Engineering Non‐Repetitive Codon‐Optimized HP <i>crtW</i> Integration With Inducible Regulation for Canthaxanthin Biosynthesis in <i>Yarrowia lipolytica</i>

This study aimed to engineer Yarrowia lipolytica for efficient and high-yield canthaxanthin production. We evaluated five heterologous β-carotene ketolase (CrtW) genes from various sources and identified HPcrtW from Haematococcus pluvialis for canthaxanthin biosynthesis. The strain YCan101, expressing HPcrtW, produced 61.52 mg/L of canthaxanthin. Further improvements were achieved by introducing a second copy of HPcrtW, increasing titer by 60% to 98.65 mg/L. To overcome β-carotene supply limitation, a strategy of co-expressing the CarRP-R98A (AGA → GCG) mutant with CrtB was employed. The strains co-expressing these two genes exhibited a significant increase in both β-carotene and total carotenoid accumulation. Three nonrepetitive codon-optimized HPcrtW were further utilized to improve strain stability and facilitate the integration of multiple gene copies, resulting in higher canthaxanthin production. Additionally, the inducible promoter pEYK-5AB was employed to partially mitigate the metabolic burden of the exogenous pathway on cell growth during fed-batch fermentation. The integration of nine copies of HPcrtW through nonrepetitive codon optimization and three cycles of homologous recombination, resulted in a final canthaxanthin production of 457 mg/L in flask fermentation and 3.08 g/L in fed-batch fermentation. This study provides valuable insights for optimizing metabolic flux in industrial-scale carotenoid production, offering a sustainable alternative to chemical synthesis.