Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting.

Koji Yamada,Osamu Iwata,Takuto Takeuchi,Hideyuki Suzuki,Kengo Suzuki,Tomoko Abe,Keisuke Goda,Sharbanee Mitra,Yusuke Kazama

doi:10.1038/srep26327

Abstract

Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions–a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY505/515, and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.

Highlights

Inefficient mutations in its chloroplast genomes[16]
The breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY505/515, and Fluorescence-activated cell sorting (FACS)-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT)
We investigated the autofluorescence of E. gracilis cells, in particular the WT (Z strain) cultured in an autotropic and heterotrophic condition and a chloroplast-less mutant strain, SM-ZK, cultured in a heterotrophic condition when exciting them with laser light at 350 nm, 488 nm, and 635 nm

Summary

Introduction

Inefficient mutations in its chloroplast genomes[16]. its killing curve by UV irradiation indicates that E. gracilis is polyploid, making it difficult to alter its nuclear genome[17,18]. For evaluation and characterization of E. gracilis, while FACS has been used to analyze its cell cycle by staining with propidium iodide or Hoechst 33258 after fixation[30,31], it has been difficult to apply FACS to live E. gracilis cells due to the toxicity of the staining reagents and its low cell viability after FACS In this Article, we present a method that overcomes the aforementioned technical difficulties and enables efficient selective breeding of live oil-rich E. gracilis with FACS. The breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY505/515, and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). We anticipate that a further understanding of lipid metabolism combined with a further optimization of the breeding method will enable rapid, cost-effective, energy-efficient production of biofuel

Methods

Results

Conclusion