Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• Engineering cellular metabolism relies on manipulating metabolic pathways.
• Understanding and optimizing these pathways is crucial for producing valuable compounds.

Purpose of the Study:

• To present a framework for rewriting central metabolic pathways, specifically for malonyl-CoA biosynthesis in yeast.
• To develop a method for assessing malonyl-CoA output through DNA reconstruction and colorimetric screening.

Main Methods:

• Applied pathway-scale DNA reconstruction and colorimetric screening (Pracs) to generate and test millions of enzyme variants.
• Introduced genetic mutations into genes encoding the malonyl-CoA biosynthetic pathway.
• Integrated beneficial enzyme variants into synthetic pathways to assess phenotypic diversity.

Main Results:

• Identified hundreds of beneficial enzyme mutants that increase malonyl-CoA output.
• Generated synthetic pathways with significant phenotypic diversity.
• Observed enhanced production of malonyl-CoA, carotenoids, and betaxanthin in engineered yeast.

Conclusions:

• Pracs is a versatile tool for efficiently orchestrating central metabolism to optimize the production of various chemicals.
• The developed framework advances the understanding and engineering of cellular metabolism.
• This method has broad utility for metabolic engineering applications.