Genetic engineering and strain optimization of Clostridium ljungdahlii for the production of biobutanol by syngas fermentation

在世界范围内，能源需求不断增加，而化石能源载体的来源有限。必须开发生产可再生能源、运输燃料和大宗商品的路线。今天使用的最突出的生物燃料是从甘蔗、玉米或小麦(第一代燃料)中生产的乙醇。木质纤维素生物质被认为在克服土地利用冲突等与第一代燃料相关的问题方面发挥了重要作用，而不是将这些食用植物用于生产生物燃料。此外，正丁醇作为乙醇的替代品具有更高的能量密度和更好的燃烧性能。从木质纤维素生产第二代生物燃料有两个平台：糖和合成气(合成气)平台。在糖平台中，木质纤维素通过加热、化学物质和酶的广泛预处理转化为糖和木质素，并进一步发酵成生物醇。合成气平台(热化学路线)使用气化或缓慢热解消化的木质纤维生物质来生产合成气，合成气主要是一氧化碳(CO)、氢(H2)、二氧化碳(CO2)和氮气(N2)的混合物。然后，所产生的合成气可以在热化学费托工艺中转化为中到长链醇。在这个过程中，化学催化剂被气体中的杂质毒化，产物取决于混合气体的组成，或者，羧基营养细菌可以在称为合成气发酵的过程中专门产生多碳化合物，如醋酸酯、乙醇或正丁醇。利用合成气平台，利用羧基营养的同产乙酸菌发酵合成气为生物醇和/或乙酸酯，在反应器设计方面取得了可观的进展。正丁醇的生产仍然具有挑战性。另一个巨大的挑战是缺乏足够的遗传工具来通过基因工程来优化这些生物。拟议的工作旨在建立合成气发酵生产正丁醇作为第二代生物燃料和/或平台化学品，并通过以下几个方面优化工艺：1)通过基因工程优化合成气发酵生物永达氏梭菌；2)优化发酵条件，如合成气和羧化底物正丁酸酯的共加。

Worldwide the energy demands are increasing and sources of fossil energy carriers are limited. Routes to produce renewable energy, transportation fuels and commodities have to be developed. The most prominent biofuel used today is ethanol produced from sugar cane, corn or wheat (1st generation fuel). Instead of using these food plants for the production of biofuels, lignocellulosic biomass is considered to play an important role in overcoming problems associated with 1st generation fuels such as land use conflicts. Furthermore, n-butanol as an alternative to ethanol offers advantages such as a higher energy density and better combustion properties. There are two platforms described for the production of 2nd generation biofuels from lignocellulose: the sugar and the synthesis gas (syngas) platform. In the sugar platform, lignocellulose is converted into sugars and lignin by extensive pretreatment with heat, chemicals, and enzymes, and the sugars are further fermented into bioalcohols. The syngas platform (thermochemical route) uses gasification or slow pyrolysis of the indigestive lignocellulosic biomass to produce syngas, a mixture of mainly carbon monoxide (CO), hydrogen (H2), carbon dioxide (CO2) and nitrogen (N2). The produced syngas can then be converted into medium to long chain alcohols in the thermo-chemical Fischer-Tropsch process. While in this process chemical catalysts are poisoned by impurities in the gas and the products are depending on the gas mixture composition, alternatively, carboxydotrophic bacteria can be used to specifically produce multicarbon compounds, such as acetate, ethanol, or n-butanol, in a process called syngas fermentation.Considerable progress is made in reactor design to use carboxidotrophic, homoacetogenic bacteria for the fermentation of syngas into bioalcohols and/ or acetate using the syngas platform. The production of n-butanol is still challenging. Another big challenge is the lack of sufficient genetic tools to optimize these organisms via genetic engineering.The proposed work aims to establish syngas fermentation to produce n-butanol as a 2nd generation biofuel and/ or platform chemical and optimize the process through: 1) optimization of the syngas fermenting organism Clostridium ljungdahlii by genetic engineering; and 2) the optimization of fermentation conditions such as co-feeding of syngas and the carboxylate substrate n-butyrate.