低场核磁共振技术快速检测小球藻油脂含量及其在高通量选育中的应用

Low field nuclear magnetic resonance for rapid quantitation of microalgae lipid and its application in high throughput screening

摘要:

为了建立一个高效的高产油微藻诱变育种流程，微藻中油脂含量快速和准确的测定在其中具有重要作用。在本研究中，首先利用低场核磁共振技术，建立了直接检测干藻粉和培养液中小球藻油脂含量的方法，其信号强度与细胞中油脂含量存在特异的线性关系，干藻粉和藻液中油脂含量与信号值拟合的R2均高于0.99，说明该方法用于小球藻油脂含量的检测是准确和可行的。同时该方法与传统油脂测量方法相比，具有快速、简便和准确等优点。但其通量不及尼罗红染色法，因此，我们开发了将尼罗红染色法用于初筛，低场核磁共振技术用于复筛的新型高通量藻种复合筛选方法，并将此筛选方法应用于一种异养高产油原壳小球藻的诱变育种过程中。首先从3 098株诱变藻种中初筛得到108株具有较高油脂含量的藻株，然后利用低场核磁共振技术复筛得到9株高产油性能的藻株，其中一株甘油三酯含量超过20%，比原始藻株提高1倍，培养168 h后培养液油脂浓度达到5 g/L，证明此诱变育种流程不仅提高了筛选的效率还可靠且可行。

Abstract:

A rapid and accurate determination method of lipids in microalgae plays a significant role in an efficient breeding process for high-lipid production of microalgae. Using low field nuclear magnetic resonance (LF-NMR), we developed a direct quantitative method for cellular lipids in Chlorella protothecoides cells. The LF-NMR signal had a linear relationship with the lipid content in the microalgae cells for both dry cell samples and algal broth samples (R2>0.99). These results indicated that we could use this method for accurate determination of microalgal lipids. Although LF-NMR is a rapid and easy lipid determination method in comparison to conventional methods, low efficiency would limit its application in high throughput screening. Therefore, we developed a novel combined high throughput screening method for high-lipid content mutants of C. protothecoides. Namely, we initially applied Nile red staining method for semi-quantification of lipid in the pre-screening process, and following with LF-NMR method for accurate lipid determination in re-screening process. Finally, we adopted this novel screening method in the breeding process of high-lipid content heterotrophic cells of C. protothecoides. From 3 098 mutated strains 108 high-lipid content strains were selected through pre-screening process, and then 9 mutants with high-lipid production were obtained in the re-screening process. In a consequence, with heterotrophical cultivation of 168 h, the lipid concentration could reach 5 g/L, and the highest lipid content exceeded 20% (W/W), which was almost two-fold to that of the wild strain. All these results demonstrated that the novel breeding process was reliable and feasible for improving the screening efficiency.