Increased Mevalonate Production Using Engineered Citrate Synthase and Phosphofructokinase Variants of Escherichia...

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biochemical Engineering

Background:

• Mevalonate is a crucial precursor for isoprenoid biosynthesis.
• Native metabolic pathways in Escherichia coli compete for acetyl-CoA, a key substrate for mevalonate synthesis.
• Hydroxymethylglutaryl-CoA reductase, the final enzyme in mevalonate formation, requires NADPH, potentially limiting production.

Purpose of the Study:

• To enhance mevalonate yield and productivity in E. coli by engineering chromosomal genes involved in acetyl-CoA metabolism and NADPH formation.
• To investigate the impact of modifying citrate synthase (GltA) and phosphofructokinase (PfkA) on mevalonate production.

Main Methods:

• Engineered nine variants of citrate synthase (GltA) and four variants of phosphofructokinase (PfkA) in E. coli strains overexpressing heterologous mevalonate pathway genes.
• Compared mevalonate production of engineered strains against wild-type and knockout strains in shake flask and controlled 1-liter bioreactor experiments.
• Analyzed the effects of casamino acids and nitrogen limitation on mevalonate yield and productivity.

Main Results:

• GltA variants generally improved mevalonate yield compared to wild-type, particularly in the absence of casamino acids.
• PfkA variants resulted in lower mevalonate production than the wild-type PfkA strain.
• The GltA[K167A] variant demonstrated an optimal balance between yield (0.20 g/g) and productivity (0.87 g/L·h).
• A nitrogen-limited process with GltA[K167A] achieved a final concentration of 36.9 g/L mevalonate with a yield of 0.31 g/g in 31 hours.

Conclusions:

• Chromosomal modification of GltA is an effective strategy to modulate intracellular acetyl-CoA pools for enhanced production of acetyl-CoA derived compounds like mevalonate.
• Acetyl-CoA availability, rather than NADPH, is identified as the primary limiting factor for mevalonate production in this system.
• The GltA[K167A] variant represents a promising strain for high-yield and high-productivity mevalonate manufacturing.