Protocol for connector-enabled multigene pathway assembly using Golden Gate Assembly and yeast recombination.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Yeast Genetics

Background:

• Multigene pathway construction is crucial for metabolic engineering and synthetic biology.
• Traditional methods can be complex, time-consuming, and require specialized expertise.
• Standardized and efficient protocols are needed to accelerate yeast-based research.

Purpose of the Study:

• To present a streamlined protocol for multigene pathway construction in Saccharomyces cerevisiae.
• To integrate Golden Gate Assembly with yeast homologous recombination for enhanced efficiency.
• To reduce complexity and operator dependence in assembling multiple transcriptional units.

Main Methods:

• Developed a connector-enabled workflow for assembling Level 1 transcriptional unit (TU) fragments.
• Utilized standardized connectors for seamless Golden Gate Assembly.
• Co-transformed assembled TUs into Saccharomyces cerevisiae for Level 2 multigene plasmid formation.

Main Results:

• The protocol significantly reduces procedural complexity compared to conventional methods.
• Minimized the dependence on specialized operator expertise.
• Substantially accelerated workflow timelines for multigene construct assembly.

Conclusions:

• The presented protocol offers a simplified, faster, and more accessible approach for multigene pathway construction in yeast.
• This method facilitates rapid assembly of complex genetic constructs in Saccharomyces cerevisiae.
• The connector-enabled workflow enhances the efficiency and reproducibility of yeast synthetic biology applications.