Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• L-Malic acid is a versatile chemical used in food additives, pharmaceuticals, and cosmetics.
• Efficient biosynthesis of L-malic acid is crucial for industrial applications.
• Current production methods require optimization for yield and productivity.

Purpose of the Study:

• To engineer a microbial strain for enhanced L-malic acid biosynthesis.
• To improve the L-malic acid transport system for increased efflux.
• To optimize the metabolic pathway for high-yield L-malic acid production.

Main Methods:

• Combinatorial overexpression of pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) to establish the reductive tricarboxylic acid (rTCA) pathway.
• Engineering of the L-malic acid transporter (Spmae) by removing ubiquitination sites to enhance efflux.
• Fine-tuning gene expression levels for pathway optimization.

Main Results:

• Achieved L-malic acid concentration of 30.25 g L⁻¹, yield of 0.30 g g⁻¹, and productivity of 0.32 g L⁻¹ h⁻¹ in strain W4209.
• Maintained high pyruvic acid levels (30.75 g L⁻¹, 0.31 g g⁻¹, 0.32 g L⁻¹ h⁻¹) with CaCO₃ as a neutralizing agent.
• Demonstrated successful reconstruction and optimization of the L-malic acid biosynthesis and transport system.

Conclusions:

• The developed metabolic engineering strategy significantly enhances L-malic acid production.
• The engineered strain W4209 shows high efficiency in L-malic acid biosynthesis and transport.
• This approach provides a valuable framework for the industrial production of L-malic acid and other chemicals.