Engineering acid-tolerant non-conventional yeast Pichia kudriavzevii E1 for butyric acid production

Butyric acid is an important organic acid with diverse applications; however, microbial production is hampered by the scarcity of acid-tolerant hosts. Here, we established a cytosolic reverse β-oxidation pathway in the acid-tolerant non-conventional yeast Pichia kudriavzevii E1 for butyric acid production. Strategies to enhance cytosolic acetyl-CoA availability were evaluated, including glyoxylate cycle disruption, acetyl-CoA synthetase overexpression, mobilization of peroxisomal acetyl units, and cytosolic expression of the pyruvate dehydrogenase complex. Cytosolic expression of the pyruvate dehydrogenase complex increased butyric acid production 1.7-fold, highlighting the importance of acetyl-CoA compartmentalization. Evaluation of pathway enzymes identified trans-enoyl-CoA reductase (Ter) as a potential rate-limiting enzyme. A previously uncharacterized Ter enzyme, designated LpTer, was identified from a strong-aroma Baijiu pit-mud metagenome. Optimizing LpTer copy number and cytosolic NADH utilization increased the butyric acid titer to 339.07 ± 8.30 mg/L, representing an approximately 68-fold improvement over the initial strain. This study represents the first systematic engineering of the acid-tolerant yeast P. kudriavzevii for de novo butyric acid production without pH adjustment through pathway optimization and enzyme mining.