Advertisement

ATP limitation in a pyruvate formate lyase mutant of Escherichia coli MG1655 increases glycolytic flux to d-lactate

  • José Utrilla
  • Guillermo Gosset
  • Alfredo Martinez
Original Paper

Abstract

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.

Keywords

d-Lactate Escherichia coli ATP Glycolytic flux 

Notes

Acknowledgments

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.

References

  1. 1.
    Beall DS, Ohta K, Ingram LO (1991) Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli. Biotechnol Bioeng 38:296–303. doi: 10.1002/bit.260380311 PubMedCrossRefGoogle Scholar
  2. 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
  3. 3.
    Clark DP (1989) The fermentation pathways of Escherichia coli. FEMS Microbiol Rev 63:223–234. doi: 10.1016/0168-6445(89)90033-8 CrossRefGoogle Scholar
  4. 4.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645. doi: 10.1073/pnas.120163297 PubMedCrossRefGoogle Scholar
  5. 5.
    Dien BS, Nichols NN, Bothast RJ (2001) Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars. J Ind Microbiol Biotechnol 27:259–264. doi: 10.1038/sj.jim.7000195 Google Scholar
  6. 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
  7. 7.
    Grabar TB, Zhou S, Shanmugam KT, Yomano LP, Ingram LO (2006) Methylglyoxal bypass identified as source of chiral contamination in L(+) and D(−) lactate fermentations by recombinant Escherichia coli. Biotechnol Lett 28:1527–1535. doi: 10.1007/s10529-006-9122-7 Google Scholar
  8. 8.
    Kim Y, Ingram LO, Shanmugam KT (2007) Construction of an Escherichia coli K-12 mutant for homoethanologenic fermentation of glucose or xylose without foreign genes. Appl Environ Microbiol 73(6):1766–1771PubMedCrossRefGoogle Scholar
  9. 9.
    Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 184:3909–3916. doi: 10.1128/JB.184.14.3909-3916.2002 PubMedCrossRefGoogle Scholar
  10. 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
  11. 11.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  12. 12.
    Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  13. 13.
    Martinez A, Grabar TB, Shanmugam KT, Yomano LP, York SW, Ingram LO (2007) Low salt medium for lactate and ethanol production by recombinant Escherichia coli B. Biotechnol Lett 29:397–404. doi: 10.1007/s10529-006-9252-y PubMedCrossRefGoogle Scholar
  14. 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. 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. 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
  17. 17.
    Zhou S, Grabar TB, Shanmugan KT, Ingram LO (2006) Betaine tripled the volumetric productivity of D-lactate by Escherichia coli strain SZ132 in mineral salts medium. Biotechnol Lett 28:671–676. doi: 10.1007/s10529-006-0033-4 Google Scholar
  18. 18.
    Zhou S, Iverson AG, Grayburn WS (2008) Engineering a native homoethanol pathway in Escherichia coli B for ethanol production. Biotechnol Lett 30:335–342. doi: 10.1007/s10529-007-9544-x PubMedCrossRefGoogle Scholar
  19. 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
  20. 20.
    Zhou S, Shanmugam KT, Yomano LP, Grabar TB, Ingram LO (2006) Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium. Biotechnol Lett 28:663–670. doi: 10.1007/s10529-006-0032-5 PubMedCrossRefGoogle Scholar
  21. 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
  22. 22.
    Zhu J, Zhimizu K (2004) The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Appl Microbiol Biotechnol 64:367–375. doi: 10.1007/s00253-003-1499-9 Google Scholar

Copyright information

© Society for Industrial Microbiology 2009

Authors and Affiliations

  • José Utrilla
    • 1
  • Guillermo Gosset
    • 1
  • Alfredo Martinez
    • 1
  1. 1.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

Personalised recommendations