葡萄糖酸（GA）是一种增值化学品，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​​细胞间变异是自然固有的，表明培养物中同时存在高性能和低性能细胞。低性能的细胞会导致营养浪费和生产效率低下。此外，肌醇加氧酶（MIOX）是GA生产中的关键限速酶，存在稳定性和活性低的问题。因此，消除电池之间的差异并提高 MIOX 稳定性可以选择高性能电池并提高 GA 产量。本研究基于GA生物传感器和四环素外排泵蛋白TetA构建了体内GA生物选择器，以持续选择GA高效生产菌株。此外，通过核糖体结合位点优化，将GA生物传感器的检测上限提高至40 g/L，实现了GA高性能细胞的高效富集。一种小型泛素样修饰剂 (SUMO) 增强了 MIOX 的稳定性和活性。总体而言，我们在补料分批 GA 生产中使用 GA 生物选择器和 SUMO-MIOX 融合体，在大肠杆菌中获得了 5.52 g/L 的滴度，比原始菌株高 17 倍。主要通过化学方法合成的有毒有价值的产品。由于GA生物合成存在非选择性、低效率和环境污染等问题，引起了人们的广泛关注。细胞间的非遗传变异和 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滴度，本工作通过基于GA生物传感器和TetA构建的GA生物选择器消除了细胞间的变异，并通过将SUMO与MIOX融合，提高了MIOX在GA生物合成途径中的稳定性和活性。最后，这些方法在 65 小时时将 GA 产量提高了 17 倍，达到 5.52 g/L。这项研究代表了大肠杆菌中 GA 生物合成途径工业应用的重要一步。

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, myo-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 in vivo 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 Escherichia coli, 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 E. coli.