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Glucaric acid is a valuable chemical with applications in the detergent, polymer, pharmaceutical and food industries. In this study, two key enzymes for glucaric acid biosynthesis, MIOX4 (myo-inositol oxygenase) and Udh (uronate dehydrogenase), were fused and expressed with different peptide linkers. It was found that a strain harboring the fusion protein MIOX4-Udh linked by the peptide (EA3K)3 produced the highest glucaric acid titer and thereby resulted in glucaric acid production that was 5.7-fold higher than that of the free enzymes. Next, the fusion protein MIOX4-Udh linked by (EA3K)3 was integrated into delta sequence sites of the Saccharomyces cerevisiae opi1 mutant, and a strain, GA16, that produced a glucaric acid titer of 4.9 g/L in a shake flask fermentation was identified by a high-throughput screening method using an Escherichia coli glucaric acid biosensor. Strain improvement by further engineering was performed to regulate the metabolic flux of myo-inositol to increase the supply of glucaric acid precursors. The downregulation of ZWF1 and the overexpression of INM1 and ITR1 increased glucaric acid production significantly, and glucaric acid production was increased to 8.49 g/L in the final strain GA-ZII in a shake flask fermentation. Finally, in a 5-L bioreactor, GA-ZII produced a glucaric acid titer of 15.6 g/L through fed-batch fermentation. IMPORTANCE Glucaric acid is a value-added dicarboxylic acid that was synthesized mainly through the oxidation of glucose chemically. Due to the problems of the low selectivity, by-products, and highly polluting waste of this process, producing glucaric acid biologically has attracted great attention. The activity of key enzymes and the intracellular myo-inositol level were both rate-limiting factors for glucaric acid biosynthesis. To increase glucaric acid production, this work improved the activity of the key enzymes in the glucaric acid biosynthetic pathway through the expression of a fusion of Arabidopsis thaliana MIOX4 and Pseudomonas syringae Udh as well as a delta sequence-based integration. Furthermore, intracellular myo-inositol flux was optimized by a series of metabolic strategies to increase the myo-inositol supply, which improved glucaric acid production to a higher level. This study provided a way for constructing a glucaric acid-producing strain with good synthetic performance, making glucaric acid production biologically in yeast cells much more competitive. [ABSTRACT FROM AUTHOR]