Anaerobic poly-3-d-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductaseReportar como inadecuado




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Microbial Cell Factories

, 15:197

First Online: 18 November 2016Received: 01 September 2016Accepted: 10 November 2016

Abstract

BackgroundPoly-3-d-hydroxybutyrate PHB that is a promising precursor for bioplastic with similar physical properties as polypropylene, is naturally produced by several bacterial species. The bacterial pathway is comprised of the three enzymes β-ketothiolase, acetoacetyl-CoA reductase AAR and PHB synthase, which all together convert acetyl-CoA into PHB. Heterologous expression of the pathway genes from Cupriavidus necator has enabled PHB production in the yeast Saccharomyces cerevisiae from glucose as well as from xylose, after introduction of the fungal xylose utilization pathway from Scheffersomyces stipitis including xylose reductase XR and xylitol dehydrogenase XDH. However PHB titers are still low.

ResultsIn this study the acetoacetyl-CoA reductase gene from C. necator CnAAR, a NADPH-dependent enzyme, was replaced by the NADH-dependent AAR gene from Allochromatium vinosum AvAAR in recombinant xylose-utilizing S. cerevisiae and PHB production was compared. A. vinosum AAR was found to be active in S. cerevisiae and able to use both NADH and NADPH as cofactors. This resulted in improved PHB titers in S. cerevisiae when xylose was used as sole carbon source 5-fold in aerobic conditions and 8.4-fold under oxygen limited conditions and PHB yields 4-fold in aerobic conditions and up to 5.6-fold under oxygen limited conditions. Moreover, the best strain was able to accumulate up to 14% of PHB per cell dry weight under fully anaerobic conditions.

ConclusionsThis study reports a novel approach for boosting PHB accumulation in S. cerevisiae by replacement of the commonly used AAR from C. necator with the NADH-dependent alternative from A. vinosum. Additionally, to the best of our knowledge, it is the first demonstration of anaerobic PHB synthesis from xylose.

KeywordsSaccharomyces cerevisiae Poly-3-d-hydroxybutyrate PHB Xylose NADH NADPH Acetoacetyl-CoA reductase AbbreviationsAcetyl-CoAacetyl coenzyme A

AARacetyl-coA reductase

NAD-Hnicotinamide adenine dinucleotide

NADP-Hnicotinamide adenine dinucleotide phosphate

PCRpolymerase chain reaction

XDHxylitol dehydrogenase

XRmutmutated xylose reductase

XRwtwild type xylose reductase

YsXyield of biomass on xylose

YsEtOHyield of ethanol on xylose

YsAcyield of acetate on xylose

YsXylitolyield of xylitol on xylose

YsGlyyield of glycerol on xylose

YsPHByield of PHB on xylose

Electronic supplementary materialThe online version of this article doi:10.1186-s12934-016-0598-0 contains supplementary material, which is available to authorized users.

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Autor: Alejandro Muñoz de las Heras - Diogo J. Portugal-Nunes - Nathasha Rizza - Anders G. Sandström - Marie F. Gorwa-Grauslun

Fuente: https://link.springer.com/







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