Antonie van Leeuwenhoek

, Volume 105, Issue 4, pp 687–696 | Cite as

Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability

  • Sara Centeno-Leija
  • Gerardo Huerta-Beristain
  • Martha Giles-Gómez
  • Francisco Bolivar
  • Guillermo Gosset
  • Alfredo Martinez
Original Paper

Abstract

The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHBAv). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHBAv). In comparison with the wild type strain expressing pPHBAv, the specific accumulation of PHB (gPHB/gDCW) in MG1655 Δack-pta/pTrcgapN/pPHBAv increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.

Keywords

Escherichia coli NAD(P)+-dependent glyceraldehyde-3-phosphate-dehydrogenase NAD(P)H Acetic acid Acetyl-CoA pool Poly-3-hydroxybutyrate 

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Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Sara Centeno-Leija
    • 1
  • Gerardo Huerta-Beristain
    • 1
    • 3
  • Martha Giles-Gómez
    • 2
  • Francisco Bolivar
    • 1
  • Guillermo Gosset
    • 1
  • Alfredo Martinez
    • 1
  1. 1.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Departamento de Biología, Facultad de QuímicaUniversidad Nacional Autónoma de MéxicoMexicoMexico
  3. 3.Unidad Académica de Ciencias Químico BiológicasUniversidad Autónoma de GuerreroChilpancingoMexico

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