Highly efficient neutralizer-free l-malic acid production using engineered Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Industrial Microbiology
• Metabolic Engineering

Background:

• Industrial organic acid production incurs high costs due to neutralizer use.
• Environmental concerns associated with traditional production methods necessitate sustainable alternatives.

Purpose of the Study:

• To develop a novel Saccharomyces cerevisiae strain for high-yield l-malic acid production.
• To engineer a cost-effective and environmentally friendly bioprocess for l-malic acid synthesis.

Main Methods:

• Adaptive laboratory evolution to isolate a low-pH tolerant yeast mutant (TAMC).
• Overexpression of malate dehydrogenase 3 (MDHΔSKL) and pyruvate carboxylase 2 (PYC2) for enhanced cytoplasmic synthesis.
• Design and implementation of the malic acid transporter SpMae1 for improved extracellular secretion.
• Optimization of precursor pyruvate synthesis pathways.

Main Results:

• Achieved a 5.6-fold increase in l-malic acid titer through gene overexpression.
• Increased extracellular l-malic acid titer from 7.3 to 73.6 g/L by introducing SpMae1.
• Reached a titer of 81.8 g/L by optimizing pyruvate synthesis.
• Attained the highest reported l-malic acid titer of 232.9 g/L in a 3-L bioreactor without neutralizers.

Conclusions:

• The engineered yeast strain demonstrates significant potential for large-scale, green l-malic acid production.
• This approach offers a sustainable and economically viable alternative to conventional methods.
• The developed strain and process pave the way for reduced industrial costs and environmental burden.