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Engineering of Saccharomyces cerevisiae for the production of poly-3-d-hydroxybutyrate from xylose.

Sandström AG, Muñoz de Las Heras A, Portugal-Nunes D, Gorwa-Grauslund MF - AMB Express (2015)

Bottom Line: In the present study, the robust baker's yeast Saccharomyces cerevisiae was engineered to produce PHB from xylose, the main pentose found in lignocellulosic biomass.The PHB pathway genes from the well-characterized PHB producer Cupriavidus necator were introduced in recombinant S. cerevisiae strains already capable of pentose utilization by introduction of the fungal genes for xylose utilization from the yeast Scheffersomyces stipitis.PHB production from xylose was successfully demonstrated in shake-flasks experiments, with PHB yield of 1.17 ± 0.18 mg PHB g(-1) xylose.

View Article: PubMed Central - PubMed

Affiliation: Division of Applied Microbiology, Department of Chemistry, Lund University, PO Box 124, Lund, SE-221 00 Sweden.

ABSTRACT
Poly-3-d-hydroxybutyrate (PHB) is a promising biopolymer naturally produced by several bacterial species. In the present study, the robust baker's yeast Saccharomyces cerevisiae was engineered to produce PHB from xylose, the main pentose found in lignocellulosic biomass. The PHB pathway genes from the well-characterized PHB producer Cupriavidus necator were introduced in recombinant S. cerevisiae strains already capable of pentose utilization by introduction of the fungal genes for xylose utilization from the yeast Scheffersomyces stipitis. PHB production from xylose was successfully demonstrated in shake-flasks experiments, with PHB yield of 1.17 ± 0.18 mg PHB g(-1) xylose. Under well-controlled fully aerobic conditions, a titer of 101.7 mg PHB L(-1) was reached within 48 hours, with a PHB yield of 1.99 ± 0.15 mg PHB g(-1) xylose, thereby demonstrating the potential of this host for PHB production from lignocellulose.

No MeSH data available.


Related in: MedlinePlus

A condensed view of the glycolytic pathway steps, as well as the XR and PHB pathway steps that are relevant for the PHB accumulation in the constructed strain. Abbreviations: EtOH, ethanol; Ac-CoA, acetyl coenzyme A; G3P, glyceraldehyde 3-phosphate; PPP, pentose phosphate pathway The parts marked in red are the introduced xylose assimilation pathway and the overexpressed PPP. The parts marked in blue are the components of the PHB metabolic pathway.
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Fig4: A condensed view of the glycolytic pathway steps, as well as the XR and PHB pathway steps that are relevant for the PHB accumulation in the constructed strain. Abbreviations: EtOH, ethanol; Ac-CoA, acetyl coenzyme A; G3P, glyceraldehyde 3-phosphate; PPP, pentose phosphate pathway The parts marked in red are the introduced xylose assimilation pathway and the overexpressed PPP. The parts marked in blue are the components of the PHB metabolic pathway.

Mentions: The final titer reached a total of 45.0 ± 3.5 mg L−1 of PHB for TMB 4443 (PHB+) in shake flask conditions, and increased to 101.7 ± 7.1 mg L−1 of PHB in well-aerated bioreactor. By-product accumulation was much lower in the fully aerobic and pH-controlled bioreactor set-up than in the shake-flask experiment, indicating increased respiratory metabolism that directly translated into higher biomass. This may explain the positive impact on PHB production as more cells were available to synthesize PHB while less carbon was dissipated into reduced byproducts. This observation was made despite the fact that low levels of acetate, that is required for PHB production (Figure 4), were generated. In contrast, unusually high NADP+-catalyzed acetate levels were observed in the shake flask experiments. In that case, the limited respiration may trigger higher availability of acetaldehyde precursor; this, combined with the need for additional routes for NADPH generation for biomass synthesis and for xylose utilization via the NAD(P)H-dependent xylose reductase, could explain the dramatic increase in acetate production. It may also explain why xylose consumption stopped before completion as pH decreased over time (data not shown). The accumulation of acetate also indicates that, under these conditions, PHB generation may rather be controlled by reactions downstream of acetate (Figure 4). This includes the acetyl-CoA synthesis that has been shown to be limited by the availability of ATP and by the efficiency of the acetyl-CoA synthetase (Shiba et al. 2007), or the acetoacetyl-CoA reductase-catalyzed reduction that could be limited by NADPH shortage. Overall, the results indicate that a threshold in oxygenation may be necessary to provide sufficient resources for biomass synthesis while enabling sufficient formation and further conversion of the acetate precursor.Figure 4


Engineering of Saccharomyces cerevisiae for the production of poly-3-d-hydroxybutyrate from xylose.

