Acetate metabolism during xylose fermentation enhances 3-hydroxypropionic acid production in engineered acid-tolerant Issatchenkia orientalis

Efficient bioconversion of acetate-rich lignocellulosic biomass into value-added chemicals remains a major challenge due to the toxicity of acetic acid. In this study, we engineered an acid-tolerant Issatchenkia orientalis strain (IoDY01H) capable of producing 3-hydroxypropionic acid (3-HP), a key bioplastic precursor, from glucose, xylose, and acetate. Using a Cas9-based genome editing system with a hygromycin B resistance marker, we introduced heterologous genes encoding xylose utilization and β-alanine-based 3-HP biosynthetic pathways into the I. orientalis genome. Metabolomic analysis revealed that acetate supplementation redirected metabolic flux toward amino acid and lipid metabolism while reducing TCA cycle intermediates. Acetate enhanced 3-HP production by promoting accumulation of β-alanine, but also revealed β-alanine–pyruvate aminotransferase as a metabolic bottleneck under acidic conditions. Using pretreated hemp stalk hydrolysate as a feedstock, the engineered strain achieved a 3-HP titer of 8.7 g/L via separate hydrolysis and fermentation (SHF), outperforming simultaneous saccharification and fermentation (SSF). These findings demonstrate the feasibility of producing 3-HP from acetate-rich biomass using engineered non-conventional yeast and highlight I. orientalis as a promising microbial chassis for industrial bioconversion.