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。目前使用的最主要的生物燃料是从甘蔗、玉米或小麦中提取的乙醇（第一代燃料）。木质纤维素生物质被认为在克服与第一代燃料相关的问题（如土地使用冲突）方面发挥了重要作用，而不是使用这些粮食植物生产生物燃料。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等优点。从木质纤维素生产第二代生物燃料有两个平台：糖和合成气平台。在糖平台中，木质纤维素通过热、化学和酶的广泛预处理转化为糖和木质素，糖进一步发酵成生物醇。合成气平台（热化学路线）利用气化或缓慢热解消化木质纤维素生物质生产合成气，主要是一氧化碳（CO）、氢气（H2）、二氧化碳（CO2）和氮气（N2）的混合物。产生的合成气可以在热化学费托法中转化为中链到长链醇。在这个过程中，化学催化剂被气体中的杂质毒害，产物取决于气体混合物的组成，或者，在一个称为合成气发酵的过程中，羧营养细菌可以用来专门生产多碳化合物，如醋酸盐、乙醇或正丁醇。在反应器设计方面取得了相当大的进展，利用羧营养、同产丙酮的细菌，利用合成气平台将合成气发酵成生物醇和/或乙酸。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优化这些生物体。本研究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，并通过以下途径对该工艺进行优化：1)通过基因工程对合成气发酵菌隆达氏梭菌进行优化；2)发酵条件的优化，如合成气与羧酸底物正丁酸酯共投料。

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.