Matrix regulation: a plug-and-tune method for combinatorial regulation in Saccharomyces cerevisiae

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• CRISPR Technology

Background:

• Optimizing transcriptional regulation of complex metabolic pathways in Saccharomyces cerevisiae is challenging.
• Existing methods lack the precision and scalability for fine-tuning multiple genes simultaneously.

Purpose of the Study:

• To develop a novel CRISPR-mediated method for precise transcriptional fine-tuning of long metabolic pathways.
• To enable high-throughput screening of gene expression combinations for pathway optimization.

Main Methods:

• Developed Matrix Regulation (MR), a CRISPR system for combinatorial gRNA assembly using efficient tRNA processing.
• Expanded CRISPR target recognition by characterizing dCas9 variants for NG PAM compatibility.
• Screened and enhanced activation domains to increase regulatory dynamic range.

Main Results:

• Generated combinatorial strain libraries for mevalonate and heme biosynthesis pathways.
• Achieved a 37-fold increase in squalene production and a 17-fold increase in heme production.
• Demonstrated the versatility and effectiveness of MR in Saccharomyces cerevisiae.

Conclusions:

• Matrix Regulation (MR) offers a powerful platform for optimizing complex metabolic pathways through precise transcriptional control.
• The method significantly enhances the production of target compounds, showcasing its potential for industrial biotechnology and fundamental research.