Type and capacity of glucose transport influences succinate yield in two-stage cultivations

Background: Glucose is the main carbon source of E. coli and a typical substrate in production processes. The main glucose uptake system is the glucose specific phosphotransferase system (Glc-PTS). The PTS couples glucose uptake with its phosphorylation. This is achieved by the concomitant conversion of phosphoenolpyruvate (PEP) to pyruvate. The Glc-PTS is hence unfavorable for the production of succinate as this product is derived from PEP. Results: We studied, in a systematic manner, the effect of knocking out the Glc-PTS and of replacing it with the glucose facilitator (Glf) of Zymomonas mobilis on succinate yield and productivity. For this study a set of strains derived from MG1655, carrying deletions of ackA-pta, adhE and ldhA that prevent the synthesis of competing fermentation products, were constructed and tested in two-stage cultivations. The data show that inactivation of the Glc-PTS achieved a considerable increase in succinate yield and productivity. On the other hand, aerobic growth of this strain on glucose was strongly decreased. Expression of the alternative glucose transporter, Glf, in this strain enhanced aerobic growth but productivity and yield under anaerobic conditions were slightly decreased. This decrease in succinate yield was accompanied by pyruvate production. Yield could be increased in both Glc-PTS mutants by overexpressing phosphoenolpyruvate carboxykinase (Pck). Productivity on the other hand, was decreased in the strain without alternative glucose transporter but strongly increased in the strain expressing Glf. The experiments were complemented by flux balance analysis in order to check the observed yields against the maximal theoretical yields. Furthermore, the phosphorylation state of EIIA(Glc) was determined. The data indicate that the ratio of PEP to pyruvate is correlating with pyruvate excretion. This ratio is affected by the PTS reaction as well as by further reactions at the PEP/pyruvate node. Conclusions: The results show that for optimization of succinate yield and productivity it is not sufficient to knock out or introduce single reactions. Rather, balancing of the fluxes of central metabolism most important at the PEP/pyruvate node is important.