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Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli.

Zhang Y, Lin Z, Liu Q, Li Y, Wang Z, Ma H, Chen T, Zhao X - Microb. Cell Fact. (2014)

Bottom Line: Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.This work demonstrates a novel strategy for improving PHB production in E. coli.The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, People's Republic of China. zhangyan12@tju.edu.cn.

ABSTRACT

Background: Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources.

Results: In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP(+)] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.

Conclusions: Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

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Schematic representation of the SD and ED metabolic pathways in PHB accumulation recombinantE.coli. Dashed lines indicate multiple enzymatic steps. The bold lines indicate the enzymes of SD pathway including reactions catalyzed by SerACB and SdaA. The enzymes that had been overexpressed in this work were shown in boldface. G6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glycerahyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphate-gluconate. Enzymes are as follows: Zwf, glucose 6-phosphate-1-dehydrogenase; Edd, phosphogluconate dehydratase; Eda, 2-keto-3-deoxygluconate 6-phosphate aldolase; Pgk, phosphoglycerate kinase; SerA, D-3-phosphoglycerate dehydrogenase; SerB, phosphoserine phosphatase; SerC, 3-phosphoserine aminotransferase; SdaA, L-serine deaminase I; PoxB, pyruvate oxidase; Pta, phosphate acetyltransferase; Ack, acetate kinase; PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHB synthase.
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Fig1: Schematic representation of the SD and ED metabolic pathways in PHB accumulation recombinantE.coli. Dashed lines indicate multiple enzymatic steps. The bold lines indicate the enzymes of SD pathway including reactions catalyzed by SerACB and SdaA. The enzymes that had been overexpressed in this work were shown in boldface. G6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glycerahyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphate-gluconate. Enzymes are as follows: Zwf, glucose 6-phosphate-1-dehydrogenase; Edd, phosphogluconate dehydratase; Eda, 2-keto-3-deoxygluconate 6-phosphate aldolase; Pgk, phosphoglycerate kinase; SerA, D-3-phosphoglycerate dehydrogenase; SerB, phosphoserine phosphatase; SerC, 3-phosphoserine aminotransferase; SdaA, L-serine deaminase I; PoxB, pyruvate oxidase; Pta, phosphate acetyltransferase; Ack, acetate kinase; PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHB synthase.

Mentions: In the majority of native PHB-accumulating species, PHB is synthesized from acetyl-CoA by a sequence of three enzyme reactions catalyzed by β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, encoded by phaA, phaB and phaC, respectively (Figure 1). Recombinant E. coli harboring the exogenous PHB synthetic pathway was one of the most frequently used hosts for biopolymer production because of its advantages such as having a wide range of utilizable carbon sources, accumulating of large amounts of polymers with a high level of productivity, high cell density fermentation, and lacking PHA degradation system.Figure 1


Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli.

Zhang Y, Lin Z, Liu Q, Li Y, Wang Z, Ma H, Chen T, Zhao X - Microb. Cell Fact. (2014)

Schematic representation of the SD and ED metabolic pathways in PHB accumulation recombinantE.coli. Dashed lines indicate multiple enzymatic steps. The bold lines indicate the enzymes of SD pathway including reactions catalyzed by SerACB and SdaA. The enzymes that had been overexpressed in this work were shown in boldface. G6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glycerahyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphate-gluconate. Enzymes are as follows: Zwf, glucose 6-phosphate-1-dehydrogenase; Edd, phosphogluconate dehydratase; Eda, 2-keto-3-deoxygluconate 6-phosphate aldolase; Pgk, phosphoglycerate kinase; SerA, D-3-phosphoglycerate dehydrogenase; SerB, phosphoserine phosphatase; SerC, 3-phosphoserine aminotransferase; SdaA, L-serine deaminase I; PoxB, pyruvate oxidase; Pta, phosphate acetyltransferase; Ack, acetate kinase; PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHB synthase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279783&req=5

Fig1: Schematic representation of the SD and ED metabolic pathways in PHB accumulation recombinantE.coli. Dashed lines indicate multiple enzymatic steps. The bold lines indicate the enzymes of SD pathway including reactions catalyzed by SerACB and SdaA. The enzymes that had been overexpressed in this work were shown in boldface. G6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glycerahyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphate-gluconate. Enzymes are as follows: Zwf, glucose 6-phosphate-1-dehydrogenase; Edd, phosphogluconate dehydratase; Eda, 2-keto-3-deoxygluconate 6-phosphate aldolase; Pgk, phosphoglycerate kinase; SerA, D-3-phosphoglycerate dehydrogenase; SerB, phosphoserine phosphatase; SerC, 3-phosphoserine aminotransferase; SdaA, L-serine deaminase I; PoxB, pyruvate oxidase; Pta, phosphate acetyltransferase; Ack, acetate kinase; PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHB synthase.
Mentions: In the majority of native PHB-accumulating species, PHB is synthesized from acetyl-CoA by a sequence of three enzyme reactions catalyzed by β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, encoded by phaA, phaB and phaC, respectively (Figure 1). Recombinant E. coli harboring the exogenous PHB synthetic pathway was one of the most frequently used hosts for biopolymer production because of its advantages such as having a wide range of utilizable carbon sources, accumulating of large amounts of polymers with a high level of productivity, high cell density fermentation, and lacking PHA degradation system.Figure 1

Bottom Line: Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.This work demonstrates a novel strategy for improving PHB production in E. coli.The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, People's Republic of China. zhangyan12@tju.edu.cn.

ABSTRACT

Background: Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources.

Results: In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP(+)] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.

Conclusions: Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

Show MeSH
Related in: MedlinePlus