Production of metabolites in recombinant Escherichia coli via protein protein scaffold

Abstract

Metabolic engineering has become an attractive alternative to chemical synthesis. They have the potential to provide environmentallysafe and cost-effective routes for synthesizing a range of compounds, from high-value specialty compounds such as therapeutics to bulk commodities including plastics and biofuels.However, engineered metabolic pathways often suffer from flux imbalances such as typically lack the regulatory mechanisms characteristic of natural metabolism. The interaction among different enzymes in the same metabolic pathway plays an important role in the metabolic processes. Consequently, protein scaffolds were being created to adopt a spatial co-localization in metabolic engineering, drawing inspiration from natural pathways.In this study, we focus on ongoing efforts to improvepathway efficiency through engineered enzyme complex formation using synthetic scaffolds. Overview of Dissertation The dissertation on developing recombinant strains by synthetic biotechnology strategies for production of itaconic acid, L-serine and 1 propanol. This dissertation as follows: Chapter 1 cover the introduction of basic knowledge related to synthesis scaffold protein and their effect on metabolic engineering. Chapter 2 describes Efficient itaconic acid production via protein-protein scaffold introduction between GltA, AcnA, and CadA in recombinant Escherichia coli The recombinant E. coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein-protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/l of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold Chapter 3 describes Improvement of itaconic acid production by introduction of synthesis scaffold between CadA and AcnA in recombinant Escherichia coli. The synthetic scaffold complex was contructed between CadA from A.tereus and AcnA from E. coli havs made efficient production of itaconic acid. In this system, SH3 ligand and SH3 domain were incorporated with pathway enzyme CadA and AcnA respectively and formed a synthetic scaffold complex. Chapter 4 describes High yield fermentation of L-serine in recombinant Escherichia coli via scaffolding protein. In order to achieve the higher titer of L-serine, PSP co-localized along with EamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance, by using scaffold protein.These results suggest that carbon flux was successfully directed to the L-serine secretion pathway without knock-out of a competing pathway or addition of expensive glycine. Chapter 5 describes Improving L-serine productionin Escherichia coli via synthetic protein scaffold of SerB, SerC, and EamA. To achieve L-serine production with high efficiency, three enzymes (SerB, SerC, and EamA) were physically re-localized by using a scaffold system GBD:SH3:PDZ. Such strategy was highly effective in improving the production of L-serine in Escherichia coli. Chapter 6 describesMetabolic engineering ofEscherichia coliusing synthetic protein scaffold for the production of 1 propanol and furure work for high yield 1 propanol from E.coli