Engineered citrate synthase improves citramalic acid generation in Escherichia coli

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Citramalic acid (citramalate) is a key five-carbon precursor for methacrylic acid synthesis.
• Escherichia coli produces citramalate via citramalate synthase, competing with citrate synthase for acetyl-CoA.
• Citrate synthase (gltA) deletion requires glutamate for growth, redirecting carbon flux.

Purpose of the Study:

• To engineer Escherichia coli citrate synthase to reduce its affinity for acetyl-CoA.
• To enhance microbial production of citramalate by redirecting carbon flux.
• To improve the synthesis of commodity chemicals through protein engineering.

Main Methods:

• Point mutations were introduced into the E. coli citrate synthase gene (gltA).
• Cell growth, enzyme activity, and citramalate production were compared in engineered variants.
• Fed-batch fermentation with an exponential feeding strategy was employed.

Main Results:

• The GltA[F383M] variant facilitated cell growth without glutamate.
• Citramalate production increased by 125% compared to the control strain in batch fermentation.
• Fed-batch fermentation yielded over 60 g/L citramalate with a yield of 0.53 g/g glucose.

Conclusions:

• Protein engineering of citrate synthase is effective for redirecting carbon flux.
• Reduced enzyme affinity can enhance the microbial production of valuable chemicals.
• This approach offers a viable strategy for producing traditional commodity chemicals.