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

ABSTRACT 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 HP crtW from Haematococcus pluvialis for canthaxanthin biosynthesis. The strain YCan101, expressing HP crtW , produced 61.52 mg/L of canthaxanthin. Further improvements were achieved by introducing a second copy of HP crtW , 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 HP crtW 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 HP crtW 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.