SusChEM: Installing the Wood-Ljungdahl pathway in a clostridium platform organism for enhanced metabolite production

[1511660] Papoutsakis, Eleftherios T.该项目的目标是设计工业生物乙酰丁酸梭菌，使其能够利用二氧化碳、一氧化碳和氢气作为生长和产物形成的唯一底物。这将使在发酵系统中从废气中生产生物燃料（例如丁醇）和其他有用的化学物质成为可能，这种系统可以很容易地用于工业生产，并且具有卓越的效率。从基本的角度来看，在不同的生物体中安装这种复杂的原始途径的能力构成了合成生物学的重大进步，这将为其他工业微生物的途径工程和合成方法开辟新的视野。非光合化养碳利用的最有效途径是由醋酸菌（一种将大量二氧化碳固定在地球生物圈中的梭状芽孢杆菌）采用的Wood-Ljungdahl途径（WLP）。结果表明，这3种微生物的WLP基因均集中在各自基因组的高度保守区域。根据已发表的文献和研究者实验室的工作，我们选择了模型醋酸C. ljungdahlii作为大部分WLP基因的来源，这些基因将用于在C. acetobutylicum中安装WLP。该计划是将一些基因整合到染色体中，并在两个大的兼容/共存的质粒上表达其他基因。染色体整合将基于最近开发的双交叉等位基因DNA交换技术，该技术可实现无标记染色体基因缺失和DNA整合。构建的菌株将通过体外试验、生长实验和基于碳-13的碳固定试验来评估其WLP功能。该奖项由CBET部门的生物技术和生化工程项目颁发，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

1511660 Papoutsakis, Eleftherios T. The goal of this project is to engineer the industrial organism Clostridium acetobutylicum to make it capable of utilizing carbon dioxide, carbon monoxide and hydrogen, as sole substrates for growth and product formation. This will enable the production of biofuels (e.g. butanol) and other useful chemicals from waste gases in a fermentation system that can be easily scaled for industrial production, and with exceptional efficiencies. From the fundamental point of view, the ability to install this complex, primordial pathway in a different organism constitutes a major advance in synthetic biology that would open new horizons for pathway engineering and synthetic approaches in other industrial microorganisms. The most efficient pathway for non-photosynthetic chemotrophic carbon utilization is the Wood-Ljungdahl pathway (WLP) employed by acetogens, which are Clostridium organisms that fix enormous quantities of carbon dioxide in the geobiosphere. Comparative genomic analysis of the three sequenced genomes of mesophilic acetogens showed that the WLP genes of all three organisms are concentrated in a highly conserved region in their respective genomes. Based on the published literature and work from the investigator's laboratory, the model acetogen C. ljungdahlii was chosen as the source of most of the WLP genes that will be used to install the WLP in C. acetobutylicum. The plan is to integrate a number of genes into the chromosome and express other genes on two large compatible/co-existing plasmids. Chromosomal integrations will be based on a recently developed technology for double-crossover allelic DNA exchange that enables markerless chromosomal gene deletions and DNA integration. The constructed strains will be assessed for the WLP functionality through in vitro assays, growth experiments and carbon-13 based carbon-fixation assays.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.