Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i>
Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, <i>Chlamydomonas reinhardtii</i> has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs...
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| Format: | Article |
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MDPI AG
2023-04-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/14/4/948 |
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| author | Justus Niemeyer Laura Fischer Frank O’Neill Aylward Michael Schroda |
| author_facet | Justus Niemeyer Laura Fischer Frank O’Neill Aylward Michael Schroda |
| author_sort | Justus Niemeyer |
| collection | DOAJ |
| description | Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, <i>Chlamydomonas reinhardtii</i> has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect <i>Chlamydomonas</i>, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of <i>Chlamydomonas reinhardtii</i> field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in <i>Chlamydomonas</i>. We used <i>ble</i>, <i>NanoLUC</i>, and <i>mCherry</i> as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in <i>Chlamydomonas</i>. We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5′-UTR of <i>βTUB2</i> instead of the 5′-UTRs of <i>PSAD</i> or <i>RBCS2</i>. Apparently, the <i>βTUB2</i> 5′-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the <i>βTUB2</i> 5′-UTR and sequences downstream of the first AUG in the <i>mCherry</i> reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning. |
| first_indexed | 2024-03-11T04:58:57Z |
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| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-11T04:58:57Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-55e76b355b2f4200919ff565dfbd4cc02023-11-17T19:24:54ZengMDPI AGGenes2073-44252023-04-0114494810.3390/genes14040948Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i>Justus Niemeyer0Laura Fischer1Frank O’Neill Aylward2Michael Schroda3Molecular Biotechnology & Systems Biology, RPTU Kaiserslautern-Landau, Paul-Ehrlich-Straße 23, 67663 Kaiserslautern, GermanyMolecular Biotechnology & Systems Biology, RPTU Kaiserslautern-Landau, Paul-Ehrlich-Straße 23, 67663 Kaiserslautern, GermanyDepartment of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USAMolecular Biotechnology & Systems Biology, RPTU Kaiserslautern-Landau, Paul-Ehrlich-Straße 23, 67663 Kaiserslautern, GermanyMicroalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, <i>Chlamydomonas reinhardtii</i> has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect <i>Chlamydomonas</i>, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of <i>Chlamydomonas reinhardtii</i> field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in <i>Chlamydomonas</i>. We used <i>ble</i>, <i>NanoLUC</i>, and <i>mCherry</i> as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in <i>Chlamydomonas</i>. We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5′-UTR of <i>βTUB2</i> instead of the 5′-UTRs of <i>PSAD</i> or <i>RBCS2</i>. Apparently, the <i>βTUB2</i> 5′-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the <i>βTUB2</i> 5′-UTR and sequences downstream of the first AUG in the <i>mCherry</i> reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning.https://www.mdpi.com/2073-4425/14/4/948synthetic biologygolden gate cloninggiant virusviral promotersmCherry isoforms5′-UTR |
| spellingShingle | Justus Niemeyer Laura Fischer Frank O’Neill Aylward Michael Schroda Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i> Genes synthetic biology golden gate cloning giant virus viral promoters mCherry isoforms 5′-UTR |
| title | Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i> |
| title_full | Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i> |
| title_fullStr | Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i> |
| title_full_unstemmed | Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i> |
| title_short | Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in <i>Chlamydomonas</i> |
| title_sort | analysis of viral promoters for transgene expression and of the effect of 5 utrs on alternative translational start sites in i chlamydomonas i |
| topic | synthetic biology golden gate cloning giant virus viral promoters mCherry isoforms 5′-UTR |
| url | https://www.mdpi.com/2073-4425/14/4/948 |
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