Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• Yeast organelles offer unique environments for bioproduct synthesis.
• Mitochondria are attractive for producing terpenoids and branched-chain alcohols due to abundant acetyl-CoA, ATP, and cofactors.
• This study investigates mitochondrial potential for 3-hydroxypropionate (3-HP) production.

Purpose of the Study:

• To explore the potential of yeast mitochondria for 3-HP synthesis.
• To maximize 3-HP production through cofactor engineering and flux control at the acetyl-CoA node.

Main Methods:

• Metabolic modeling to compare mitochondrial vs. cytosolic 3-HP synthesis.
• Targeting malonyl-CoA reductase (MCR) to mitochondria.
• Enhancing MCR enzyme expression.
• Optimizing mitochondrial NADPH supply by overexpressing POS5 and IDP1.
• Inducing expression of a mutated ACC1 in mitochondria.
• Evaluating engineered strains in shake flask and fed-batch fermentations.

Main Results:

• Mitochondrial 3-HP synthesis via the malonyl-CoA pathway showed higher yield and lower oxygen consumption than cytosolic synthesis.
• Mitochondrial MCR expression increased 3-HP production to 0.27 g/L (vs. 0.09 g/L in cytosol).
• Enhanced MCR expression reached 4.42 g/L 3-HP.
• Optimized NADPH supply increased titer to 5.11 g/L.
• Induced ACC1 mutant expression led to 6.16 g/L 3-HP in shake flasks.
• Fed-batch fermentation achieved a record 71.09 g/L 3-HP with 0.71 g/L/h productivity and 0.23 g/g glucose yield.

Conclusions:

• Yeast mitochondria are a promising compartment for 3-HP production.
• The engineered strain achieved the highest reported 3-HP titer in Saccharomyces cerevisiae.
• This demonstrates the potential of yeast mitochondria for developing advanced cell factories.