co-production of 1,3 propanediol and 3HP from glycerol by clostridium pasteurianum

为了与现有的基于石油化工的化学制造工艺竞争，用于生物发酵工艺的低成本原料是必不可少的，因为原料通常相当于总生产成本的60%以上。甘油是一种多用途的碳和能源，目前它作为生物柴油工业的主要副产品（10%w/w）大规模生产，现在基本上是一种废物。梭菌属物种，特别是巴氏梭菌，可以在甘油作为唯一的碳源和能源上生长得特别好，并将其转化为许多有用的化学品，如1，3-丙二醇（PDO）、丙酮、丁醇、乙醇等。到目前为止，由于缺乏基因工程工具，使用梭菌属菌株从甘油高水平生产化学品的机会受到限制。然而，SBRC诺丁汉的梭菌研究小组已经开发出优化巴氏梭菌丁醇生产所需的代谢途径所需的必要工具箱。本博士项目的目的是利用合成生物学方法和诺丁汉大学的工具箱来产生C.巴氏杆菌能够使用甘油作为原料过量生产许多有用的化学品。最初的重点将是重要的平台化学品3-羟基丙酸（3-HP），它可以作为生产丙烯酸（与相应的酯一起代表100亿美元的市场）、丙烯酰胺、丙二酸、聚（羟基丙酸酯）、1，3-丙二醇和丙内酯的起点。所采取的方法将是既引入编码必需酶的基因，又敲除指定竞争途径的基因。这将涉及对现有工具进行必要的改进，包括生成无限制宿主和使用CRISPR/Cas9技术。之后，其他产品可能成为目标。

To compete with existing petrochemical-based chemical manufacturing processes, low cost feedstocks for biological fermentation processes are essential, since the feedstock typically equates to over 60% of the overall production cost. Glycerol is a versatile carbon and energy source and presently it is produced in large scale as the principle by-product (10% w/w) of the biodiesel industry and it is now essentially a waste product. Clostridial species, and in particular Clostridium pasteurianum, can grow particularly well on glycerol as a sole carbon and energy source and transform it into a number of useful chemicals such as 1,3-propanediol (PDO), acetone, butanol, ethanol etc. Until now, the opportunity to use Clostridium strains for the high level production of chemicals from glycerol has been limited due to the lack of genetic engineering tools. However, the requisite tool box needed for the optimisation of the metabolic pathways required for butanol production by Clostridium pasteurianum has been developed within the Clostridia Research Group of SBRC Nottingham. It is the purpose of this PhD project to use synthetic biology approaches and the University of Nottingham tool box to generate an engineered strain of C. pasteurianum able to over-produce a number of useful chemicals using glycerol as a feedstock. The initial focus will be the important platform chemical 3-hydroxypropanoic acid (3-HP) which can serve as a starting point for production of acrylic acid (representing a $ 10 billion market together with the respective esters), acrylamide, malonic acid, poly(hydroxypropionate), 1,3-propanediol, and propiolactone. The approach taken will be to both introduce genes encoding the requisite enzymes and to knock-out genes specifying competing pathways. This will involve bringing about essential refinements to the tools available, including the generation of restriction minus hosts and the use of CRISPR/Cas9 technology. Thereafter, other products may be targeted.