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[Display omitted] • The poor utilization of sucrose limits the CM application in Schizochytrium sp. • Overexpression of SH enhanced the utilization of sucrose by 2.57-fold. • The DHA titer of evolved strain from CM was 42.69% higher than from glucose. • Proteomics confirmed that CM enhanced DHA biosynthesis and lipid storage. • 25.26 g/L DHA with 51.45% content were obtained from CM by fed-batch. Cane molasses (CM) is a sugar-rich agro-industrial byproduct. The purpose of this study is to synthesize docosahexaenoic acid (DHA) in Schizochytrium sp. by using CM. The single factor analysis showed that sucrose utilization was the main factor limiting the utilization of CM. Therefore, the endogenous sucrose hydrolase (SH) was overexpressed in Schizochytrium sp., which enhanced the sucrose utilization rate 2.57-fold compared to the wild type. Furthermore, adaptive laboratory evolution was used to further improve sucrose utilization from CM. Comparative proteomics and RT-qPCR were used out to analyze the metabolic differences of evolved strain grown on CM and glucose, respectively. Finally, a constant flow rate CM feeding strategy was implemented, whereby the DHA titer and lipid yield of the final strain OSH-end reached 25.26 g/L and 0.229 g/g sugar, respectively. This study demonstrated the CM is a cost-effective carbon source for industrial DHA fermentation. [ABSTRACT FROM AUTHOR]