单细胞水平高产虾青素雨生红球藻高通量筛选新方法研究

As an excellent antioxidant in nature, the natural astaxanthin shows great application in disease prevention and treatment. Haematococcus pluvialis (H. pluvialis) is the richest source of astaxanthin in nature and has been employed as microbial cell factory for producing astaxanthin. The screening of astaxanthin-hyperproducing strains is helpful to obtain the astaxanthin effectively, yet the low-throughput and low-accuracy are the fatal defects of the existing methods. This project proposes a solution to solve these key issues by developing a Raman cytometry sorting platform to realize the screening of astaxanthin-hyperproducing strains with high-throughput and high-accuracy. Rapid Raman detection will be employed to characterize and measure the content of astaxanthin precisely at single-cell level under high-speed flow state without extended cell culture, which could settle the key obstacle that the existing methods could not identify the astaxanthin precisely in live H. pluvialis and further resolve the issue of long period of screening due to the low growth rate of H. pluvialis. Meanwhile, droplet manipulation technology is applied to synchronize the on-line single-cell encapsulation and dielectrophoresis-based high-throughput and high-accuracy sorting. Finally, the screening of astaxanthin-hyperproducing H. pluvialis strains at the unit of live individual cells will be accomplished with non-invasive, high-throughput and high-accuracy. In Summary, this project will greatly improve the efficiency and of obtaining superior H. pluvialis strains and moreover should provide a single-cell analysis platform of common value for screening other kinds of microbial cell factories.

天然虾青素是自然界中已知的最强的抗氧化剂，在疾病的预防和治疗等领域具有广泛的应用价值。雨生红球藻是自然界中虾青素含量最高的生物资源，利用其提取虾青素具有广阔的发展前景。高产虾青素藻株的选育是提高虾青素获取效率的有效手段，但现有的选育方法存在通量低、准确性差等问题。本项目拟针对上述瓶颈问题发展基于拉曼流式分选技术的高产虾青素藻株高通量、高准确性筛选平台。利用拉曼快速识别技术，在单细胞水平上准确识别高速流动状态下高产虾青素细胞，克服现有方法无法精确识别活体雨生红球藻中虾青素的难题并有效解决由于雨生红球藻生长缓慢导致的筛选周期长的问题。同时借助液滴操控技术，实现单细胞在线液滴包裹和介电高通量、高准确性分选。最终实现活体单细胞水平高产虾青素雨生红球藻无损、高通量、高准确性筛选。项目的实施将为高产虾青素雨生红球藻的选育提供方法学平台，同时服务于其他能源微生物的种质选育。

天然虾青素是自然界中已知的最强的抗氧化剂，在疾病的预防和治疗等领域具有广泛的应用价值。雨生红球藻是自然界中虾青素含量最高的生物资源，利用其提取虾青素具有广阔的发展前景。高产虾青素藻株的选育是提高虾青素获取效率的有效手段，但现有的选育方法存在通量低、准确性差等问题。针对该瓶颈问题，本项目发展了高通量拉曼激活液滴分选技术，实现了高速流动状态下单细胞雨生红球藻细胞中虾青素的快速拉曼识别，克服了现有方法无法精确识别活体雨生红球藻中虾青素的难题并有效解决由于雨生红球藻生长缓慢导致的筛选周期长的问题；通过进一步耦合液滴微流控技术，实现了目标细胞的在线液滴包裹和介电高通量、高准确性分选。通过该拉曼激活液滴分选平台，实现了高产虾青素雨生红球藻细胞的高通量分选（260个细胞每分钟）、高准确率（98%）分选，而且分选后仍有92%细胞保持活性，与未分选细胞相比无明显差异。该平台将为高产虾青素雨生红球藻的选育提供方法学平台，同时服务于其他能源微生物的种质选育。