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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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Comparison of CDW, PHB concentration and PHB content in recombinantE. colistrains. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
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Fig2: Comparison of CDW, PHB concentration and PHB content in recombinantE. colistrains. Histogram shows the mean of three biological replicates, and error bars show standard deviations.

Mentions: Under aerobic or anaerobic conditions, acetyl-CoA was derived mostly from the decarboxylation of pyruvate which respectively catalyzed by the PDH complex or pyruvate-formate lyase in E.coli [15,16]. Pyruvate was formed mostly from several glycolysis pathways. Moreover, L-serine derived from D-3-phosphoglycerate can be catalyzed to pyruvate and ammonias by L-serine deaminase. However, L-serine also can be cleaved into glycine and one carbon unit by serine hydroxymethyltransferase (SHMT, encoded by glyA), and be used as a building block for protein synthesis. Considering the competitive pathways existing at L-serine node, we firstly overexpressed the L-serine deaminase to enhance the conversion of L-serine into pyruvate. In E. coli, L-serine is deaminated by three L-serine deaminases, which are encoded by sdaA, sdaB and tdcG, respectively. L-serine deaminases I (SdaA) which is encoded by sdaA gene, is responsible for L-serine degradation in minimal media [14]. Moreover, a previous report has shown that sdaA-overexpressing Corynebacterium glutamicum could grow in the medium using L-serine as the sole carbon source [17]. Thus, in order to activate the SD pathway, sdaA was selected to be overexpressed by replacing the native sdaA promoter with a strong constitutive promoter trc in JM109, resulting in strain SD01 (JM109, Ptrc-sdaA). To test the effect of overexpressing sdaA on PHB production, SD01 and JM109 were both transformed with the plasmid pBHR68 which consists of the PHB biosynthesis genes from Ralstonia eutropha, creating strains SD01 (pBHR68) and JM109 (pBHR68). The PHB production of SD01 (pBHR68) was 3.58 g/L, 1.72-fold of that of JM109 (pBHR68) (Figure 2), and the PHB content is 73.8% of the cell dry weight (CDW) (Figure 2). These results suggested that overexpression of sdaA obviously “pulled” more L-serine to pyruvate and led to the improvement of PHB production. Thus, SD01 was chosen as the host for subsequent modifications to improve PHB production.Figure 2


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)

Comparison of CDW, PHB concentration and PHB content in recombinantE. colistrains. 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

Fig2: Comparison of CDW, PHB concentration and PHB content in recombinantE. colistrains. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
Mentions: Under aerobic or anaerobic conditions, acetyl-CoA was derived mostly from the decarboxylation of pyruvate which respectively catalyzed by the PDH complex or pyruvate-formate lyase in E.coli [15,16]. Pyruvate was formed mostly from several glycolysis pathways. Moreover, L-serine derived from D-3-phosphoglycerate can be catalyzed to pyruvate and ammonias by L-serine deaminase. However, L-serine also can be cleaved into glycine and one carbon unit by serine hydroxymethyltransferase (SHMT, encoded by glyA), and be used as a building block for protein synthesis. Considering the competitive pathways existing at L-serine node, we firstly overexpressed the L-serine deaminase to enhance the conversion of L-serine into pyruvate. In E. coli, L-serine is deaminated by three L-serine deaminases, which are encoded by sdaA, sdaB and tdcG, respectively. L-serine deaminases I (SdaA) which is encoded by sdaA gene, is responsible for L-serine degradation in minimal media [14]. Moreover, a previous report has shown that sdaA-overexpressing Corynebacterium glutamicum could grow in the medium using L-serine as the sole carbon source [17]. Thus, in order to activate the SD pathway, sdaA was selected to be overexpressed by replacing the native sdaA promoter with a strong constitutive promoter trc in JM109, resulting in strain SD01 (JM109, Ptrc-sdaA). To test the effect of overexpressing sdaA on PHB production, SD01 and JM109 were both transformed with the plasmid pBHR68 which consists of the PHB biosynthesis genes from Ralstonia eutropha, creating strains SD01 (pBHR68) and JM109 (pBHR68). The PHB production of SD01 (pBHR68) was 3.58 g/L, 1.72-fold of that of JM109 (pBHR68) (Figure 2), and the PHB content is 73.8% of the cell dry weight (CDW) (Figure 2). These results suggested that overexpression of sdaA obviously “pulled” more L-serine to pyruvate and led to the improvement of PHB production. Thus, SD01 was chosen as the host for subsequent modifications to improve PHB production.Figure 2

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