Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii

Kasai, Seitaro; Yoshimura, Satomi; Ishikura, Kiyohide; Takaoka, Yasuko; Kobayashi, Kei; Kato, Ko; SHINMYO, ATSUHIKO

doi:10.1016/s1389-1723(03)80029-4

Cited by 35 publications

(25 citation statements)

References 17 publications

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“…4c with 5c). This result supports previous studies [34, 35] done on the reporter gene uidA in C. reinhardtii chloroplasts. We also found that, overall, heterologous transcripts accumulate to noticeably lower levels when cultures were grown at low cell density/high light conditions (Figs.…”

Section: Discussionsupporting

confidence: 93%

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

et al. 2010

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In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.

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Section: Discussionsupporting

confidence: 93%

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

et al. 2010

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“…The amounts of either protein that we obtained with the P ⁄ 5¢UTR of psbA were relatively low compared to those that were reported for other transgenes with the same P ⁄ 5¢UTR, suggesting that vapA and acrV are particularly difficult to express Surzycki et al, 2009). It was previously reported that inclusion of the beginning of the coding sequence of a native chloroplast gene in a chimeric construct can improve expression in Chlamydomonas (Kasai et al, 2003) or in higher plants (Maliga, 2002;Lenzi et al, 2008). To test whether this would apply to our transgenes, we compared psaA::vapA or psaA::acrV, which were fused at the AUG start codon of psaA, to constructs where the transgenes were fused to the coding region of psaA after codon 20 or codon 30.…”

Section: Discussioncontrasting

confidence: 55%

“…Thus, the beneficial effects that were reported apply to particular chloroplast gene ⁄ transgene pairs, but are not universally applicable. In our constructs with the P ⁄ 5¢UTR of atpA, the fusion is at position 26 of the coding sequence, similar to what was reported to be optimal (Kasai et al, 2003). However, this construct was not investigated further, because expression was lower than with the fusion to the AUG start codon of psaA-exon1.…”

Section: Discussionmentioning

confidence: 84%

“…It has been reported that higher levels of expression can be reached if the transgene is fused to a small part of the coding region of a chloroplast gene rather than to its AUG translation start codon (Kasai et al, 2003). In the atpA::vapA lines, the transgene is fused to the first 26 codons of the atpA gene and is expressed at comparatively high levels ( Figure 2a; Table S1).…”

Section: Comparison Of Chloroplast Promoters and 5¢utrsmentioning

confidence: 95%

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Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas

Michelet

Lefebvre-Legendre

Burr

et al. 2010

Plant Biotechnology Journal

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SummaryChloroplast transformation in microalgae offers great promise for the production of proteins of pharmaceutical interest or for the development of novel biofuels. For many applications, high level expression of transgenes is desirable. We have transformed the chloroplast of Chlamydomonas reinhardtii with two genes, acrV and vapA, which encode antigens from the fish pathogen Aeromonas salmonicida. The promoters and 5¢ untranslated regions of four chloroplast genes were compared for their ability to drive expression of the bacterial genes. The highest levels of expression were obtained when they were placed under the control of the cis-acting elements from the psaA-exon1 gene. The expression of these chimeric genes was further increased when a nuclear mutation that affects a factor involved in psaA splicing was introduced in the genetic background of the chloroplast transformants. Accumulation of both the chimeric mRNAs and the recombinant proteins was dramatically increased, indicating that negative feedback loops limit the expression of chloroplast transgenes. Our results demonstrate the potential of manipulating anterograde signalling to alter negative regulatory feedback loops in the chloroplast and improve transgene expression.

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“…Post-transcriptional control mechanisms can also have synergistic or antagonistic effects. Kasai et al (2003) used the GUS reporter controlled by various endogenous 3′- and 5′-UTRs to determine that there is an inverse correlation between protein accumulation and transcript stability, suggesting a feedback mechanism. Together all of these data suggest that regulation of mRNA translation has the greatest impact on recombinant protein accumulation, and is clearly an area where additional research seems likely to identify mechanisms for increased recombinant protein accumulation.…”

Section: Genetic Tools and Techniquesmentioning

confidence: 99%

Micro-algae come of age as a platform for recombinant protein production

2010

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A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins.

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Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii

Cited by 35 publications

References 17 publications

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas

Micro-algae come of age as a platform for recombinant protein production

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