Engineering Non-Repetitive Codon-Optimized HPcrtW Integration With Inducible Regulation for Canthaxanthin...

Area of Science:

• Biotechnology
• Metabolic Engineering
• Microbial Production

Background:

• Canthaxanthin is a valuable carotenoid with applications in food, feed, and pharmaceuticals.
• Current production methods often rely on chemical synthesis, which can be costly and environmentally impactful.
• Engineering microbial hosts like Yarrowia lipolytica offers a sustainable and scalable alternative for canthaxanthin biosynthesis.

Purpose of the Study:

• To engineer Yarrowia lipolytica for efficient and high-yield production of canthaxanthin.
• To identify and optimize key enzymes and genetic elements for enhanced carotenoid biosynthesis.
• To develop robust fermentation strategies for industrial-scale canthaxanthin manufacturing.

Main Methods:

• Screening of five heterologous β-carotene ketolase (CrtW) genes, identifying HPcrtW from Haematococcus pluvialis.
• Strain engineering involving gene copy number amplification, codon optimization, and co-expression strategies (e.g., CarRP-R98A mutant with CrtB).
• Utilizing an inducible promoter (pEYK-5AB) to manage metabolic burden and employing homologous recombination for stable integration of multiple gene copies.

Main Results:

• The engineered YCan101 strain produced 61.52 mg/L of canthaxanthin, increased to 98.65 mg/L with a second HPcrtW copy.
• Co-expression of CarRP-R98A and CrtB significantly enhanced β-carotene and total carotenoid accumulation.
• Final engineered strains achieved 457 mg/L in flask fermentation and a remarkable 3.08 g/L in fed-batch fermentation.

Conclusions:

• Successful engineering of Yarrowia lipolytica for high-titer canthaxanthin production.
• Demonstrated the effectiveness of metabolic flux optimization and genetic engineering strategies for industrial carotenoid synthesis.
• Established a viable microbial platform for sustainable and cost-effective canthaxanthin production, reducing reliance on chemical synthesis.