Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Co-expression of multiple genes is crucial for synthetic biology but limited by traditional monodirectional promoters (MDPs).
• Nature utilizes bidirectional promoters (BDPs) for efficient gene co-expression, offering a model for improved synthetic tools.

Purpose of the Study:

• To create a comprehensive library of synthetic BDPs for enhanced gene co-expression in the yeast Komagataella phaffii.
• To provide a flexible platform for optimizing genetic circuits and metabolic pathways.

Main Methods:

• Generated a library of 168 synthetic BDPs in Komagataella phaffii (syn. Pichia pastoris).
• Leveraged naturally occurring BDPs as a source for synthetic parts.
• Applied modular design strategies for BDP construction.

Main Results:

• The synthetic BDP library enables rapid screening of diverse expression profiles and ratios.
• Successfully optimized gene co-expression for metabolic pathways, including taxadiene and β-carotene production.
• Demonstrated the utility of BDPs for metabolic engineering and synthetic biology.

Conclusions:

• The developed synthetic BDP library offers a versatile tool for advancing synthetic biology and metabolic engineering.
• The modular design approach for BDPs can be adapted for use in other eukaryotic hosts.
• This work expands the toolkit for precise control of gene expression in biotechnological applications.