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 {beta}-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 {beta}-alanine, but also revealed {beta}-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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/649204v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c04442org.highwire.dtl.DTLVardef@c16b4aorg.highwire.dtl.DTLVardef@6af6d9org.highwire.dtl.DTLVardef@743029_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIEngineered I. orientalis co-utilized xylose and acetate to produce 3-HP. C_LIO_LIAcetate addition enhanced 3-HP production during xylose fermentation. C_LIO_LIAcetate redirected metabolic flux toward 3-HP biosynthesis pathways. C_LIO_LIEngineered I. orientalis achieved 8.7 g/L 3-HP from hemp stalk hydrolysate via SHF. C_LI