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

Abstract

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 developed 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 tricarboxylic acid (TCA) cycle intermediates. Acetate enhanced 3-HP production; however, the accumulation of β-alanine suggests that the activity of β-alanine-pyruvate aminotransferase may have been limited under acidic conditions. Consistent with this, fermentation at pH 5.5 resulted in higher 3-HP titers than at pH 3.5. 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.