Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by 8-carotene hydroxylase (crtZ) and 8-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.

Automated summary

This study improved the biosynthesis pathway of astaxanthin in yeast by utilizing directed evolution. By reducing the accumulation of intermediate products and increasing the content of astaxanthin, the fusion enzyme of crtZ-crtW with a 2 X GGGGS peptides linker showed promising results. Through directed evolution, 9 variant fusion mutants with improved astaxanthin production were generated, and a variant, L95S + I206L, was identified to have a significantly increased astaxanthin content compared to the control strain. Structural analysis suggested that the mutations altered the interaction between the substrate and the enzyme's pocket. These findings provide an efficient strategy to reduce the accumulation of intermediate products in complex biosynthesis pathways.