3-hydroxypropionic acid production from Brewer's spent grain with an engineered Issatchenkia orientalis.

3-Hydroxypropionic acid (3-HP) is a versatile platform chemical with wide-ranging industrial applications. This study presents a proof-of-concept approach for producing 3-HP from brewer's spent grain (BSG) using a novel one-pot CaO pretreatment method and an engineered, acid-tolerant Issatchenkia orientalis IoDY01H strain. The effects of acid type for pH adjusting of pretreated slurry, nitrogen supplementation, NaHCO3 addition, and BSG deproteinization were evaluated. Results showed that using pure glucose and xylose as carbon sources, yeast extract-peptone (YPDX) medium enhanced 3-HP titers by 13.3 % compared to synthetic complete (SCDX) medium. NaHCO3 supplementation further boosted the 3-HP titer to 7.8 g/L, representing a 7.8 % increase over YPDX alone. In BSG hydrolysates, pH adjustment with H2SO4 resulted in higher 3-HP production (7.4 g/L) compared to H3PO4 (6.8 g/L). Moreover, supplementation with NaHCO3 further increased the 3-HP titer to 7.7 g/L, while yeast extract-peptone reduced the 3-HP titer to 6.7 g/L by diverting flux toward strain growth and ethanol formation. Notably, deproteinization of BSG improved xylose recovery and achieved the highest 3-HP titer of 8.7 g/L without nitrogen supplementation. This work provides insights into integrating one-pot pretreatment and hydrolysis and robust fermentation without external nitrogen to produce value-added products from biomass feedstocks.