Screening and characterization of a high-astaxanthin-producing Xanthophyllomyces dendrorhous strain TP-4 via an efficient evolutionary-enrichment system.

Xanthophyllomyces dendrorhous (anamorph form Phaffia rhodozyma) is a key microbial host for industrial astaxanthin production, where yield depends on both the efficiency of the biosynthetic pathway and overall metabolic robustness. To develop superior industrial strains, we established an efficient evolutionary-enrichment system and an accelerated evolutionary workflow that couples astaxanthin production with cell growth under optimized oxidative stress conditions. This strategy enabled the enrichment of mutants exhibiting both high astaxanthin production and strong industrial fitness. Using this approach, we employed a high astaxanthin-producing X. dendrorhous strain A06 as the parental strain for evolution. After several rounds of screening over 2,000 colonies, we successfully identified the top-performing mutant, TP-4. TP-4 demonstrated a stable 20% increase in astaxanthin production relative to the parental strain, along with faster growth and enhanced fermentation performance. The genome of TP-4 was sequenced, and comparative genomic analysis against the parent strain A06 and a low-astaxanthin-producing control strain, C01, revealed multiple mutations potentially underlying its superior phenotype. These findings provide new insights into the metabolic and genetic basis of astaxanthin overproduction. This work establishes an efficient evolutionary platform for strain improvement and provides a mechanistic understanding of astaxanthin overproduction in X. dendrorhous, laying the groundwork for further optimization and industrial-scale application.