Rational Engineering of Advanced Clostridia for Transformational Improvements in Fermentation (REACTIF)

正丁醇是一种有价值的平台化学品，市场规模很大（> 300万吨/年）。它被用作各种聚合物、塑料和溶剂的化学前体，全球市场价值约80亿美元。平均增长率约为3.2%，需求集中在N。美国（28%）、西欧（23%）和东北亚（35%）。目前几乎所有的正丁醇都是合成的，由丙烯（从石油中提取）生产。发酵法生产丁醇是一种古老的工业工艺，1912年在英国发明，并迅速传播到美国。美国、南非、日本和俄罗斯。它依赖于一种名为梭菌的细菌，这种细菌发酵富含糖的液体并将其转化为丁醇，类似于酵母用于酿造乙醇的方式。商业丁醇发酵工艺在20世纪50年代中期变得不经济，因为它不再能与来自低成本油的石油衍生等价物竞争，并逐渐消失。最近，该发酵已在中国重新商业化，至少有6个新工厂，产能超过300，000吨溶剂/年。然而，这些植物正在挣扎，因为技术不够先进，细菌菌株产生少量丁醇（由于丁醇毒性），并且它们依赖昂贵的富含淀粉的植物材料作为发酵的糖源。除了这些原料的高昂成本之外，它们用于此目的与作为人类消费的食物的可能用途相冲突。因此，需要从非食用植物来源的材料（所谓的木质纤维素组分）获得所用的糖。木质纤维素是植物中最丰富的碳源。然而，它特别难以分解，所需的治疗（酸、碱或蒸汽爆破）转化为糖通常会导致形成抑制细菌生长的副产物，即所谓的“发酵剂”。目的和益处如今，发酵技术的进步和廉价原料的利用，与用于菌株改良的新的基于基因的方法一起具有改变发酵过程经济学的潜力。我们已经召集了一批杰出的学术和工业合作伙伴，他们在梭菌发酵方面具有特定的专业知识。工业合作伙伴为商业供应链（从原料到丁醇生产）搭建桥梁，而学术团体则专注于使用一系列先进的分子生物学技术提供先进的微生物和上级发酵性能。我们的目标是：1。鉴定和鉴定耐受性性状（对高丁醇滴度和木质纤维素衍生的抑制剂），在四十多年来商业使用的历史菌株和在实施新的定向进化策略后的当前生产菌株中;2.将所鉴定的等位基因转移到当前的、稳健的生产菌株中，连同合理的代谢工程以提高产物滴度，以及;3.在实验室和中试规模上评估菌株对纤维素原料的性能应用和效益该项目将提供先进的梭菌菌株，为丁醇生产的发酵性能和工艺经济性提供转型性变化。先进的菌株不仅可以提高发酵性能，还可以使工厂业主转向更低成本和更环保的纤维素原料。与化石燃料相比，从可再生植物生物质生产丁醇将净减少温室气体排放，有助于减少全球变暖并减少环境污染对人类健康的影响。生物丁醇有可能直接取代所有合成丁醇，满足化学工业中对成本效益高的可再生化学品替代品的特定市场需求，特别是在欧洲和美国。美国

CONTEXT n-Butanol is a valuable platform chemical with a large market (>3 M tonne/yr). It is used as a chemical precursor for a variety of polymers, plastics and solvents with a global market worth ~$8 billion). Average growth is about 3.2% with demand concentrated in N. America (28%), Western Europe (23%) and North East Asia (35%). Virtually all n-butanol today is synthetic and produced from propylene (derived from oil). The fermentation route to butanol is an old industrial process that was developed in the UK in 1912 and spread quickly to the N. America, S.Africa, Japan and Russia. It is reliant on a bacteria called Clostridia, which ferments sugar-rich liquors and converting them to butanol, in a similar way to which yeasts is employed in brewing to make ethanol. The commercial butanol fermentation process became uneconomic in the mid 1950's when it could no longer compete with petro-derived equivalents from low cost oil and gradually disappeared. More recently, the fermentation has been re-commercialized in China with at least 6 new plants and capacity for >300,000 T solvent/yr. However, these plants are struggling because the technology is not sufficiently advanced, the bacterial strains produce low amounts of butanol (due to butanol toxicity) and they rely on expensive starch-rich plant material as the source of sugar for the fermentation. Aside from the prohibitive cost of such feedstocks, their use for this purpose conflicts with a possible use as a food for human consumption. There is, therefore, a need to derive the sugar used from non-food plant derived material, the so-called lignocellulose component. Lignocellulose is the most abundant source of carbon on the plant. However, it is particularly recalcitrant to breakdown, and the treatments required (acid, alkali or steam explosion) to its conversion into sugar frequently leads to the formation of by-products that inhibit bacterial growth, so-called 'inhibitors'.AIMS & OBJECTIVESToday, advances in fermentation technology and the utilization of cheaper feedstocks, together with novel gene-based methods for strain improvement have the potential to transform fermentation process economics. We have assembled an exceptional group of academic and industrial partners, with specific expertise in the Clostridia fermentation. The industrial partners bridge the commercial supply chain (from feedstock to butanol production) whilst the academic groups focus on delivery of advanced microbes and superior fermentation performance using an array of advanced molecular biology techniques. Our objectives are to:-1. identify and characterise tolerance traits (to high butanol titres and lignocellulose-derived inhibitors), both in historical strains that were used commercially over four decades and in current production strains following the implementation of novel directed evolution strategies;2. transfer the identified alleles into current, robust production strains, together with rationale metabolic engineering to improve product titres, and;3. assessment of strain(s) performance on cellulosic feedstocks at lab and pilot scaleAPPLICATIONS AND BENEFITSThe project will deliver advanced Clostridia strains that offer a transformational change to fermentation performance and process economics of butanol production. Advanced strains not only improve fermentation performance but they will enable plant owners to switch to lower cost and more environmentally friendly cellulosic feedstocks. The production of butanol from renewable plant biomass as opposed to fossil fuels will provide a net reduction in greenhouse gas emission, contributing to a reduction in global warming and reducing the effects of environmental pollution on human health. Biobutanol has the potential to directly replace all synthetic butanol addressing a specific market need in the chemical industry for cost efficient renewable chemical replacements, particularly in Europe & N. America