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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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Results of relative transcriptional level. A. The transcription level of the genes in SD pathway; B. The transcription level of the genes in ED pathway; C. The transcription level of the genes in PDH. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
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Fig3: Results of relative transcriptional level. A. The transcription level of the genes in SD pathway; B. The transcription level of the genes in ED pathway; C. The transcription level of the genes in PDH. Histogram shows the mean of three biological replicates, and error bars show standard deviations.

Mentions: Considering the instability of plasmid system and the metabolic burden resulted from plasmid replication [24,25], the pgk-serABC operon together with the fragment Trc-162 was integrated into the choromosome of SD01 at serC site, creating strain SD02 (SD01, PTrc-162-SerABC). The transcription levels of genes in SD pathway in strain SD02 were compared with that of the reference strain JM109 through RT-PCR analysis. As shown in Figure 3A, the genes of SD pathway have been successfully overexpressed. SD02 (pBHR68) accumulated 4.86 g/L PHB, which was almost the same as that of SD01 (pBHR68/pTPSer), but had a higher PHB content of 77.2% CDW (Figure 2). Therefore SD02 was selected for further engineering.Figure 3


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)

Results of relative transcriptional level. A. The transcription level of the genes in SD pathway; B. The transcription level of the genes in ED pathway; C. The transcription level of the genes in PDH. 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

Fig3: Results of relative transcriptional level. A. The transcription level of the genes in SD pathway; B. The transcription level of the genes in ED pathway; C. The transcription level of the genes in PDH. Histogram shows the mean of three biological replicates, and error bars show standard deviations.
Mentions: Considering the instability of plasmid system and the metabolic burden resulted from plasmid replication [24,25], the pgk-serABC operon together with the fragment Trc-162 was integrated into the choromosome of SD01 at serC site, creating strain SD02 (SD01, PTrc-162-SerABC). The transcription levels of genes in SD pathway in strain SD02 were compared with that of the reference strain JM109 through RT-PCR analysis. As shown in Figure 3A, the genes of SD pathway have been successfully overexpressed. SD02 (pBHR68) accumulated 4.86 g/L PHB, which was almost the same as that of SD01 (pBHR68/pTPSer), but had a higher PHB content of 77.2% CDW (Figure 2). Therefore SD02 was selected for further engineering.Figure 3

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