Sandström AG, Muñoz de Las Heras A, Portugal-Nunes D, Gorwa-Grauslund MF - AMB Express (2015)

A condensed view of the glycolytic pathway steps, as well as the XR and PHB pathway steps that are relevant for the PHB accumulation in the constructed strain. Abbreviations: EtOH, ethanol; Ac-CoA, acetyl coenzyme A; G3P, glyceraldehyde 3-phosphate; PPP, pentose phosphate pathway The parts marked in red are the introduced xylose assimilation pathway and the overexpressed PPP. The parts marked in blue are the components of the PHB metabolic pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385036&req=5

Fig4: A condensed view of the glycolytic pathway steps, as well as the XR and PHB pathway steps that are relevant for the PHB accumulation in the constructed strain. Abbreviations: EtOH, ethanol; Ac-CoA, acetyl coenzyme A; G3P, glyceraldehyde 3-phosphate; PPP, pentose phosphate pathway The parts marked in red are the introduced xylose assimilation pathway and the overexpressed PPP. The parts marked in blue are the components of the PHB metabolic pathway.
Mentions: The final titer reached a total of 45.0 ± 3.5 mg L−1 of PHB for TMB 4443 (PHB+) in shake flask conditions, and increased to 101.7 ± 7.1 mg L−1 of PHB in well-aerated bioreactor. By-product accumulation was much lower in the fully aerobic and pH-controlled bioreactor set-up than in the shake-flask experiment, indicating increased respiratory metabolism that directly translated into higher biomass. This may explain the positive impact on PHB production as more cells were available to synthesize PHB while less carbon was dissipated into reduced byproducts. This observation was made despite the fact that low levels of acetate, that is required for PHB production (Figure 4), were generated. In contrast, unusually high NADP+-catalyzed acetate levels were observed in the shake flask experiments. In that case, the limited respiration may trigger higher availability of acetaldehyde precursor; this, combined with the need for additional routes for NADPH generation for biomass synthesis and for xylose utilization via the NAD(P)H-dependent xylose reductase, could explain the dramatic increase in acetate production. It may also explain why xylose consumption stopped before completion as pH decreased over time (data not shown). The accumulation of acetate also indicates that, under these conditions, PHB generation may rather be controlled by reactions downstream of acetate (Figure 4). This includes the acetyl-CoA synthesis that has been shown to be limited by the availability of ATP and by the efficiency of the acetyl-CoA synthetase (Shiba et al. 2007), or the acetoacetyl-CoA reductase-catalyzed reduction that could be limited by NADPH shortage. Overall, the results indicate that a threshold in oxygenation may be necessary to provide sufficient resources for biomass synthesis while enabling sufficient formation and further conversion of the acetate precursor.Figure 4

Bottom Line: In the present study, the robust baker's yeast Saccharomyces cerevisiae was engineered to produce PHB from xylose, the main pentose found in lignocellulosic biomass.The PHB pathway genes from the well-characterized PHB producer Cupriavidus necator were introduced in recombinant S. cerevisiae strains already capable of pentose utilization by introduction of the fungal genes for xylose utilization from the yeast Scheffersomyces stipitis.PHB production from xylose was successfully demonstrated in shake-flasks experiments, with PHB yield of 1.17 ± 0.18 mg PHB g(-1) xylose.

View Article: PubMed Central - PubMed

Affiliation: Division of Applied Microbiology, Department of Chemistry, Lund University, PO Box 124, Lund, SE-221 00 Sweden.

ABSTRACT
Poly-3-d-hydroxybutyrate (PHB) is a promising biopolymer naturally produced by several bacterial species. In the present study, the robust baker's yeast Saccharomyces cerevisiae was engineered to produce PHB from xylose, the main pentose found in lignocellulosic biomass. The PHB pathway genes from the well-characterized PHB producer Cupriavidus necator were introduced in recombinant S. cerevisiae strains already capable of pentose utilization by introduction of the fungal genes for xylose utilization from the yeast Scheffersomyces stipitis. PHB production from xylose was successfully demonstrated in shake-flasks experiments, with PHB yield of 1.17 ± 0.18 mg PHB g(-1) xylose. Under well-controlled fully aerobic conditions, a titer of 101.7 mg PHB L(-1) was reached within 48 hours, with a PHB yield of 1.99 ± 0.15 mg PHB g(-1) xylose, thereby demonstrating the potential of this host for PHB production from lignocellulose.

No MeSH data available.


Related in: MedlinePlus