Jeong Deokyeol, Lee Dahye, Liu Junli, Kim Soo Rin, Jin Yong-Su, Zhao Jikai, Oh Eun Joong
Department of Food Science, Purdue University, West Lafayette, IN 47907, USA; Whistler Center for Carbohydrate Research, Purdue University, West Lafayette, IN 47907, USA.
Department of Agriculture and Biological Engineering, Purdue University, West Lafayette, IN 47907, USA; Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907, USA.
Bioresour Technol. 2025 Dec;437:133113. doi: 10.1016/j.biortech.2025.133113. Epub 2025 Aug 6.
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.
由于乙酸的毒性，将富含乙酸的木质纤维素生物质高效生物转化为高附加值化学品仍然是一项重大挑战。在本研究中，我们开发了一种耐酸东方伊萨酵母菌株（IoDY01H），该菌株能够从葡萄糖、木糖和乙酸中生产关键生物塑料前体3-羟基丙酸（3-HP）。我们使用基于Cas9的基因组编辑系统和潮霉素B抗性标记，将编码木糖利用和基于β-丙氨酸的3-HP生物合成途径的异源基因引入东方伊萨酵母基因组。代谢组学分析表明，添加乙酸可使代谢通量转向氨基酸和脂质代谢，同时减少三羧酸（TCA）循环中间体。乙酸提高了3-HP的产量；然而，β-丙氨酸的积累表明，β-丙氨酸-丙酮酸转氨酶的活性在酸性条件下可能受到限制。与此一致的是，在pH 5.5下发酵产生的3-HP滴度高于在pH 3.5下。使用预处理的大麻茎水解物作为原料，通过单独水解和发酵（SHF），工程菌株实现了8.7 g/L的3-HP滴度，优于同步糖化发酵（SSF）。这些发现证明了使用工程化非常规酵母从富含乙酸的生物质中生产3-HP的可行性，并突出了东方伊萨酵母作为工业生物转化的有前景的微生物底盘。
