in the UK. Therefore, methacrylate manufacturing and use provides an important contribution to the UK economy. The dependence on petrochemical feedstocks is an increasing risk factor for the future sustainability of the acrylics industry. Oil reserves are rapidly being depleted, and this is already causing increased feedstock costs. Longer term, there are concerns over the availability of feedstock supplies. Therefore, a transition to renewable feedstocks will be required to 'future-proof' the supplies of methacrylate monomers. Lucite is exploring the potential to introduce a new Hybrid Bio- and Chemocatalytic Process to produce methacrylic acid from renewable feedstocks. The proposed process involves production of organic acids by fermentation, followed by base-catalysed decarboxylation to produce methacrylic acid in near- or supercritical water. The great advantage is that both processes operate in water. This avoids the need to separate the organic acid from the fermentation broth, which would otherwise require a costly crystallization process. The chemocatalytic stage has already been developed successfully in an EPSRC-funded CASE project at the University of Nottingham, using itaconic acid and citramalic acid as substrates. The aim of this project is to develop an improved route to produce itaconic acid by fermentation and to demonstrate that raw fermentation broth can be fed directly into the hot water process, after removing the cells. The traditional itaconic acid fermentation depends on the use of filamentous fungi, and is reasonably efficient. However, there are mass transfer problems both within the fermentation process (because the fungi grow as pellets) and at the intracellular level, because aconitate has to be transferred from the mitochondria to the cytoplasm. Both problems contribute to limited productivity. Furthermore, acid pH is required in the fungal process, whereas the base-catalysed Hybrid Process requires the neutral salt. Therefore, we shall develop engineered E. coli strains to produce itaconic acid. The project will include overexpression of citrate synthase, aconitate hydratase, and aconitate decarboxylase in E. coli for initial proof of concept. Subsequently, a two stage fermentation process will be developed, with initial, rapid growth to produce the biocatalyst under aerobic conditions, followed by a switch to anaerobic conditions to produce itaconate using non-growing cells. Although this will automatically suppress the downstream reactions of the TCA cycle, further metabolic engineering will be needed to develop a robust manufacturing process. Therefore, metabolic modelling will be used to design strains which produce itaconic acid precursors efficiently, and which do not divert the precursors and product into unproductive metabolism. Some of the preliminary designs will be constructed and tested in the Hybrid Process for methacrylic acid production. This will provide a platform for future follow-on projects to construct metabolically engineered biocatalysts, based on the designs, and to develop a fully integrated Hybrid Process