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The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by β-carotene hydroxylase ( crtZ ) and β-carotene ketolase ( crtW ) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae . The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)