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Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

Kasai Y, Oshima K, Ikeda F, Abe J, Yoshimitsu Y, Harayama S - Biotechnol Biofuels (2015)

Bottom Line: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system.These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates.Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.

ABSTRACT

Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.

Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.

Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

No MeSH data available.


Related in: MedlinePlus

Construction of the plasmids used in this study. The genes and loci are abbreviated as follows: cPeUMPS PeUMPS cDNA; lacP, the promoter and operator regions of the E. coli beta-galactosidase gene (lacZ); nptII, the gene for neomycin phosphotransferase II to confer G418 resistance; TUBP, the promoter region of PeTUBULIN1; ACTT, the terminator region of PeACT1; RBCSP, the promoter region of PeRBCS; RBCST, the terminator region of PeRBCS; RBCS 1st Int, the first intron of RBCS
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Fig1: Construction of the plasmids used in this study. The genes and loci are abbreviated as follows: cPeUMPS PeUMPS cDNA; lacP, the promoter and operator regions of the E. coli beta-galactosidase gene (lacZ); nptII, the gene for neomycin phosphotransferase II to confer G418 resistance; TUBP, the promoter region of PeTUBULIN1; ACTT, the terminator region of PeACT1; RBCSP, the promoter region of PeRBCS; RBCST, the terminator region of PeRBCS; RBCS 1st Int, the first intron of RBCS

Mentions: A 1.5-kb PeUMPS cDNA was cloned into pUC119 to construct pUC_cPeUMPS (Fig. 1). The cloned PeUMPS cDNA sequence was 100 % identical to that predicted from the genomic sequence of P. ellipsoidea strain Obi. The pTV-cPeUMPS plasmid (Fig. 1) was constructed to express PeUMPS cDNA in two uracil auxotrophic mutants of Escherichia coli: JD24489 (an OPRTase mutant) and JW1273 (an ODCase mutant). Both E. coli mutants did not grow on glucose M9 minimal medium, whereas clones that possessed pTV-cPeUMPS grew efficiently, suggesting that the PeUMPS cDNA was expressed functionally in E. coli.Fig. 1


Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

Kasai Y, Oshima K, Ikeda F, Abe J, Yoshimitsu Y, Harayama S - Biotechnol Biofuels (2015)

Construction of the plasmids used in this study. The genes and loci are abbreviated as follows: cPeUMPS PeUMPS cDNA; lacP, the promoter and operator regions of the E. coli beta-galactosidase gene (lacZ); nptII, the gene for neomycin phosphotransferase II to confer G418 resistance; TUBP, the promoter region of PeTUBULIN1; ACTT, the terminator region of PeACT1; RBCSP, the promoter region of PeRBCS; RBCST, the terminator region of PeRBCS; RBCS 1st Int, the first intron of RBCS
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4489027&req=5

Fig1: Construction of the plasmids used in this study. The genes and loci are abbreviated as follows: cPeUMPS PeUMPS cDNA; lacP, the promoter and operator regions of the E. coli beta-galactosidase gene (lacZ); nptII, the gene for neomycin phosphotransferase II to confer G418 resistance; TUBP, the promoter region of PeTUBULIN1; ACTT, the terminator region of PeACT1; RBCSP, the promoter region of PeRBCS; RBCST, the terminator region of PeRBCS; RBCS 1st Int, the first intron of RBCS
Mentions: A 1.5-kb PeUMPS cDNA was cloned into pUC119 to construct pUC_cPeUMPS (Fig. 1). The cloned PeUMPS cDNA sequence was 100 % identical to that predicted from the genomic sequence of P. ellipsoidea strain Obi. The pTV-cPeUMPS plasmid (Fig. 1) was constructed to express PeUMPS cDNA in two uracil auxotrophic mutants of Escherichia coli: JD24489 (an OPRTase mutant) and JW1273 (an ODCase mutant). Both E. coli mutants did not grow on glucose M9 minimal medium, whereas clones that possessed pTV-cPeUMPS grew efficiently, suggesting that the PeUMPS cDNA was expressed functionally in E. coli.Fig. 1

Bottom Line: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system.These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates.Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.

ABSTRACT

Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.

Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.

Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

No MeSH data available.


Related in: MedlinePlus