Engineering yeast artificial core promoter with designated base motifs

Area of Science:

• Synthetic biology
• Molecular biology
• Metabolic engineering

Background:

• Synthetic biology demands versatile promoters for precise gene expression control in cell factories.
• Yeast promoters feature variable core regions (20-80 bp) between the TATA-box and transcriptional start site (TSS), allowing for artificial promoter design.
• Optimizing these core regions requires specific base motifs to maintain or enhance promoter strength.

Purpose of the Study:

• To design and screen base motifs for yeast artificial core promoters.
• To evaluate the impact of different artificial sequences on gene expression levels.
• To identify promoters that enhance the production of carotenoids like lycopene and beta-carotene.

Main Methods:

• Designed and screened various 30-base artificial sequences for yeast core promoters.
• Tested promoter activities by measuring CrtY enzyme expression, which influences lycopene-carotene composition.
• Utilized upstream sequences from strong promoters (PEXP1, PGP D) and characterized promoter sequences based on T-rich or G/C-rich motifs.
• Assessed promoter performance by observing colony color spectrum (red-orange-yellow) and quantifying beta-carotene production.

Main Results:

• Variable expression levels of the CrtY enzyme were observed with different 30-base artificial sequences.
• Distinct colony-color distributions indicated varying lycopene-carotene compositions based on promoter designs.
• A champion promoter achieved a 5.5-fold enhancement in lycopene-carotene transformation.
• Another selected promoter resulted in a peak beta-carotene production of 7.4 mg/g dry cell weight (DCW).

Conclusions:

• The core promoter region can be effectively designed with a 30-base sequence.
• Specific base motifs significantly influence artificial core promoter strength, enhancing or weakening gene expression.
• T-rich elements and higher percentage of T (%T) with lower G/C percentage generally benefit artificial core promoter strength.