13TSB_CRD: HIGH PRODUCTIVITY HOMOFERMENTATIVE PROCESS for BUTANOL (HIPHOP)

丙酮-丁醇-乙醇（ABE）发酵使用来自梭菌属的厌氧细菌以典型的A：B：E为3：6：1的比例将糖或淀粉发酵成溶剂混合物。从历史上看，发酵被用来制造溶剂和化学品，但当石油工业开发出更便宜的方法来制造这些化学品时，它就失宠了。随着人们对石油安全和全球变暖的日益关注，ABE发酵现在正在经历一场大规模的复兴。如今，丁醇是人们的首选产品，因为它可以用作生物燃料、溶剂和制造多种化学品的中间体。目前，丁醇发酵效率低下，因为积累的丁醇会毒害细菌，最终导致发酵停止。此外，生产丁醇的梭菌属物种在遗传上是不稳定的，因此发酵只能在关闭和从新鲜细胞重新启动之前运行很短的时间。回收丁醇也存在问题，因为产品流是丁醇、丙酮和乙醇在水中的稀释混合物。蒸馏提供了唯一简单的方法来回收产品，但需要大量的能源。我们将使用合成生物学来生产新的梭菌菌株，这些菌株可以生产丁醇，而不会形成丙酮和乙醇-同型发酵菌株。绿色生物公司的科学家们已经利用传统的诱变技术开发出了同型发酵突变体，并对它们的DNA进行了测序，以确定突变的基因。在这个项目中，我们将选择最重要的突变，并在商业生产菌株中重新创造它们，以开发一种遗传稳定、高产丁醇的微生物。新的微生物将产生更清洁的产品流，允许开发基于液-液萃取的新分离工艺。这包括将生长的培养物与水不混溶的溶剂混合，该溶剂比水更有效地溶解丁醇。因此，丁醇将转移到溶剂相中，溶剂相可以通过使两种不混溶的液相沉降出来（如油和水）而容易地分离。这为解决丁醇毒性问题提供了一个非常简洁的方法，因为丁醇被从细胞周围的直接环境中去除，所以细胞不会暴露在有毒的产品中。这使得丁醇的生产可以持续到溶剂相饱和，这样细胞就可以形成非常高浓度的丁醇。原位溶剂萃取依赖于找到一种与水不混溶的液体，这种液体不仅是丁醇的良好溶剂，而且对细胞无毒。大多数传统溶剂难以从水中提取丁醇，而且与丁醇本身一样有毒。然而，诺丁汉大学的科学家们发现，一种名为离子液体（IL）的新型溶剂可以从水中提取丁醇，并且对活细胞无毒。离子液体是由在室温下熔化的盐制成的，因此不像传统的溶剂那样具有挥发性。因此，丁醇可以很容易地回收，只需分离IL相，然后煮沸丁醇，留下IL用于再利用。总体而言，该项目将合成生物学和创新的生物分离技术结合起来，开发单一产品，高生产率丁醇发酵，以及简单，低能耗的产品纯化工艺。该项目的最后一部分将把这些技术结合起来，开发一种长期稳定运行的丁醇连续生产工艺。该工艺将利用新的工程菌株的遗传稳定性、简化的丁醇分离和通过原位丁醇回收来减轻产物抑制。新工艺将提供比传统分批发酵显著更高的生产率，从而改变丁醇生产的经济性

The acetone-butanol-ethanol (ABE) fermentation uses anaerobic bacteria from the genus Clostridium to ferment sugars orstarch to solvent mixtures in a typical A:B:E ratio of 3:6:1. Historically, the fermentation was used to manufacture solventsand chemicals, but fell out of favour when the oil industry developed cheaper ways to make these chemicals. With growingconcerns about oil security and global warming, the ABE fermentation is now undergoing a massive revival. Today, butanolis the preferred product, since it can be used as a biofuel, a solvent and an intermediate to manufacture a wide range ofchemicals.At present, butanol fermentations are inefficient because the accumulating butanol poisons the bacteria, ultimately causingthe fermentation to stop. Furthermore, butanol-producing Clostridium species are genetically unstable, so the fermentationcan only be run for a short time before shutting down and restarting from fresh cells. There are also problems withrecovering the butanol, because the product stream is a dilute mixture of butanol, acetone and ethanol in water. Distillationprovides the only easy way to recover the products, but requires a lot of energy.We will use synthetic biology to produce new Clostridium strains that produce butanol without forming acetone and ethanol- homofermentative strains. Scientists at Green Biologics have already developed homofermentative mutants usingtraditional mutagenesis techniques and have sequenced their DNA, to identify the mutated genes. In this project, we willselect the most important mutations and recreate them in a commercial production strain to develop a genetically stable,high productivity butanol-producing organism.The new organisms will produce much cleaner product streams, allowing development of new separation processes, basedon liquid-liquid extraction. This involves mixing the growing culture with a water-immiscible solvent that dissolves thebutanol more efficiently than water. As a result, the butanol will transfer into the solvent phase, which can easily beseparated by allowing the two immiscible liquid phases to settle out (like oil and water). This provides a very neat way tosolve problems with butanol toxicity, because the butanol is removed from the immediate environment surrounding thecells, so the cells are not exposed to the poisonous product. This allows butanol production to continue until the solventphase is saturated, so that the cells can form very high butanol concentrations.In situ solvent extraction depends on finding a water-immiscible liquid that is not only a good solvent for butanol but is alsonot poisonous to the cells. Most conventional solvents struggle to extract butanol from water and are just as poisonous asbutanol itself. However, scientists at the University of Nottingham have discovered that a new class of solvents called ionicliquids (ILs) can extract butanol from water and are not poisonous to living cells. ILs are made from salts that are molten atroom temperature, and so are non-volatile, unlike conventional solvents. Therefore, the butanol can easily be recovered,simply by separating the IL phase and then boiling off the butanol, leaving the IL behind for re-use.Overall, this project brings together synthetic biology and innovative bioseparations to develop a single-product, highproductivity butanol fermentation, together with a simple, low energy process for product purification. The last part of theproject will bring these technologies together to develop a continuous process for butanol production with stable operationover long periods. This process will exploit the genetic stability of the new, engineered strains, the simplified butanolseparation and the relief of product inhibition by in situ butanol recovery. The new process will provide significantly greaterproductivity than conventional batch fermentations, thus transforming the economics of butanol production