Presentation Information
[2Brew-17]Engineering Kluyveromyces marxianus yeast for the production of bio-pigments
○Yu-Zhen Li1, Xin-Qing Zhao1, Kai Li1, Jun Li2 (1. Shanghai Jiao Tong University (China), 2. Shanghai CHANDO Group Co., Ltd (China))
Keywords:
Kluyveromyces marxianus,β-carotene,metabolic engineering,carotenoids,fermentation optimization
[Purpose] Carotenoids are lipid-soluble pigments produced by plants, algae, and certain microorganisms. Due to their strong antioxidant activity and coloring properties, they are widely used in food, feed, pharmaceuticals, and cosmetics. As demand grows, metabolic engineering and synthetic biology have become essential strategies for carotenoid production. Kluyveromyces marxianus is a promising host for industrial biotechnology due to its rapid growth, thermotolerance, and ability to utilize diverse carbon sources. However, studies on carotenoid production using this yeast remain limited. This study aimed to construct and optimize a β-carotene-producing K. marxianus strain through metabolic engineering and fermentation optimization. We also explore its application in skin care.
[Methods] To establish the β-carotene biosynthetic pathway, heterologous genes (crtE, crtBY, and crtI) from two different species were introduced into K. marxianus. Different promoters were evaluated to modulate gene expression levels. Shake-flask fermentation experiments were conducted to assess β-carotene production under various culture conditions. Key parameters, including carbon source composition, C/N ratio, and cultivation temperature, were systematically optimized. The proliferation-promoting effect of the fermentation products on cells was measured using the CCK-8 kit.
[Results] The engineered K. marxianus strain produced β-carotene, suggesting that the newly discovered enzyme is active. Among the tested conditions, the use of glucose and an optimized C/N ratio of 84 led to enhanced production. The engineered strain achieved a β-carotene titer up to 50 mg/L. Furthermore, cultivation at 30 ℃ improved both cell growth and production. These results demonstrate that metabolic pathway optimization combined with a fermentation strategy is effective in enhancing β-carotene production in K. marxianus. 0.5% (v/v) of the fermentation product can increase the viability of keratinocytes by 30.5% and fibroblasts by 28.7%.
[Consideration] The results indicate that balancing the expression of pathway genes is critical for efficient β-carotene biosynthesis in K. marxianus. Promoter strength plays a key role in coordinating metabolic flux toward carotenoid production. Furthermore, optimization of culture conditions, particularly carbon source utilization and C/N ratio, significantly influenced product accumulation, likely by affecting precursor supply and cellular metabolism. Compared with conventional yeast hosts, K. marxianus may offer unique advantages for carotenoid and other pigment production due to its rapid growth and thermotolerance, which could reduce fermentation time and contamination risks. The function of keratinocytes and fibroblasts indicates potential for in-depth research into skin barrier protection and anti-aging.
[Conclusion] A β-carotene-producing K. marxianus strain was successfully developed through metabolic engineering. Combined pathway optimization and fermentation strategies significantly improved carotenoid production. These findings demonstrate the potential of K. marxianus as an efficient and robust platform for β-carotene biosynthesis and provide a foundation for further development of high-value carotenoid production in non-conventional yeasts. At the same time, it has great potential for application as a raw material in skin care products.
[Methods] To establish the β-carotene biosynthetic pathway, heterologous genes (crtE, crtBY, and crtI) from two different species were introduced into K. marxianus. Different promoters were evaluated to modulate gene expression levels. Shake-flask fermentation experiments were conducted to assess β-carotene production under various culture conditions. Key parameters, including carbon source composition, C/N ratio, and cultivation temperature, were systematically optimized. The proliferation-promoting effect of the fermentation products on cells was measured using the CCK-8 kit.
[Results] The engineered K. marxianus strain produced β-carotene, suggesting that the newly discovered enzyme is active. Among the tested conditions, the use of glucose and an optimized C/N ratio of 84 led to enhanced production. The engineered strain achieved a β-carotene titer up to 50 mg/L. Furthermore, cultivation at 30 ℃ improved both cell growth and production. These results demonstrate that metabolic pathway optimization combined with a fermentation strategy is effective in enhancing β-carotene production in K. marxianus. 0.5% (v/v) of the fermentation product can increase the viability of keratinocytes by 30.5% and fibroblasts by 28.7%.
[Consideration] The results indicate that balancing the expression of pathway genes is critical for efficient β-carotene biosynthesis in K. marxianus. Promoter strength plays a key role in coordinating metabolic flux toward carotenoid production. Furthermore, optimization of culture conditions, particularly carbon source utilization and C/N ratio, significantly influenced product accumulation, likely by affecting precursor supply and cellular metabolism. Compared with conventional yeast hosts, K. marxianus may offer unique advantages for carotenoid and other pigment production due to its rapid growth and thermotolerance, which could reduce fermentation time and contamination risks. The function of keratinocytes and fibroblasts indicates potential for in-depth research into skin barrier protection and anti-aging.
[Conclusion] A β-carotene-producing K. marxianus strain was successfully developed through metabolic engineering. Combined pathway optimization and fermentation strategies significantly improved carotenoid production. These findings demonstrate the potential of K. marxianus as an efficient and robust platform for β-carotene biosynthesis and provide a foundation for further development of high-value carotenoid production in non-conventional yeasts. At the same time, it has great potential for application as a raw material in skin care products.
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