Isolation and characterization of a motility-defective mutant of Euglena gracilis

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, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\mathrm{M}}_{3}^{-}$\end{document}M3−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.

157 50 μmol photons m -2 s -1 of fluorescent light, and shaded with aluminum foil, except at the 158 closing end. After 2 weeks of static horizontal incubation in the tubes, the cells proliferated more 159 than 1,000 times. Most cells were gathered at the unshaded end to seek light. We retrieved about 160 1,000 of the cells that showed no movement from the initial inoculation position and cultured 161 them for an additional two weeks. 162 The proliferated cells were randomly isolated using fluorescence-activated cell sorting 163 (MoFlo XDP; Beckman Coulter, Brea, CA, USA) to establish clonal lines in individual wells of 164 96-well plates (Tissue Culture Test Plate; TPP, Trasadingen, CHE) filled with 200 μL of KH 165 medium. Populations without cell motility were selected by microscopic observations at 26 °C 166 and identified as the motility-defective strains. The above-mentioned screening was performed 167 for two independently mutagenized populations. During the screening process, cells proliferated 168 more than 10 6 times; therefore, each population included many genetically identical cells. Based 169 on this possibility, one mutant strain was established from each population, i.e., two strains were 170 established from the two independent populations. 171 172 Morphological characterization 173 The cells were observed under an upright light microscope (DM2500B; Leica, Wetzlar, DEU) 174 equipped with a differential interference contrast module. Scanning electron microscopy (SEM) 175 was conducted using a field emission scanning electron microscope (SU8200; Hitachi, Tokyo, 176 JPN) after following a general sample preparation. Briefly, 1 mL of culture in KH medium was 177 centrifuged at 2,000 × g for 1 min to collect the cells. The precipitated cells were pre-fixed by 178 adding a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.1 M sodium 179 cacodylate, and fixed again using a solution containing 1% osmium tetroxide and 0.1 M sodium 180 cacodylate. The fixed samples were completely dried using a critical point dryer, sputter-coated 181 with osmium, and then subjected to SEM photographing.

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For quantifying the proportion of cells with flagella, the cells were cultured in CM or 183 KH medium and used in their logarithmic growth phase for analysis. For the CM medium, the 184 cells were cultured in 50 mL of medium using a 100 mL volume test tube aerated with 50 mL 185 min -1 of air containing 5% CO 2 . For the KH medium, each strain was cultured in 50 mL of 186 medium using a 100 mL volume conical flask with rotary shaking at 100 rpm. Each culture was 187 conducted at 26 °C with 100 µmol m -2 s -1 of constant illumination and sub-cultured every week 188 to maintain a stable proliferation. The cells were then fixed with glutaraldehyde (0.025%) and 189 observed for the presence of flagella under an inverted microscope (CKX41; Olympus, Tokyo, 190 JPN) equipped with a phase contrast module. To exclude subjective judgment, short and intact 191 flagella were not differentiated and were counted as cells with flagella.

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193 Growth tests 194 The algae growth rate was evaluated in 100 mL test tubes containing 50 mL of medium. The 195 cells were inoculated with an initial optical density (OD) of 0.1 and precultured for three days in 196 100 mL volume conical flasks containing KH medium with continuous shaking (100 rpm at 26 220 221 Sedimentation analysis 222 Cellular sedimentation rates in KH and CM media at 26 °C were evaluated in 1.5 mL 223 microtubes. The culture was prepared as indicated above for the morphology analysis. In brief, 224 when CM medium was used, cells were cultured in 100 mL volume test tubes with aeration, 225 whereas with KH medium, they were cultured in a 100 mL volume conical flask with rotary 226 shaking at 100 rpm. Each culture was conducted with constant light illumination (100 µmol m -2 227 s -1 ). Culture ODs average at λ 680 nm were 23.4 and 22.0 by KH culture, and 4.1 and 4.4 by 228 CM culture of wild-type and M -3 ZFeL strains, respectively. Images of the cell suspension in 229 each microtubule were taken at 1-5 min intervals with a general compact digital camera. The 230 transparent supernatant area was detected and quantified from the images using the Image J 231 software, with the threshold values set as 0 to 50 after converting to greyscale. Identical 232 rectangular regions were selected inside the images of microtube images, and the transparent 233 area inside each region was quantified.

