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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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Related in: MedlinePlus

Intracellular acetyl-CoA concentrations of recombinantE. coli. The average cell dry weight for all of the strains was 0.38 g/liter per optical-density (OD600) unit of culture. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
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Fig4: Intracellular acetyl-CoA concentrations of recombinantE. coli. The average cell dry weight for all of the strains was 0.38 g/liter per optical-density (OD600) unit of culture. Histogram shows the mean of three biological replicates, and error bars show standard deviations.

Mentions: The ED pathway joins the oxidative pentose phosphate pathway to EMP pathway via two enzyme-catalyzed reactions. The two critical enzymes are phosphogluconate dehydratase and 2-keto-3-deoxygluconate 6-phosphate aldolase, which were encoded by edd and eda, respectively. In E. coli, the ED pathway has been shown to be inactive with glucose as the carbon source [27]. Thus, the upstream regulated region of the edd-eda operon in SD02 was replaced with a constitutive promoter J23119 (http://partsregistry.org/Part:BBa_J23119) for eliminating the repression of the ED pathway and activating the ED pathway, resulting in SD03 (SD02 PJ23119-edd). Unexpectedly, strain SD03 (pBHR68) produced 2.90 g/L PHB which was a significant decrease compared with that of SD02 (pBHR68) (Table 1). Then fragment Trc-162 was inserted at the upstream of zwf gene in SD03 to further enhance the flux of ED pathway, creating strain SD04 (SD03, PTrc-162-zwf). The transcription levels of zwf, edd and eda genes in SD04 were up-regulated by 5.46, 4.57 and 2.16-fold, respectively (Figure 3B). PHB was accumulated to 3.39 g/L in SD04 (pBHR68), a 16.9% increase compared with that of SD03 (pBHR68), but was still lower than that of the parent strain SD02 (pBHR68) (Table 1). In addition, a small amount of pyruvate and acetate had been detected in the media of strains SD03 (pBHR68) and SD04 (pBHR68), but not detected in SD02 (pBHR68) (Table 1). This might be due to the improvement of glucose consumption after co-overexpressing the edd, eda, zwf genes (Table 1), which resulted in pyruvate accumulation in the medium. Moreover, the acetate was produced directly from pyruvate by pyruvate oxidase (encoded by poxB) in the JM109 derivative strains [28]. Thus it was possible that the accumulation of pyruvate and acetate retarded the cell growth and led to lower PHB production, although the intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio was increased (Figure 4, Table 2). So we tried to enhance the conversion of pyruvate to acetyl-CoA to eliminate the pyruvate accumulation and increase the PHB production.Table 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)

Intracellular acetyl-CoA concentrations of recombinantE. coli. The average cell dry weight for all of the strains was 0.38 g/liter per optical-density (OD600) unit of culture. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Intracellular acetyl-CoA concentrations of recombinantE. coli. The average cell dry weight for all of the strains was 0.38 g/liter per optical-density (OD600) unit of culture. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
Mentions: The ED pathway joins the oxidative pentose phosphate pathway to EMP pathway via two enzyme-catalyzed reactions. The two critical enzymes are phosphogluconate dehydratase and 2-keto-3-deoxygluconate 6-phosphate aldolase, which were encoded by edd and eda, respectively. In E. coli, the ED pathway has been shown to be inactive with glucose as the carbon source [27]. Thus, the upstream regulated region of the edd-eda operon in SD02 was replaced with a constitutive promoter J23119 (http://partsregistry.org/Part:BBa_J23119) for eliminating the repression of the ED pathway and activating the ED pathway, resulting in SD03 (SD02 PJ23119-edd). Unexpectedly, strain SD03 (pBHR68) produced 2.90 g/L PHB which was a significant decrease compared with that of SD02 (pBHR68) (Table 1). Then fragment Trc-162 was inserted at the upstream of zwf gene in SD03 to further enhance the flux of ED pathway, creating strain SD04 (SD03, PTrc-162-zwf). The transcription levels of zwf, edd and eda genes in SD04 were up-regulated by 5.46, 4.57 and 2.16-fold, respectively (Figure 3B). PHB was accumulated to 3.39 g/L in SD04 (pBHR68), a 16.9% increase compared with that of SD03 (pBHR68), but was still lower than that of the parent strain SD02 (pBHR68) (Table 1). In addition, a small amount of pyruvate and acetate had been detected in the media of strains SD03 (pBHR68) and SD04 (pBHR68), but not detected in SD02 (pBHR68) (Table 1). This might be due to the improvement of glucose consumption after co-overexpressing the edd, eda, zwf genes (Table 1), which resulted in pyruvate accumulation in the medium. Moreover, the acetate was produced directly from pyruvate by pyruvate oxidase (encoded by poxB) in the JM109 derivative strains [28]. Thus it was possible that the accumulation of pyruvate and acetate retarded the cell growth and led to lower PHB production, although the intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio was increased (Figure 4, Table 2). So we tried to enhance the conversion of pyruvate to acetyl-CoA to eliminate the pyruvate accumulation and increase the PHB production.Table 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