Metabolic engineering of escherichia coli for the production of diamines

Abstract

A diamine is a chemical that contains two amino groups. Diamines are important raw materials with wide applications in the nitrogen containing chemical industry. For instance, they have been used in the production of pharmaceuticals, agrochemicals, surfactants and other additives. The linear carbon chain diamines with 3 to 5 carbon atoms are particularly interesting. Therefore, this thesis aims to the production of C3 1,3-diaminopropane, C4 1,4-diaminobutane (also referred to as putresine), and C5 1,5-diaminopentane (also referred to as cadaverine). Currently, the three diamines are industrially produced mainly from chemical synthesis. The chemical routes are undesirable from an environmental standpoint. Therefore, there exists substantial need for biotechnological production of those diamines. Here, the bacterium Escherichia coli was engineered to overproduce the diamines from a renewable carbon source (glucose). The thesis focuses on putrescine because it is most important in terms of the market. Putrescine is a natural product found only inside the cells of wildtype E. coli at physiological levels (approximately 30 millimolar). High-titer production of putrescine in the culture media has been achieved through rational pathway design and systems metabolic engineering of E. coli. First, a base strain was constructed by inactivating the putrescine degradation and utilization pathways, and decreasing the metabolic flux through the bypass pathway (arginine biosynthesis). Next, ornithine decarboxylase, which converts ornithine to putrescine, was amplified by a combination of plasmid- and chromosome-overexpression of the coding genes. Furthermore, the ornithine biosynthetic genes (argC-E) were overexpressed from the strong trc promoter through genome engineering. Finally, the global regulator of the general stress response, RNA polymerase subunit RpoS, was deleted. The final engineered E. coli strain produced putrescine with a yield of 0.17 g per gram of glucose, ...