Limits...
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

Growth of transgenic strains in MA5 minimal medium. aP. ellipsoidea strain Obi (closed circles), M4 in the presence of 1 mM uracil (open circles), M4-1A (closed squares), M4-1B (open squares), M4-1C (closed triangles), and M4-1D (open triangles). bP. ellipsoidea strain Obi (closed circles), M4-2A (closed squares), M4-2B (open squares), M4-2D (closed triangles), and M4-2E (open triangles). Experiments were performed in triplicate; bars represent the standard deviation
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Growth of transgenic strains in MA5 minimal medium. aP. ellipsoidea strain Obi (closed circles), M4 in the presence of 1 mM uracil (open circles), M4-1A (closed squares), M4-1B (open squares), M4-1C (closed triangles), and M4-1D (open triangles). bP. ellipsoidea strain Obi (closed circles), M4-2A (closed squares), M4-2B (open squares), M4-2D (closed triangles), and M4-2E (open triangles). Experiments were performed in triplicate; bars represent the standard deviation

Mentions: Analysis of the growth of the transgenic strains in MA5 minimal liquid medium showed that transgenic strains containing the pUT1 sequence started to grow with a lag phase of 2–8 days and slower growth rates compared with the wild-type strain (P. ellipsoidea strain Obi) and strain M4 in the presence of 1 mM uracil (Fig. 5a). In contrast, two of the transgenic strains carrying the pUT2 sequence (4 M-2B and 4 M-2D) grew in MA5 minimal liquid culture at growth rates equivalent to wild-type P. ellipsoidea strain Obi, whereas the other two transgenic strains (4 M-2A and 4 M-2E) grew slower than the wild-type strain, despite high levels of transgene expression (Fig. 5b). Although the reason for the slower growth of strains 4 M-2A and 4 M-2E is unclear, it is possible that the insertion of a copy of PeUMPS cDNA or the G428-resistance gene into the genomes of these strains disrupted gene(s) that are essential to the rapid growth of P. ellipsoidea.Fig. 5


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)

Growth of transgenic strains in MA5 minimal medium. aP. ellipsoidea strain Obi (closed circles), M4 in the presence of 1 mM uracil (open circles), M4-1A (closed squares), M4-1B (open squares), M4-1C (closed triangles), and M4-1D (open triangles). bP. ellipsoidea strain Obi (closed circles), M4-2A (closed squares), M4-2B (open squares), M4-2D (closed triangles), and M4-2E (open triangles). Experiments were performed in triplicate; bars represent the standard deviation
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Growth of transgenic strains in MA5 minimal medium. aP. ellipsoidea strain Obi (closed circles), M4 in the presence of 1 mM uracil (open circles), M4-1A (closed squares), M4-1B (open squares), M4-1C (closed triangles), and M4-1D (open triangles). bP. ellipsoidea strain Obi (closed circles), M4-2A (closed squares), M4-2B (open squares), M4-2D (closed triangles), and M4-2E (open triangles). Experiments were performed in triplicate; bars represent the standard deviation
Mentions: Analysis of the growth of the transgenic strains in MA5 minimal liquid medium showed that transgenic strains containing the pUT1 sequence started to grow with a lag phase of 2–8 days and slower growth rates compared with the wild-type strain (P. ellipsoidea strain Obi) and strain M4 in the presence of 1 mM uracil (Fig. 5a). In contrast, two of the transgenic strains carrying the pUT2 sequence (4 M-2B and 4 M-2D) grew in MA5 minimal liquid culture at growth rates equivalent to wild-type P. ellipsoidea strain Obi, whereas the other two transgenic strains (4 M-2A and 4 M-2E) grew slower than the wild-type strain, despite high levels of transgene expression (Fig. 5b). Although the reason for the slower growth of strains 4 M-2A and 4 M-2E is unclear, it is possible that the insertion of a copy of PeUMPS cDNA or the G428-resistance gene into the genomes of these strains disrupted gene(s) that are essential to the rapid growth of P. ellipsoidea.Fig. 5

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