In this study, we demonstrated that Ptet was useful for controlling essential gene expressions. Subsequently, we have constructed a high pyruvate producing strain LAFCPCPt-accBC-aceE. After optimal culture conditions for pyruvate production were determined, the final pyruvate concentration reached 26.1 g L?1 after 72 h with a theoretical yield of 55.6 %. In our knowledge, this yield was similar compared with the yields of another engineered E. coli strains (Table 1). However, our pyruvate producing strain was not required supplemental carbon additives and special expertise for pyruvate production, which was easy to perform. These results indicate that strain LAFCPCPt-accBC-aceE also has an industrial potential for pyruvate production.
With pyruvate production using strain LAFCPCPt-accBC-aceE, we think the culture pH is the most important factor. pH is known to have a significant effect on gene expression in E. coli cells, and expression of aceE is promoted at acidic pH (Maurer et al. 2005). On the other hand, after deletion of the ackA–pta pathway, cell growth is accelerated at acidic pH and the activity of the poxB gene product is enhanced (Dittrich et al. 2005). Similarly, pyruvate productivity in a poxB-deleted strain is higher at acidic pH than neutral pH (Dittrich et al. 2005). Taken together, these observations suggest that both the production and degradation of pyruvate is stimulated at acidic pH, and thus pH 5.7 was optimal in our study. Note that pyruvate degradation is limited in our study due to gene manipulation.
We also observed that both stirrer speed and airflow rate influenced pyruvate production. Transcription of several genes involved in the gluconeogenesis and anaplerosis pathways (pckA, ppsA, ppc and sfcA), the TCA cycle (gltA), the glyoxylate cycle (aceA), and acetate metabolic pathways (acs, ackA, pta and poxB) are affected by the dissolved oxygen concentration (Phue and Shiloach 2005). In our study, more aerobic conditions showed improved growth rate (Fig. 5), presumably to support facilitate overall carbon flux (Matsuoka and Shimizu 2013).
We found that the optimal culture temperature was 35 °C (Fig. 3a), though the reason is unclear. This temperature has no effect on expression of the genes involved in glycolysis or the pentose phosphate pathway (Gadgil et al. 2005). Perhaps this temperature contributes to enhancing the growth rate. For example, when E. coli strain ML30G was cultured in glucose minimal medium at various temperatures, the maximum growth rate was observed at 35 °C (Shehata and Marr 1975).