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To evaluate the sedimentation speed at 20 cm depth, the E. gracilis wild type and the M -235 3 ZFeL mutant strains were cultured in cuboid acryl beakers (10 × 10 × 30 cm) using 2 L of CM 236 medium at 29 °C and 1,300 µmol m -2 s -1 of overhead illumination (light-dark ratio of 12:12 h). 237 Beakers were filled with approximately the same concentrations (g L -1 ) of cells, 0.37 and 0.32 g 238 L -1 for the wild-type and M -3ZFeL strains, respectively. After stirring the culture, sedimentation 239 was periodically observed for 3.5 h, and supernatant was carefully removed to obtain a 240 concentrated culture. Sediment concentration was then quantified by weighing the dried cells 241 after filtration with a glass fiber filter (GA-55; ADVANTEC, Tokyo, JPN). The paramylon was subsequently separated from the residual components using 249 centrifugation, boiled for 30 min in 10 mL of 1% sodium dodecyl sulfate aqueous solution, and 250 washed twice with 10 mL of water. The extracted paramylon was then quantified using the 251 phenol-sulfuric acid method (Montgomery 1957), which can quantify total carbohydrates. 252 Similarly, neutral lipids were extracted from 100 mg of dried cells using n-hexane as a solvent; 253 10 mL of n-hexane was added to the dried cells in 50 mL glass centrifuge tubes. The suspension 254 was then homogenized for 90 s using a sonicator (UD-201; TOMY, Tokyo, JPN) with dial 255 setting at 4, and then filtered with a piece of glass fiber filter paper (GF/C; Whatman, Little 256 Chalfont, Buckinghamshire, UK), followed by an additional step of residue extraction. After 257 evaporating the collected organic solvent dissolving lipids, the weight of the residue left in the 258 flask was quantified as that of the extracted total neutral lipid.

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260 Statistical analyses 261 The results with error bars, except that of motility quantification, are represented as mean ± 262 standard error from three independent experiments. The results of motility quantification are 263 represented as mean ± standard deviation (SD) for the whole measured time points. Statistical 264 significance was analyzed using Student's t-test. For multiple comparisons, Bonferroni's 265 corrections were applied. p < 0.05 was considered significant.  Manuscript to be reviewed 276 The E. gracilis M -3 ZFeL strain showed non-motile phenotype with few defects in 277 proliferation. This phenotype was identified by the formation of colonies from single-cell 278 inoculation in liquid culture. The M -3 ZFeL strain showed colony formation in liquid KH culture 279 medium after a week, whereas wild-type cells were dispersed in the same culture medium (Fig.  280 1A and B). As judged by both light and electron microscopy, the M -3 ZFeL cells were more 281 rounded than were the wild-type cells. In addition, the M -3 ZFeL flagella were shorter than those 282 of the wild type ( Fig. 1C-F). Moreover, a significantly higher proportion of M -3 ZFeL cells 283 lacked flagella both in the CM (p = 5.6 × 10 -5 , t (4) = 15.1) and KH (p = 0.04, t (4) = 2.29) 284 media ( Fig. 1G and H).

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The autotrophic and heterotrophic growth rates of the wild-type and M -3 ZFeL strains were 287 evaluated using KH and CM culture media (Fig. 2). The mutant strain showed slightly slower 288 and faster growth than did the wild type in the KH and CM culture, respectively. The M -3 ZFeL 289 culture ODs at the third (p = 0.020, t (4) = -5.81) and fourth (p = 1.5 × 10 -4 , t (4) = -20.48) days 290 of KH culture, which included high amounts of glucose, were significantly lower than those of 291 the wild type ( Fig. 2A). On the other hand, the ODs on the second (p = 0.020, t (4) = -6.13), 292 fourth (p = 0.042, t (4) = -4.98), and eleventh (p = 0.036, t (4) = -5.18) days of CM culture were 293 significantly higher than those of the wild type (Fig. 2B).

