State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, China.
Zhejiang Provincial Key Laboratory of Resources Protection and Innovation of Traditional Chinese Medicine, Zhejiang A&F University, Hangzhou, China.
Appl Environ Microbiol. 2024 Jun 18;90(6):e0014924. doi: 10.1128/aem.00149-24. Epub 2024 May 29.
Glucaric acid (GA) is a value-added chemical and can be used to manufacture food additives, anticancer drugs, and polymers. The non-genetic cell-to-cell variations in GA biosynthesis are naturally inherent, indicating the presence of both high- and low-performance cells in culture. Low-performance cells can lead to nutrient waste and inefficient production. Furthermore, -inositol oxygenase (MIOX) is a key rate-limiting enzyme with the problem of low stability and activity in GA production. Therefore, eliminating cell-to-cell variations and increasing MIOX stability can select high-performance cells and improve GA production. In this study, an GA bioselector was constructed based on GA biosensor and tetracycline efflux pump protein TetA to continuously select GA-efficient production strains. Additionally, the upper limit of the GA biosensor was improved to 40 g/L based on ribosome-binding site optimization, achieving efficient enrichment of GA high-performance cells. A small ubiquitin-like modifier (SUMO) enhanced MIOX stability and activity. Overall, we used the GA bioselector and SUMO-MIOX fusion in fed-batch GA production and achieved a 5.52-g/L titer in , which was 17-fold higher than that of the original strain.IMPORTANCEGlucaric acid is a non-toxic valuable product that was mainly synthesized by chemical methods. Due to the problems of non-selectivity, inefficiency, and environmental pollution, GA biosynthesis has attracted significant attention. The non-genetic cell-to-cell variations and MIOX stability were both critical factors for GA production. In addition, the high detection limit of the GA biosensor was a key condition for performing high-throughput screening of GA-efficient production strains. To increase GA titer, this work eliminated the cell-to-cell variations by GA bioselector constructed based on GA biosensor and TetA, and improved the stability and activity of MIOX in the GA biosynthetic pathway through fusing the SUMO to MIOX. Finally, these approaches improved the GA production by 17-fold to 5.52 g/L at 65 h. This study represents a significant step toward the industrial application of GA biosynthetic pathways in .
葡萄糖醛酸（GA）是一种高附加值的化学品，可用于制造食品添加剂、抗癌药物和聚合物。GA 生物合成中的非遗传细胞间变异是自然存在的，这表明培养物中存在高表现和低表现细胞。低表现细胞会导致营养浪费和生产效率低下。此外，肌醇氧化酶（MIOX）是一种关键的限速酶，在 GA 生产中存在稳定性和活性低的问题。因此，消除细胞间变异并提高 MIOX 的稳定性可以选择高表现细胞并提高 GA 的产量。在本研究中，基于 GA 生物传感器和四环素外排泵蛋白 TetA 构建了 GA 生物选择器，以连续选择 GA 高效生产菌株。此外，通过核糖体结合位点优化将 GA 生物传感器的上限提高到 40 g/L，实现了 GA 高产细胞的高效富集。一种小泛素样修饰物（SUMO）增强了 MIOX 的稳定性和活性。总体而言，我们在 GA 分批补料生产中使用 GA 生物选择器和 SUMO-MIOX 融合，在 65 小时内达到 5.52 g/L 的滴度，比原始菌株高 17 倍。
葡萄糖醛酸是一种无毒的有价值产品，主要通过化学方法合成。由于非选择性、低效性和环境污染等问题，GA 生物合成引起了广泛关注。非遗传细胞间变异和 MIOX 稳定性都是 GA 生产的关键因素。此外，GA 生物传感器的高检测限是进行 GA 高效生产菌株高通量筛选的关键条件。为了提高 GA 的产量，本工作通过基于 GA 生物传感器和 TetA 的 GA 生物选择器消除了细胞间变异，并通过将 SUMO 融合到 MIOX 中提高了 GA 生物合成途径中 MIOX 的稳定性和活性。最终，在 65 小时内将 GA 的产量提高到 5.52 g/L，提高了 17 倍。这项研究代表了朝着 GA 生物合成途径在工业应用中的重要一步。
