Splitting the E. coli metabolism for the production of fructose-6-P derived chemicals

In the past, coupling product formation with growth has proven to be a highly successful metabolic engineering strategy for the biotechnological production of numerous molecules, e.g., succinate (Hong, 2002). The guaranteed (high) product yield, the fast recuperation of cofactors and abundance of intermediates are some of the main advantages.Such a strategy was developed for the production of fructose-6-phosphate derived molecules in E. coli, as many high added value molecules, such as for example chito-oligomers, are derived from fructose-6-P. However, compared to previous examples of this strategy, fructose-6-phosphate is a key metabolite in central metabolism, that intervenes in the glycolysis, the pentose phosphate shunt, etc. which renders such a strategy intrinsically more challenging.The coupling between fructose-6-phosphate production and growth was achieved by using the heteromeric substrate sucrose, which is cleaved by a heterologously expressed sucrose phosphorylase into glucose-1-phosphate and fructose. A knock-out strategy was developed using metabolic models and C13 labeled flux analysis to preserve the fructose moiety for the production of fructose-6-phosphate derived molecules, while growth is fueled by metabolizing glucose-1-phosphate, hence, resulting in guaranteed product yields and a reasonable productivity.To achieve this split metabolism, since fructose-6-P is a key intermediate in the central metabolism, the fluxes of the central carbon metabolism have to be redirected severely, e.g., the glycolysis has to be split by knocking out the genes pgi and pfkAB, which prevents the flow from fructose-6-phosphate through the glycolysis to the Krebs cycle. Different additional knockouts were evaluated in order to achieve a complete split metabolism. C13 labeled flux analysis, using OpenFlux, was performed to evaluate the fluxome of the various knock-out mutants, which ultimately showed that the fructose moiety of sucrose can be completely preserved for the formation of fructose-6-P derived products, rendering such a strain the ideal base strain for the development of a wide variety of production hosts.In the past, coupling product formation with growth has proven to be a highly successful metabolic engineering strategy for the biotechnological production of numerous molecules, e.g., succinate (Hong, 2002). The guaranteed (high) product yield, the fast recuperation of cofactors and abundance of intermediates are some of the main advantages.Such a strategy was developed for the production of fructose-6-phosphate derived molecules in E. coli, as many high added value molecules, such as for example chito-oligomers, are derived from fructose-6-P. However, compared to previous examples of this strategy, fructose-6-phosphate is a key metabolite in central metabolism, that intervenes in the glycolysis, the pentose phosphate shunt, etc. which renders such a strategy intrinsically more challenging.The coupling between fructose-6-phosphate production and growth was achieved by using the heteromeric substrate sucrose, which is cleaved by a heterologously expressed sucrose phosphorylase into glucose-1-phosphate and fructose. A knock-out strategy was developed using metabolic models and C13 labeled flux analysis to preserve the fructose moiety for the production of fructose-6-phosphate derived molecules, while growth is fueled by metabolizing glucose-1-phosphate, hence, resulting in guaranteed product yields and a reasonable productivity.To achieve this split metabolism, since fructose-6-P is a key intermediate in the central metabolism, the fluxes of the central carbon metabolism have to be redirected severely, e.g., the glycolysis has to be split by knocking out the genes pgi and pfkAB, which prevents the flow from fructose-6-phosphate through the glycolysis to the Krebs cycle. Different additional knockouts were evaluated in order to achieve a complete split metabolism. C13 labeled flux analysis, using OpenFlux, was performed to evaluate the fluxome of the various knock-out mutants, which ultimately showed that the fructose moiety of sucrose can be completely preserved for the formation of fructose-6-P derived products, rendering such a strain the ideal base strain for the development of a wide variety of production hosts.C