ATP limitation in a pyruvate formate lyase mutant of Escherichia coli MG1655 increases glycolytic flux to d-lactate
A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.
Keywordsd-Lactate Escherichia coli ATP Glycolytic flux
We thank Georgina Hernández for the HPLC analysis; Montserrat Orencio, Martín Patiño and Mario Trejo for technical support; and E. López and P. Gaytan for oligonucleotide synthesis. This work was supported by grants from UNAM (PAPIIT-DGAPA: IN220908) and the Mexican Council of Science and Technology (CONACyT––SAGARPA 2004-C01-224 and CONACyT––Estado de Morelos 2007-COL-80360). J.U. held a scholarship from CONACyT.
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