Table_1_Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of β-carotene Ketolase Gene.docx

Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces β-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized β-carotene ketolase gene (bkt) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1. First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial β-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 μg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin.

角黄素（Canthaxanthin）是一种红橙色的叶黄素类（xanthophyll）化合物，具有较强的抗氧化活性，且生物利用度优于胡萝卜素类，主要应用于食品、化妆品、水产养殖及制药工业。天然角黄素市场的需求激增，推动研究者通过对异源宿主开展基因工程改造以生产角黄素。卷枝毛霉（Mucor circinelloides）是一种二态性真菌，其主要类胡萝卜素产物为β-胡萝卜素，被视作产类胡萝卜素研究的模式生物。本研究通过将密码子优化后的雨生红球藻（Haematococcus pluvialis）β-胡萝卜素酮化酶基因（bkt）整合至该真菌的基因组中，并置于强启动子zrt1的调控之下，成功构建了可生产角黄素的卷枝毛霉工程菌株。研究首先构建了基础质粒以敲除类胡萝卜素生物合成的负调控因子crgA基因，该操作可显著提升β-胡萝卜素的产量，而β-胡萝卜素可作为后续酮化酶催化反应合成角黄素的前体底物。该基因工程菌株可合成（576 ± 28）μg/g的角黄素，为目前卷枝毛霉中已报道的最高产量。此外，本研究还测定了重组菌株的细胞干重：培养96小时后，其细胞干重可达9.0 g/L以上。通过逆转录实时定量PCR（RT-qPCR）分析了过表达菌株中bkt基因的mRNA表达水平，结果显示，在培养24、48及72小时时，该基因的表达量分别较对照菌株提升5.3、4.1及3倍。本研究获得的产角黄素卷枝毛霉工程菌株，为进一步通过生物技术手段优化角黄素的生产能力奠定了基础，同时也为构建虾青素等更高价值的类胡萝卜素产物提供了可行的研究策略。