食用小球藻Chlorella zofingiensis异养发酵生产虾青素的调控机制研究

Approved and recommended as a nutraceutical by the United States Food and Drug Administration, astaxanthin, an edible red pigment, is found in a variety of foods like salmon. Astaxanthin has been demonstrated to possess extremely strong antioxidant activity and be beneficial to human nutrition and health. The global market of astaxanthin has grown very fast recently. Nevertheless, due to the high production cost, astaxanthin is still very expensive. At present, Haematococcus pluvialis is used for commercial production of astaxanthin. Although Chlorella zofingiensis has relatively low cellular astaxanthin content compared to H. pluvialis, it can grow heterotrophically in the dark using high-cell density fermentation technology on a large scale. Therefore, growing C. zofingiensis for the production of astaxanthin possesses tremendous potential. The present project aims to study the relationship between the enzymatic activities and amino acids compositions of the two key enzymes, β-carotene ketolase (BKT) and β-carotene hydroxylase (CHY), which are involved in astaxanthin biosynthesis in C. zofingiesis. Coupled with the structure-function relationship analysis and site-directed mutagenesis, the functional-enhanced BKT and CHY genes will be created and introduced into the algal genome for enhanced production of astaxanthin in the organism. Furthermore, we will use metabolic engineering and fermentation technology to direct cell carbon sources towards astaxanthin biosynthesis so as to greatly enhance the production of astaxanthin in C. zofingiensis. The present study will lay a solid foundation for employing C. zofingiensis as cell factories for commercial production of high-value natural astaxanthin by fermentation technology.

作为美国FDA认可并推荐的食品营养组分，天然虾青素(可食用红色素)存在于三文鱼等多种食品中。虾青素的超级抗氧化性赋予其多种有益于人体健康的功能，但其发酵生产技术仍不成熟，导致虾青素价格昂贵，市场缺口巨大。相比虾青素生产应用较多的雨生红球藻，食用小球藻Chlorella zofingiensis能利用有机碳源高密度异养发酵积累虾青素，而且不需要光，具有规模化发酵生产虾青素的巨大潜力，但目前其虾青素的含量仍有待提高。本研究拟利用酶的定向进化分析虾青素合成的关键酶β-胡萝卜素酮化酶（BKT）和羟化酶（CHY）结构和氨基酸组成与酶活力的关系。在此基础上利用定点突变创建功能增强的BKT和CHY基因并建立具高效积累虾青素的小球藻突变株，同时结合发酵工程技术引导小球藻细胞内碳代谢流向虾青素合成，阐明发酵过程中小球藻合成积累虾青素的调控机制，为利用小球藻发酵技术工业化生产天然虾青素打下坚实的理论基础。

虾青素是世界上最强的天然抗氧化剂之一，具有多重生理功效，受到了人们的广泛关注。小球藻Chlorella zofingiensis可利用葡萄糖进行高密度培养，被认为是有前景的虾青素的重要来源之一。但其发酵细胞中虾青素含量显著低于自养细胞，仅为干重的0.1%左右，严重制约其工业应用价值。因此本项目围绕C. zofingiensis发酵细胞中虾青素含量低这一瓶颈，通过第三代测序技术获得该小球藻的全基因测序信息，根据基因组提供的虾青素合成途径上所有基因的启动子信息，绘制了C. zofingiensis全基因组精细图，并构建虾青素和油脂的代谢调控网络，并建立了含有两种内源启动子的高效遗传转化体系。同时，发现高光缺氮条件和葡萄糖浓度可以调控虾青素积累，而细胞自噬和胞内活性氧水平与虾青素积累水平密切相关。本项目通过两步法培养显著提高了虾青素产率：第一步利用分批补料发酵得到大量发酵细胞，密度达到98g L-1；第二步在室外条件下利用悬转式光生物反应器进行诱导培养，发酵细胞内虾青素含量提高50%，虾青素达到5.26mg L-1 day-1，是目前报道的最高虾青素产率。.本项目按照预期设定目标，通过全基因组测序构建了小球藻中虾青素代谢调控网络，构建了小球藻突变株，并结合发酵工程技术引导小球藻细胞内碳代谢流向虾青素合成，阐释了发酵过程中小球藻合成积累虾青素的可能的调控机制，为利用小球藻发酵技术工业化生产天然虾青素打下坚实的理论基础。该项目标注高水平SCI论文23篇，第一标注6篇，其中发表中科院JCR一区论文10篇，包括Bioresource Technology 7篇（IF：5.807）、Biotechnology for Biofuels 2篇（IF：5.497）、Journal of Materials Chemistry A 1篇（IF：9.931），申请发明专利2项，培养博士研究生3名。