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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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Strategies for chromosomal replacement. A. Genome editing cassettes are constructed by three rounds of PCR and recombinants after the first round of recombineering were selected by tetracycline; B. In the second step, the tetA marker was eliminated by simultaneous induction of I-SceI and Red recombinase expression. C. Fragments for promoter replacement or insertion; D. Fragments for Ptrc-162-pgk-serABC operon insertion. DR for duplicate region; I-sceI for I-SceI endonuclease recognition site.
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Fig5: Strategies for chromosomal replacement. A. Genome editing cassettes are constructed by three rounds of PCR and recombinants after the first round of recombineering were selected by tetracycline; B. In the second step, the tetA marker was eliminated by simultaneous induction of I-SceI and Red recombinase expression. C. Fragments for promoter replacement or insertion; D. Fragments for Ptrc-162-pgk-serABC operon insertion. DR for duplicate region; I-sceI for I-SceI endonuclease recognition site.

Mentions: The DNA fragment insertion or replacement strains were constructed by using the method reported by Lin et al with appropriate modifications [36]. The strategies of fragment construction were outlined in Figure 5. The final fragments were transformed into the competent cells with expression of the λ red recombination enzymes. The tetracycline resistant mutants were screened and confirmed by colony PCR. To induce I-SceI endonuclease expression and remove the resistance gene tetA from the genome, the positive colony was inoculated into 5 ml of LB medium with 100 μg/mL spectinomycin, 2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 0.2% w/v L–arabinose. After overnight cultivation, cultures were diluted to appropriate concentration and plated on LB agar plates. The loss of tetA was confirmed by colony PCR. The technological process in detail was displayed in Figure 5. Primers used were listed in Table 5.Figure 5


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)

Strategies for chromosomal replacement. A. Genome editing cassettes are constructed by three rounds of PCR and recombinants after the first round of recombineering were selected by tetracycline; B. In the second step, the tetA marker was eliminated by simultaneous induction of I-SceI and Red recombinase expression. C. Fragments for promoter replacement or insertion; D. Fragments for Ptrc-162-pgk-serABC operon insertion. DR for duplicate region; I-sceI for I-SceI endonuclease recognition site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Strategies for chromosomal replacement. A. Genome editing cassettes are constructed by three rounds of PCR and recombinants after the first round of recombineering were selected by tetracycline; B. In the second step, the tetA marker was eliminated by simultaneous induction of I-SceI and Red recombinase expression. C. Fragments for promoter replacement or insertion; D. Fragments for Ptrc-162-pgk-serABC operon insertion. DR for duplicate region; I-sceI for I-SceI endonuclease recognition site.
Mentions: The DNA fragment insertion or replacement strains were constructed by using the method reported by Lin et al with appropriate modifications [36]. The strategies of fragment construction were outlined in Figure 5. The final fragments were transformed into the competent cells with expression of the λ red recombination enzymes. The tetracycline resistant mutants were screened and confirmed by colony PCR. To induce I-SceI endonuclease expression and remove the resistance gene tetA from the genome, the positive colony was inoculated into 5 ml of LB medium with 100 μg/mL spectinomycin, 2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 0.2% w/v L–arabinose. After overnight cultivation, cultures were diluted to appropriate concentration and plated on LB agar plates. The loss of tetA was confirmed by colony PCR. The technological process in detail was displayed in Figure 5. Primers used were listed in Table 5.Figure 5

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