Sucrose metabolism in Escherichia coli W(ATCC 9637)

Sucrose is one of the main feedstocks for fermentation industry. Mainly produced from sugar cane, this sugar has several advantages over glucose from corn. Sucrose production uses fewer amounts of energy, water, and chemicals. It does not need a hydrolysis step that is used in the glucose production. Sucrose is also less expensive than glucose. Escherichia coli is the backbone for research in prokaryotes. This bacterium is also one of the main industrial microbes. However, most industrial E. coli strains are derived from the wild types that do not utilize sucrose. The study on this bacterium is usually performed in glucose medium. Therefore, its sucrose metabolism is relatively less understood. Consequently, the industrial application of E. coli for producing high value chemicals from sucrose is not common. Some E. coli strains posses a capable of sucrose utilization. One of these strains is E. coli W. This strain uses chromosomally encoded sucrose catabolism (csc) regulon to utilize the sugar. The regulon consists of cscA (invertase), cscB (permease), cscK (fructokinase), and cscR (repressor). In this thesis, a Systems Biology approach was applied to characterize sucrose metabolism in this strain in order to generate important knowledge for metabolic engineering. The study showed that E. coli W can grow on sucrose as fast as on glucose, but the rate was controlled by sucrose concentration. This shows that the sucrose utilization has not been optimized in this strain. A high similarity in the growth kinetic, exo-metabolome, and transcriptome was observed between the cell grow on sucrose and glucose. The role of Crp in carbon catabolite repression was confirmed. Some variations were seen in the fluxes of carbon metabolism. We found that this strain is able to co-utilize acetate and sugar (glucose or sucrose), providing advantages over mostly used K12 strains. Hence, W is a potential strain for sucrose metabolic engineering. Further charactization was conducted in W mutants capable of growth on low sucrose. Mutation in the sucrose permease was the most frequently observed, indicating the importance of this mutation for improving sucrose utilization. A study of non-csc genes revealed the role of trehalose repressor, showing the possibility sucrose transport by trehalose permease. Under optimum sucrose transport, the growth was found to be controlled by fructokinase activity, making this enzyme a potential target for sucrose metabolic engineering. Using the knowledge gained from the charactization studies, high cell density cultivation was performed for producing PHB to serve as a platform for the production of high value chemical from sucrose. The strain improvement by cscR deletion significantly improved the sucrose utilization and PHB productivity. Future study is needed to achieve PHB productivity from glucose demonstrated by optimized K12 derivative culture. In conclusion, this thesis is providing significant information on the characteristics of sucrose metabolism in E. coli W, which is essential for performing metabolic engineering. The study on sucrose mutant strains has provided an insight to the factors that may contribute to the sucrose utilization. The PHB production described in this thesis has demonstrated the potential of producing high value product from sucrose using E. coli W. All of these achievements have opened the way for specific studies on sucrose metabolism in E. coli and sucrose-based industrial application of this bacterium in the futur