Adaptive laboratory evolution of Blakeslea trispora under acetoacetanilide stress leads to enhanced β-carotene biosynthesis.

Blakeslea trispora is an important microbial producer of natural β-carotene, a valuable compound with significant nutritional and industrial applications. In the present study, an adaptive laboratory evolution (ALE) approach was applied to increase β-carotene production by exposing wild-type and UV-mutant B. trispora strains to increasing concentrations of the biosynthetic stressor acetoacetanilide. Over 95 serial transfers spanning 16 months, the adapted strain A78 showed a 45% increase in β-carotene yield (54 ± 1.95 mg/L) compared to the wild type (21.6 ± 2.11 mg/L), without a major compromise in biomass accumulation. Quantitative RT-PCR analysis revealed the upregulation of key carotenogenic genes, particularly hmgR, carRA, and SR5AL, in the adapted strains. Additionally, morphological changes, unsaturated fatty acid content, and altered antioxidant enzyme activities were investigated. The results show that chemical stress in the ALE strategy is effective in increasing metabolite production and stress tolerance of filamentous fungi and can pave the way for improving industrial strains.