Spatiotemporal Regulation of Astaxanthin Synthesis in S. cerevisiae

As a high-valued antioxidant, astaxanthin biosynthesis using microbial cell factories has attracted increasing attention. However, its lipophilic nature conflicts with the limited storage capacity for lipophilic substances of model microorganisms such as Saccharomyces cerevisiae. Expansion of lipid droplets by enhancing lipid synthesis provides more storage room while diverting the metabolic flux from the target pathway. Therefore, proper spatial regulation is required. In this study, a library of genes related to lipid metabolism were screened using the trifunctional CRISPR system, identifying opi3 and hrd1 as new engineering targets to promote astaxanthin synthesis by moderately rather than excessively upregulating lipid synthesis. The astaxanthin yield reached 9.79 mg/g DCW after lipid engineering and was further improved to 10.21 mg/g DCW by balancing the expression of β-carotene hydroxylase and ketolase. Finally, by combining spatial regulation through lipid droplet engineering and temporal regulation via temperature-responsive pathway expression, 446.4 mg/L astaxanthin was produced in fed-batch fermentation.