Abstract
Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, its development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2 or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high GFP expression. Further, we individually tested fourteen identified promoters, revealing substantial increases in GFP expression—up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome dataset.
Competing Interest Statement
SM was a founder of and holds an equity stake in Algenesis Inc, a company that could potentially benefit from this research. NJH declares financial interests in TeselaGen Biotechnologies and Ansa Biotechnologies. The remaining authors of this study declare that they have no known competing financial interests that would influence the work reported in this paper. The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abbreviations
- SAPS
- Synthetic Algal Promoters
- SPC
- Synthetic Promoter Chassis
- CRE
- Cis-regulatory Elements
- FACS
- Fluorescence Activated Cell Sorting
- NGS
- Next-generation Sequencing
- GFP
- Green Fluorescent Protein
- Hyg
- Hygromycin
- Zeo
- Zeocin
- CFU
- Colony Forming Unit
- pL0
- pLibrary-0
- pL-AR1
- pLibrary-AR1
- WT
- Wild-type
- POWRS
- POsition-sensitive WoRd Set
- STREME
- Simple, Thorough, Rapid, Enriched Motif Elicitation