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.

Cyanobacteria, which perform oxygenic photosynthesis, have drawn attention as hosts for the direct production of biofuels and commodity chemicals from CO and HO using light energy. Although cyanobacteria capable of producing diverse chemicals have been generated by metabolic engineering, anaerobic non-photosynthetic culture conditions are often necessary for their production. In this study, we conducted cell type-specific metabolic engineering of the filamentous cyanobacterium Anabaena sp. PCC 7120, which forms a terminally differentiated cell called a heterocyst with a semi-regular spacing of 10-15 cells. Because heterocysts are specialized cells for nitrogen fixation, the intracellular oxygen level of heterocysts is maintained very low even when adjacent cells perform oxygenic photosynthesis. Pyruvate decarboxylase of Zymomonas mobilis and alcohol dehydrogenase of Synechocystis sp. PCC 6803 were exclusively expressed in heterocysts. Ethanol production was concomitant with nitrogen fixation in genetically engineered Anabaena sp. PCC 7120. Engineering of carbon metabolism in heterocysts improved ethanol production, and strain ET14, with an extra copy of the invB gene expressed from a heterocyst-specific promoter, produced 130.9 mg L of ethanol after 9 days. ET14 produced 1681.9 mg L of ethanol by increasing the CO supply. Ethanol production per heterocyst cell was approximately threefold higher than that per cell of unicellular cyanobacterium. This study demonstrates the potential of heterocysts for anaerobic production of biofuels and commodity chemicals under oxygenic photosynthetic conditions.

Euglena gracilis is a green photosynthetic microalga that swims using its flagellum. This species has been used as a model organism for over half a century to study its metabolism and the mechanisms of its behavior. The development of mass-cultivation technology has led to E. gracilis application as a feedstock in various products such as foods. Therefore, breeding of E. gracilis has been attempted to improve the productivity of this feedstock for potential industrial applications. For this purpose, a characteristic that preserves the microalgal energy e.g., reduces motility, should be added to the cultivars. The objective of this study was to verify our hypothesis that E. gracilis locomotion-defective mutants are suitable for industrial applications because they save the energy required for locomotion. To test this hypothesis, we screened for E. gracilis mutants from Fe-ion-irradiated cell suspensions and established a mutant strain, ${\mathrm{M}}_{3}^{-}$ZFeL, which shows defects in flagellum formation and locomotion. The mutant strain exhibits a growth rate comparable to that of the wild type when cultured under autotrophic conditions, but had a slightly slower growth under heterotrophic conditions. It also stores 1.6 times the amount of paramylon, a crystal of β-1,3-glucan, under autotrophic culture conditions, and shows a faster sedimentation compared with that of the wild type, because of the deficiency in mobility and probably the high amount of paramylon accumulation. Such characteristics make E. gracilis mutant cells suitable for cost-effective mass cultivation and harvesting.

Euglena gracilis is a single-celled organism capable of photosynthesis and heterotrophy. Euglena sp. have long been studied in the laboratory for its metabolic pathways, cell motility, and ease of culture. The safety of E. gracilis strain eu029 (EG029) for use as a food ingredient was assessed in a bacterial reverse mutagenesis assay (Ames), rec assay, in vivo micronucleus assay, acute toxicity study in mice, 13-week toxicology in rats, and a teratology study in mice and rats. EG029 was not genotoxic. The No Observed Adverse Effect Level (NOAEL) in the 13-week study was greater than 1000 mg/kg/day, the highest dose tested. Teratogenicity studies did not find any defects in fetal development or effects to maternal health in rats at 1000 mg/kg/day, the highest dose tested.