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Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas

Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii 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. I...

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Autores principales: Niemeyer, Justus, Fischer, Laura, Aylward, Frank O’Neill, Schroda, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10138193/
https://www.ncbi.nlm.nih.gov/pubmed/37107706
http://dx.doi.org/10.3390/genes14040948
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author Niemeyer, Justus
Fischer, Laura
Aylward, Frank O’Neill
Schroda, Michael
author_facet Niemeyer, Justus
Fischer, Laura
Aylward, Frank O’Neill
Schroda, Michael
author_sort Niemeyer, Justus
collection PubMed
description Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii 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 Chlamydomonas, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of Chlamydomonas reinhardtii field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in Chlamydomonas. We used ble, NanoLUC, and mCherry 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 Chlamydomonas. 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 βTUB2 instead of the 5′-UTRs of PSAD or RBCS2. Apparently, the βTUB2 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 βTUB2 5′-UTR and sequences downstream of the first AUG in the mCherry reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning.
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spelling pubmed-101381932023-04-28 Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas Niemeyer, Justus Fischer, Laura Aylward, Frank O’Neill Schroda, Michael Genes (Basel) Brief Report Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii 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 Chlamydomonas, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of Chlamydomonas reinhardtii field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in Chlamydomonas. We used ble, NanoLUC, and mCherry 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 Chlamydomonas. 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 βTUB2 instead of the 5′-UTRs of PSAD or RBCS2. Apparently, the βTUB2 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 βTUB2 5′-UTR and sequences downstream of the first AUG in the mCherry reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning. MDPI 2023-04-21 /pmc/articles/PMC10138193/ /pubmed/37107706 http://dx.doi.org/10.3390/genes14040948 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Brief Report
Niemeyer, Justus
Fischer, Laura
Aylward, Frank O’Neill
Schroda, Michael
Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas
title Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas
title_full Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas
title_fullStr Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas
title_full_unstemmed Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas
title_short Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas
title_sort analysis of viral promoters for transgene expression and of the effect of 5′-utrs on alternative translational start sites in chlamydomonas
topic Brief Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10138193/
https://www.ncbi.nlm.nih.gov/pubmed/37107706
http://dx.doi.org/10.3390/genes14040948
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