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Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell.

Huang LF, Lin JY, Pan KY, Huang CK, Chu YK - Int J Mol Sci (2015)

Bottom Line: The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion.Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion.Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 320, Taiwan. hlf326@saturn.yzu.edu.tw.

ABSTRACT
Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

No MeSH data available.


Related in: MedlinePlus

Characterization of transgenic Chlamydomonas overexpressing PETF and FDX5, respectively; (A) Schematic illustration of plasmids pHYG3-PETF-R and pHYG3-FDX5-R prepared for electroporation. The coding sequence of either PETF or FDX5 was ligated between the β2-tubulin promoter (PT) and the 3ʹUTR of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) to generate pHYG3-PETF-R and pHYG3-Fd5-R, respectively. The transformants harboring the aph7 gene were screened by hygromycin; (B) To confirm putative transformants, specific primers were used to amplify part of the promoter and the entire FDX gene to the 3ʹUTR region. Amplification of CBLP (G-protein beta subunit-like polypeptide) was used as an internal control for genomic DNA; (C) The relative quantity (RQ) of PETF and FDX5 transcripts in algal lines. The total mRNA transcripts from endogenous and recombinant FDX genes, either PETF or FDX5, were quantified by qPCR with specific primers. Relative expression levels are the relative quantities of either PETF or FDX5 transcripts compared to the CBLP transcripts respectively, and then normalized by the value of non-transformant CC125 under normal growth conditions.
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ijms-16-19308-f001: Characterization of transgenic Chlamydomonas overexpressing PETF and FDX5, respectively; (A) Schematic illustration of plasmids pHYG3-PETF-R and pHYG3-FDX5-R prepared for electroporation. The coding sequence of either PETF or FDX5 was ligated between the β2-tubulin promoter (PT) and the 3ʹUTR of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) to generate pHYG3-PETF-R and pHYG3-Fd5-R, respectively. The transformants harboring the aph7 gene were screened by hygromycin; (B) To confirm putative transformants, specific primers were used to amplify part of the promoter and the entire FDX gene to the 3ʹUTR region. Amplification of CBLP (G-protein beta subunit-like polypeptide) was used as an internal control for genomic DNA; (C) The relative quantity (RQ) of PETF and FDX5 transcripts in algal lines. The total mRNA transcripts from endogenous and recombinant FDX genes, either PETF or FDX5, were quantified by qPCR with specific primers. Relative expression levels are the relative quantities of either PETF or FDX5 transcripts compared to the CBLP transcripts respectively, and then normalized by the value of non-transformant CC125 under normal growth conditions.

Mentions: The binary constructs pHyg3-PETF-R and pHyg3-FDX5-R (Figure 1A), which contain different ferredoxin cDNAs, PETF and FDX5, fused downstream of the B2T (β2-tubulin) promoter, were transformed into Chlamydomonas by electroporation, and at least three putative independent transgenic Chlamydomonas lines were selected for each construct. To evaluate the putative transgenic Chlamydomonas lines, PCR-based genomic DNA analysis was performed using specific primers to confirm the presence of transgenes. The transgene PETFtg was detected in the cell lines P22, P51 and P67, and FDX5tg was detected in lines F5-1, F5-81, F5-302 and F5-303, respectively. In contrast, no transgenes were detected in the wild-type line (Figure 1B). The CBLP gene, encoding a Chlamydomonas G-protein beta subunit-like polypeptide, was used as a loading control for the extracted genomic DNA (Figure 1B). The levels of total PETF and FDX5 mRNA were determined by quantitative RT-PCR in each selected transgenic line. Figure 1C shows that PETF mRNA accumulated more in lines P22, P51 and P67 than in the wild type, as did FDX5 mRNA in lines F5-1, F5-302 and F5-303. These results indicate that the selected transgenic Chlamydomonas lines were ectopically overexpressing either PETF or FDX5.


Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell.

