Metabolic engineering of the flow of reducing equivalents for the production of biochemicals in Escherichia coli.

摘要

In the present thesis, metabolic engineering principles have been applied to strategically design E. coli strains with improved characteristics for the production of biochemicals. The metabolic engineering discipline combines molecular biology techniques, such as gene inactivation or gene overexpression, with an engineering perspective to design and construct more efficient biological systems to increase product yield and productivity.Microorganisms naturally produce a wide variety of compounds of industrial interest, e.g. antioxidants, polymers, amino acids, hydroxyacids and chiral alcohols, among others. However, in many cases the production processes are not economically feasible due to low product yield, low productivity, and/or difficulties on cultivating the native producer. Product yield and productivity are affected by a variety of factors. For instance, the generation of side-products limits the amount of carbon, other nutrients and energy directed to the synthesis of the compound of interest and the requirement of reducing equivalents, NAD(P)H, in stoichoimetric quantities for many enzymatic reactions. These compounds are expensive, although, they can be regenerated in vivo, but the regeneration rate may be the limiting factor in the process. Also, the metabolic pathway used by the cells for product synthesis is critical, different pathways leading to the same product could require different precursors, and have different reducing equivalents and energy requirements.Specifically, this thesis includes the design, construction and testing of strains for the production of NADH-dependent C4 compounds that are naturally produced in low quantities in E. coli such as succinate and malate, and for the production of NADPH-dependent biochemicals naturally produced by other organisms, such as the antioxidant lycopene and epsilon-caprolactone, where the genes encoding the enzymes in the corresponding pathway were heterogously expressed in E. coli. The strategic design included the inactivation of genes involved in the synthesis of side-products, the overexpression of heterologous genes for the production of non-native compounds, the replacement of a gene involved in E. coli central metabolic pathway to increase the availability of reducing equivalents required in the synthesis of the compound of interest, and the construction of multiple engineered strains to be used to study the fundamentals of redox balance in the cell.