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

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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 (pPHB Av ). 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/pPHB Av ). In comparison with the wild type strain expressing pPHB Av , the specific accumulation of PHB (gPHB/gDCW) in MG1655 Δack-pta/pTrcgapN/pPHB Av 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.

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Acknowledgements

We gratefully acknowledge Andrea del Carmen Díaz-Marcelín, María Jose Alvarado, Mercedes Enzaldo, Ramón de Anda, Georgina Hernández-Chávez, Luz María Martínez, José Utrilla and Adriana Longoria for their technical support during the process of the work. We thank Daniel Segura for providing us with the Chromosomal DNA of A. vinelandii. This Project was supported by DGAPA/PAPIIT/UNAM Grant IT201414 and UC MEXUS-CONACYT Collaborative Grant CN-12-581. SC-L held a scholarship from CONACyT.

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The authors declare that they have no conflict of interests.

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  1. Gerardo Huerta-Beristain

    Present address: Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Av. Lazaro Cardenas S/N. Cd. Universitaria, 39070, Chilpancingo, Guerrero, Mexico

Authors and Affiliations

  1. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, 62250, Cuernavaca, Morelos, Mexico

    Sara Centeno-Leija, Gerardo Huerta-Beristain, Francisco Bolivar, Guillermo Gosset & Alfredo Martinez

  2. Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Circuito Interior, Ciudad Universitaria, Coyoacán, 04510, Mexico, DF, Mexico

    Martha Giles-Gómez

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  1. Sara Centeno-Leija
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Correspondence to Alfredo Martinez.

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Centeno-Leija, S., Huerta-Beristain, G., Giles-Gómez, M. et al. Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability. Antonie van Leeuwenhoek 105, 687–696 (2014). https://doi.org/10.1007/s10482-014-0124-5

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