Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome
Abstract
Eukaryotic green microalgae represent a sustainable, photosynthetic biotechnology platform for generating high-value products. The model green alga Chlamydomonas reinhardtii has already been used to generate high value bioproducts such as recombinant proteins and terpenoids. However, low, unstable, and variable nuclear transgene expression has limited the ease and speed of metabolic engineering and recombinant protein expression in this system. Here, novel genetic devices for transgene expression in C. reinhardtii have been developed by identifying cis-regulatory DNA elements capable of driving high transgene expression in C. reinhardtii promoters using de novo motif discovery informatics approaches. Thirteen putative motifs were synthesized as concatemers, linked to a common minimal basal promoter, and assayed for their activity to drive expression of a yellow fluorescent protein reporter gene. Following transformation of the vectors into C. reinhardtii by electroporation, in vivo measurements of yellow fluorescent protein expression by flow cytometry revealed that five of the DNA motifs analyzed displayed significantly higher reporter expression compared to the basal promoter control. Two of the concatemerized motifs, despite being much smaller minimal cis-regulatory elements, drove reporter expression at levels approaching that of the conventionally-used AR1 promoter. This analysis provides insight into C. reinhardtii promoter structure and gene regulation, and providesmore »
- Authors:
-
[1];
[2];
[1]
- Univ. of Sheffield (United Kingdom)
- Univ. of California, San Diego, CA (United States)
- Publication Date:
- Research Org.:
- Univ. of California, San Diego, CA (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office; Engineering and Physical Sciences Research Council (EPSRC); Biotechnology and Biological Sciences Research Council (BBSRC)
- OSTI Identifier:
- 1853821
- Grant/Contract Number:
- EE0008246; EP/N509735/1; EP/S020705/1; PHYCSF-03
- Resource Type:
- Accepted Manuscript
- Journal Name:
- New Biotechnology
- Additional Journal Information:
- Journal Volume: 68; Journal ID: ISSN 1871-6784
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Chlamydomonas reinhardtii; Recombinant protein expression; Synthetic promoters; Algal biotechnology; de novo motif discovery; Metabolic engineering
Citation Formats
McQuillan, Josie L., Berndt, Anthony J., Sproles, Ashley E., Mayfield, Stephen P., and Pandhal, Jagroop. Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome. United States: N. p., 2022.
Web. doi:10.1016/j.nbt.2022.01.001.
McQuillan, Josie L., Berndt, Anthony J., Sproles, Ashley E., Mayfield, Stephen P., & Pandhal, Jagroop. Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome. United States. https://doi.org/10.1016/j.nbt.2022.01.001
McQuillan, Josie L., Berndt, Anthony J., Sproles, Ashley E., Mayfield, Stephen P., and Pandhal, Jagroop. Mon .
"Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome". United States. https://doi.org/10.1016/j.nbt.2022.01.001. https://www.osti.gov/servlets/purl/1853821.
@article{osti_1853821,
title = {Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome},
author = {McQuillan, Josie L. and Berndt, Anthony J. and Sproles, Ashley E. and Mayfield, Stephen P. and Pandhal, Jagroop},
abstractNote = {Eukaryotic green microalgae represent a sustainable, photosynthetic biotechnology platform for generating high-value products. The model green alga Chlamydomonas reinhardtii has already been used to generate high value bioproducts such as recombinant proteins and terpenoids. However, low, unstable, and variable nuclear transgene expression has limited the ease and speed of metabolic engineering and recombinant protein expression in this system. Here, novel genetic devices for transgene expression in C. reinhardtii have been developed by identifying cis-regulatory DNA elements capable of driving high transgene expression in C. reinhardtii promoters using de novo motif discovery informatics approaches. Thirteen putative motifs were synthesized as concatemers, linked to a common minimal basal promoter, and assayed for their activity to drive expression of a yellow fluorescent protein reporter gene. Following transformation of the vectors into C. reinhardtii by electroporation, in vivo measurements of yellow fluorescent protein expression by flow cytometry revealed that five of the DNA motifs analyzed displayed significantly higher reporter expression compared to the basal promoter control. Two of the concatemerized motifs, despite being much smaller minimal cis-regulatory elements, drove reporter expression at levels approaching that of the conventionally-used AR1 promoter. This analysis provides insight into C. reinhardtii promoter structure and gene regulation, and provides a new toolbox of cis-regulatory elements that can be used to drive transgene expression at a variety of expression levels.},
doi = {10.1016/j.nbt.2022.01.001},
journal = {New Biotechnology},
number = ,
volume = 68,
place = {United States},
year = {Mon Jan 03 00:00:00 EST 2022},
month = {Mon Jan 03 00:00:00 EST 2022}
}
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