Enhancement of photosynthetic capacity in Euglena gracilis by expression of cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase leads to increases in biomass and wax ester production

Abstract

Background: Microalgae have recently been attracting attention as a potential platform for the production of biofuels. Euglena gracilis, a unicellular phytoflagellate, has been proposed as an attractive feedstock to produce biodiesel because it can produce large amounts of wax esters, consisting of medium-chain fatty acids and alcohols with 14:0 carbon chains. E. gracilis cells highly accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. When grown aerobically and then transferred into anaerobic conditions, E. gracilis cells degrade paramylon to actively synthesize and accumulate wax esters. Thus, the enhanced accumulation of paramylon through the genetic engineering of photosynthesis should increase the capacity for wax ester production.

Results: We herein generated transgenic Euglena (EpFS) cells expressing the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), which is involved in the Calvin cycle, to enhance its photosynthetic activity. FBP/SBPase was successfully expressed within Euglena chloroplasts. The cell volume of the EpFS4 cell line was significantly larger than that of wild-type cells under normal growth conditions. The photosynthetic activity of EpFS4 cells was significantly higher than that of wild type under high light and high CO2, resulting in enhanced biomass production, and the accumulation of paramylon was increased in transgenic cell lines than in wild-type cells. Furthermore, when EpFS cell lines grown under high light and high CO2 were placed on anaerobiosis, the productivity of wax esters was approximately 13- to 100-fold higher in EpFS cell lines than in wild-type cells.

Conclusion: Our results obtained here indicate that the efficiency of biomass production in E. gracilis can be improved by genetically modulating photosynthetic capacity, resulting in the enhanced production of wax esters. This is the first step toward the utilization of E. gracilis as a sustainable source for biofuel production under photoautotrophic cultivation.

Keywords: Biofuel; Biomass; Euglena gracilis; Fructose-1,6-/sedoheptulose-1,7-bisphosphatase; Paramylon; Photosynthesis; Wax ester.

Fig. 1

Isolation of transgenic E. gracilis cells having the FBP/SBPase gene. The construct structure of the gene using transformation of Euglena cells (a). Genomic PCR amplification of endogenous rbcS (637 bp) and FBP/SBPase (937 bp) genes from the wild-type and transgenic (EpFS) cell lines of E. gracilis (b). Western blot analysis of the crude extracts from wild-type and EpFS cell lines (c) and the intact chloroplastic fractions from wild-type and EpFS4 cells (d) using an antibody raised against the FBP/SBPase protein. Total FBPase activity in the stationary phase wild-type and EpFS cell lines grown under normal conditions (e). The photographs of wild-type and EpFS4 cells grown under normal conditions (f). Values are indicated as the mean ± standard deviation for three individual experiments. An asterisk indicates significant differences from the wild-type E. gracilis cells (*P < 0.05)

Fig. 2

Growth curves and growth rates of wild-type and EpFS4 cells grown under different cultivation conditions. Growth curves and growth rates of wild-type and EpFS4 cells grown under normal (100 μmol photons m⁻² s⁻¹ at 0.04 % CO₂) (a) and high light and high CO₂ (350 μmol photons m⁻² s⁻¹ at 0.3 % CO₂) (b), respectively. Values are indicated as the mean ± standard deviation for three to seven individual experiments. Asterisks indicate significant differences from the wild-type E. gracilis cells (**P < 0.01)

Fig. 3

Photosynthetic activities and chlorophyll contents of wild-type and EpFS cell lines. The photosynthetic activities (a) and chlorophyll contents (b) of wild-type and EpFS cell lines grown under high light and high CO₂. Values are indicated as the mean ± standard deviation for three to seven individual experiments. Different letters indicate significant differences (P < 0.05)