Modular optimization of multi-gene pathways for fumarate production

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Microbial production of fumarate from renewable feedstocks presents a sustainable alternative to petroleum-based synthesis.
• Fumarate is a valuable platform chemical with diverse industrial applications.

Purpose of the Study:

• To develop a modular engineering approach for enhancing microbial fumarate production.
• To systematically address metabolic pathway bottlenecks and improve fumarate titer in yeast.

Main Methods:

• Re-casting yeast fumarate biosynthesis into three modules: reduction, oxidation, and byproduct modules.
• Targeting modules to specific cellular compartments (cytoplasm and mitochondria).
• Constructing protein fusions (RoMDH-P160A, KGD2-SUCLG2) and optimizing gene expression (RoPYC, RoMDH-P160A, KGD2-SUCLG2, SDH1).
• Utilizing DNA-guided scaffolds and sRNA switches for further optimization.

Main Results:

• Achieved a fumarate production of 20.46 g/L through combinatorial tuning and metabolic balance optimization.
• Further increased fumarate production to 33.13 g/L by engineering the byproduct module.
• Demonstrated significant titer improvements via systematic pathway bottleneck removal.

Conclusions:

• Modular pathway engineering is an effective strategy for systematically optimizing biosynthesis pathways.
• This approach enables efficient microbial production of fumarate from renewable resources.
• The developed methods hold promise for sustainable chemical synthesis.