Huang LF, Lin JY, Pan KY, Huang CK, Chu YK - Int J Mol Sci (2015)

Characterization of transgenic Chlamydomonas overexpressing PETF and FDX5, respectively; (A) Schematic illustration of plasmids pHYG3-PETF-R and pHYG3-FDX5-R prepared for electroporation. The coding sequence of either PETF or FDX5 was ligated between the β2-tubulin promoter (PT) and the 3ʹUTR of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) to generate pHYG3-PETF-R and pHYG3-Fd5-R, respectively. The transformants harboring the aph7 gene were screened by hygromycin; (B) To confirm putative transformants, specific primers were used to amplify part of the promoter and the entire FDX gene to the 3ʹUTR region. Amplification of CBLP (G-protein beta subunit-like polypeptide) was used as an internal control for genomic DNA; (C) The relative quantity (RQ) of PETF and FDX5 transcripts in algal lines. The total mRNA transcripts from endogenous and recombinant FDX genes, either PETF or FDX5, were quantified by qPCR with specific primers. Relative expression levels are the relative quantities of either PETF or FDX5 transcripts compared to the CBLP transcripts respectively, and then normalized by the value of non-transformant CC125 under normal growth conditions.
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Related In: Results  -  Collection

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ijms-16-19308-f001: Characterization of transgenic Chlamydomonas overexpressing PETF and FDX5, respectively; (A) Schematic illustration of plasmids pHYG3-PETF-R and pHYG3-FDX5-R prepared for electroporation. The coding sequence of either PETF or FDX5 was ligated between the β2-tubulin promoter (PT) and the 3ʹUTR of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) to generate pHYG3-PETF-R and pHYG3-Fd5-R, respectively. The transformants harboring the aph7 gene were screened by hygromycin; (B) To confirm putative transformants, specific primers were used to amplify part of the promoter and the entire FDX gene to the 3ʹUTR region. Amplification of CBLP (G-protein beta subunit-like polypeptide) was used as an internal control for genomic DNA; (C) The relative quantity (RQ) of PETF and FDX5 transcripts in algal lines. The total mRNA transcripts from endogenous and recombinant FDX genes, either PETF or FDX5, were quantified by qPCR with specific primers. Relative expression levels are the relative quantities of either PETF or FDX5 transcripts compared to the CBLP transcripts respectively, and then normalized by the value of non-transformant CC125 under normal growth conditions.
Mentions: The binary constructs pHyg3-PETF-R and pHyg3-FDX5-R (Figure 1A), which contain different ferredoxin cDNAs, PETF and FDX5, fused downstream of the B2T (β2-tubulin) promoter, were transformed into Chlamydomonas by electroporation, and at least three putative independent transgenic Chlamydomonas lines were selected for each construct. To evaluate the putative transgenic Chlamydomonas lines, PCR-based genomic DNA analysis was performed using specific primers to confirm the presence of transgenes. The transgene PETFtg was detected in the cell lines P22, P51 and P67, and FDX5tg was detected in lines F5-1, F5-81, F5-302 and F5-303, respectively. In contrast, no transgenes were detected in the wild-type line (Figure 1B). The CBLP gene, encoding a Chlamydomonas G-protein beta subunit-like polypeptide, was used as a loading control for the extracted genomic DNA (Figure 1B). The levels of total PETF and FDX5 mRNA were determined by quantitative RT-PCR in each selected transgenic line. Figure 1C shows that PETF mRNA accumulated more in lines P22, P51 and P67 than in the wild type, as did FDX5 mRNA in lines F5-1, F5-302 and F5-303. These results indicate that the selected transgenic Chlamydomonas lines were ectopically overexpressing either PETF or FDX5.

Bottom Line: The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion.Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion.Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 320, Taiwan. hlf326@saturn.yzu.edu.tw.

ABSTRACT
Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

No MeSH data available.


Related in: MedlinePlus