Wang Shuai, Yu Haiying, Zhu Kun
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
AMB Express. 2025 May 6;15(1):72. doi: 10.1186/s13568-025-01880-y.
(R)-3-hydroxyalkanoic acids (R-3HAs) play a crucial role as essential chemicals serving as precursors or intermediates in the synthesis of a wide range of valuable compounds, such as pharmaceuticals, antibiotics, and food additives. Despite their significance, achieving industrial-scale production of R-3HAs, particularly medium-chain-length (mcl) R-3HAs, has been challenging due to the absence of suitable strains with efficient biosynthesis pathways. This study focuses on achieving the production of mcl R-3HA monomers by leveraging the "substrate pool" of R-3-(R-3-hydroxyalkanoyloxy) alkanoic acids (HAAs) which is synthesized by HAAs synthase RhlA. The process involved truncating the rhamnolipids synthesis pathway in Pseudomonas aeruginosa PAO1 by knocking out downstream genes rhlB and rhlC, leading to the accumulation and collection of intermediate HAAs from the culture supernatant. To enhance the production of HAAs further, a series of key genes in the β-oxidation pathway were knocked out, resulting in a titer of approximately 18 g/L. Subsequently, hydrolysis of HAAs was conducted under alkaline conditions, where the dimers could be rapidly and efficiently converted into monomers. The hydrolysis process was completed in 2.5 h at 80 °C using a 0.5 M NaOH solution. The primary hydrolysis product identified through GC-MS analysis was (R)-3-hydroxydecanoic acid (R-3HD) with a purity of 95%.
（R）-3-羟基链烷酸（R-3HAs）作为重要化学品发挥着关键作用，它们是合成多种有价值化合物（如药物、抗生素和食品添加剂）的前体或中间体。尽管它们很重要，但由于缺乏具有高效生物合成途径的合适菌株，实现R-3HAs的工业规模生产，尤其是中链长度（mcl）R-3HAs的生产一直具有挑战性。本研究的重点是利用由HAA合酶RhlA合成的R-3-（R-3-羟基烷酰氧基）链烷酸（HAAs）的“底物池”来实现mcl R-3HA单体的生产。该过程包括通过敲除下游基因rhlB和rhlC来截断铜绿假单胞菌PAO1中的鼠李糖脂合成途径，从而从培养上清液中积累和收集中间产物HAAs。为了进一步提高HAAs的产量，敲除了β-氧化途径中的一系列关键基因，产量达到了约18 g/L。随后，在碱性条件下对HAAs进行水解，其中二聚体可以快速有效地转化为单体。使用0.5 M NaOH溶液在80°C下2.5小时内完成水解过程。通过GC-MS分析确定的主要水解产物是纯度为95%的（R）-3-羟基癸酸（R-3HD）。
