ATP limitation in a pyruvate formate lyase mutant of Escherichia coli MG1655 increases glycolytic flux to d-lactate
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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.
- 2.Böck A, Sawers G (1996) Fermentation. In: Neidhardt FC et al. (eds) Escherichia coli and Salmonella. Cellular and molecular biology, vol 1. American Society for Microbiology Press, Washington, DC, pp 262–282Google Scholar
- 6.Fraenkel DG (1996) Glycolysis. In: Neidhardt FC et al. (eds) Escherichia coli and Salmonella. Cellular and molecular biology, vol 1. American Society for Microbiology Press, Washington DC, pp 262–282Google Scholar
- 10.Lara AR, Vazquez-Limón C, Gosset G, López-Munguía A, Ramirez OT (2006) Engineering Escherichia coli to improve culture performance and reduce formation of by-product during recombinant protein production under transient intermittent anaerobic conditions. Biotechnol Bioeng 94(6):1164–1175. doi: 10.1002/bit.20954 PubMedCrossRefGoogle Scholar
- 12.Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- 14.Narayanan N, Roychoudhury PK, Srivastava A (2004) L(+)-lactic acid fermentation and its product polymerization. Electron J Biotechnol 7(2):167–179Google Scholar
- 15.Tsuji F (2002) Autocatalytic hydrolysis of amorphous-made polylactides: effects of L-lactide content, tacticity, and enantiomeric polymer blending. Polymer (Guildf) 43:1789–1796. doi: 10.1016/S0032-3861(01)00752-2
- 16.Zhou S, Causey TB, Hasona A, Shanmugam KT, Ingram LO (2003) Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl Environ Microbiol 69:399–407. doi: 10.1128/AEM.69.1.399-407.2003
- 19.Zhou S, Shanmugam KT, Ingram LO (2003) Functional replacement of the Escherichia coli D-(−)-lactate dehydrogenase gene (ldhA) whith the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici. Appl Environ Microbiol 69:2237–2244. doi: 10.1128/AEM.69.4.2237-2244.2003
- 21.Zhou S, Yomano LP, Shanmugam KT, Ingram LO (2005) Fermentation of 10% (w/v) sugar to D-lactate by engineered Escherichia coli B. Biotechnol Lett 27:1891–1896. doi: 10.1007/s10529-005-3899-7