Development of a Commercially Viable Itaconic Acid Fermentation Process

开发商业上可行的衣康酸发酵工艺

• 批准号: BB/I016562/1
• 负责人: Gillian Stephens
• 金额: $ 11.71万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI016562%2F1
• 关键词: Development; Commercially; Viable; Itaconic; Acid

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

甲基丙烯酸酯目前由石化原料生产，用于制造一系列本体和专用聚合物。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的、抗紫外线的、生物相容的聚合物，可以很容易地回收。该聚合物及其共混物用于多种应用，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，从而支持大型、多样化的供应链。丙烯酸工业每年需要2000千吨甲基丙烯酸甲酯，市场规模为30亿美元。璐彩特国际公司在甲基丙烯酸酯单体、甲基丙烯酸甲酯和甲基丙烯酸的全球市场占有率超过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。对石化原料的依赖是丙烯酸工业未来可持续性的一个日益增加的风险因素。石油储量正在迅速枯竭，这已经导致原料成本增加。从长远来看，人们对原料供应的可用性感到担忧。因此，需要向可再生原料过渡，以“面向未来”的甲基丙烯酸酯单体供应。璐彩特正在探索引入一种新的混合生物和化学催化工艺的潜力，以从可再生原料中生产甲基丙烯酸。所提出的工艺涉及通过发酵生产有机酸，然后在近或超临界水中进行碱催化脱羧以生产甲基丙烯酸。最大的优点是这两个过程都在水中进行。这避免了从发酵液中分离有机酸的需要，否则将需要昂贵的结晶过程。化学催化阶段已经在诺丁汉大学EPSRC资助的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途径，并证明在去除细胞后，可以将原始发酵液直接进料到热水工艺中。传统的衣康酸发酵依赖于丝状真菌的使用，并且相当有效。然而，在发酵过程中（因为真菌作为颗粒生长）和细胞内水平都存在传质问题，因为乌头酸盐必须从线粒体转移到细胞质。这两个问题都导致生产力有限。此外，在真菌过程中需要酸性pH，而碱催化的混合过程需要中性盐。因此，我们将开发工程E。大肠杆菌菌株产衣康酸。该项目将包括在大肠杆菌中过量表达柠檬酸合酶、乌头酸水合酶和乌头酸脱羧酶。大肠杆菌进行初步的概念验证。随后，将开发两阶段发酵工艺，初始快速生长以在需氧条件下生产生物催化剂，然后切换到厌氧条件以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，但需要进一步的代谢工程来开发稳健的制造工艺。因此，代谢建模将用于设计菌株，其有效地产生衣康酸前体，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构建和测试。这将为未来的后续项目提供一个平台，以构建基于设计的代谢工程生物催化剂，并开发一个完全集成的混合过程