By Caleb Sooknanan ‘20
In genetic engineering, metabolic pathways and genetic circuits can be manipulated in microbes to produce chemicals or activate certain functions. To do this, gene expression must be fine-tuned to balance and optimize protein levels of metabolic enzymes or regulators. Manipulating these in unicellular eukaryotes often involves core promoter sequences, the minimal portion of the promoter required to initiate DNA transcription. Doctor Rui M. C. Portela and researchers at La Universidade Nova de Lisboa recently studied whether synthetic core promoters could be used interchangeably between related organisms to control gene expression strength.
The researchers designed the synthetic core promoters de novo with a genome scale data set for S. cerevisiae. 112 synthetic core promoter sequences were designed with relationships between natural core promoters, nucleosome occupancy, and motif presence. The core promoters were then fused to a P. pastoris AOX1 cis-regulatory module (CRM), an important regulatory DNA sequence for this expression system. The ten most active core promoters were eventually fused to six additional CRMs, three in P. pastoris and three in S. cerevisiae.
The results indicated that inducible cis-regulatory modules (CRMs) constructs showed higher activity, up to 176% more than natural core promoters. The researchers also compared the activity of the same synthetic core promoters fused to different CRMs, finding correlations only for organism-specific CRMS. Therefore, the researchers suggested that the degree to which a gene expression system’s components may be separated and recombined can be maintained to some extent, but only within the same type of organisms.
Limitations may include how the expression strength of S. cerevisiae may have been influenced by the CRM as well as the core promoter. The phylogenetic distance between S. cerevisiae and P. pastoris may have also affected the extent of gene expression, as core promoters from distant yeasts maintain their functionality but have lower expression.
Nevertheless, the researchers demonstrated a synthetic core promoter design that could be useful for gene expression fine-tuning. Such promoters may have future applications in eukaryotic synthetic biology and genetic engineering.
- Portela, et al., Synthetic core promoters as universal parts for fine-tuning expression in different yeast species. American Chemical Society 6, 471-484 (2017). doi: 10.1021/acssynbio.6b00178