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Our quantification of motility showed that the M -3 ZFeL TM values were less than one-296 thousandth that of the wild type. Following previous studies, cells cultured in a liquid KH 297 medium were adequately stirred and subjected to TM measurements. Fig. 3A shows the cell 298 distribution and swimming traces over approximately 8 s. The absence of swimming traces for 299 the mutant strain revealed that cells could not move using their flagella. The total TM value ± SD 300 for the wild-type strain was 11,161 ± 269 for 368 cells, whereas that for M -3 ZFeL was only 9 ± 301 6 for 467 cells, as shown in Fig. 3B. The TM value for the mutant strain cannot be distinguished 302 from the measurement noise, indicating that the M -3 ZFeL strain was almost completely non-303 motile. Although some of the M -3 ZFeL cells possessed a short flagellum (Fig. 1C-H), our results 304 indicated that the short flagellum did not function well. 305 306 Potential of M -3 ZFeL for industrial application 307 M -3 ZFeL demonstrated faster sedimentation than did the wild type in 1.5 mL microtubes. As 308 shown in Fig. 4, the M -3 ZFeL cell sedimentation rate was higher than that of the wild-type cells 309 under both heterotrophic (Fig. 4A) and autotrophic (Fig. 4B) growth conditions using KH and 310 CM media, respectively. The values at the final time point (40 min) were significantly different 311 under both heterotrophic (p = 0.010, t (4) = -3.69) and autotrophic (p = 0.011, t (4) = -3.60) 312 conditions (Fig. 4A and B).

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By analyzing sedimentation in a larger-scale culture, M -3 ZFeL was found to be 315 advantageous for industrial harvesting. To assess the effect of fast sedimentation on the harvest 316 of cells cultured using photosynthesis, wild-type and M -3 ZFeL strains were cultured using CM 317 medium and their sedimentation rates were measured in 20 cm-deep beakers. M -3 ZFeL cells 318 showed a significantly faster sedimentation rate than did the wild-type cells (Fig. 5). The M -319 3 ZFeL cell density at the bottom of the beaker (34.1 g L -1 ), was more than 10 times that of the 320 wild type (3.0 g L -1 ).

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The paramylon storage in M -3 ZFeL cells was equal to or greater than that in the wild-type 323 cells, whereas lipid accumulation in M -3 ZFeL cells was less than (autotrophic) or equal to 324 (heterotrophic) that that in the wild-type cells. As shown in Fig. 6A and B, M -3 ZFeL cells 325 produced a higher amount of paramylon (1.6 times; p = 0.033, t (4) = -2.51) than did the wild-326 type cells under autotrophic conditions, whereas there was no significant difference under 327 heterotrophic conditions. In contrast, the M -3 ZFeL strain showed a lower lipid content than did 328 the wild type, especially under autotrophic conditions (p = 0.034, t (4) = -2.47). (Fig. 6C and D).  Lebert et al. 1999). Recent research has shown that wild-type E. 335 gracilis uses mechano-sensing proteins and flagellar beating to stay at a preferential water depth 336 (Häder and Hemmersbach 2017), rather than using buoyancy control. Since M -3 ZFeL cells were 337 incapable of flagellar beating, they could not use negative gravitaxis to prevent sinking. In 338 addition, the highly accumulated paramylon, which had a particle density of approximately 1.5 in 339 M -3 ZFeL cells, may also contribute to the increase in their specific gravity and sinking under 340 autotrophic conditions. Our sedimentation tests showed that the faster sedimentation of the M -341 3ZFeL cells was achieved not only in small microtubes but also inside a deeper reservoir, thus 342 being advantageous for industrial harvesting. Typically, cells in heterotrophic cultures sediment 343 faster than those in autotrophic cultures, probably due to the higher carbohydrate accumulation. 344 However, our results in microtubes showed that both strains sedimented faster in the autotrophic 345 culture than in the heterotrophic culture. This seems to be due to the differences in cell density of 346 the cultures; stacking highly concentrated cells at the bottom of the KH culture inhibited further 347 sedimentation of the cells.

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The M -3 ZFeL strain showed slightly slower growth in the heterotrophic KH culture than 359 did the wild type, suggesting that the function of glucose metabolism was slightly disturbed in 360 the mutant. Meanwhile, the M -3 ZFeL strain showed faster growth in the autotrophic CM culture, 361 which did not include carbon sources, suggesting that the mutant strain had an intact 362 photosynthesis system and consumed the photosynthesized resources slower than the wild type. 363 Moreover, the M -3 ZFeL had no defect in paramylon production, thus indicating that M -3 ZFeL 364 may improve industrial paramylon production. Although a limited defect was observed in the 365 accumulation of lipids, M -3 ZFeL would also be applicable to lipid production. The higher 366 accumulation of paramylon in M -3 ZFeL may be partially due to the energy conservation by not 367 swimming. However, since the proliferation speed of M -3 ZFeL was not drastically faster than the 368 wild type, even in the autotrophic culture, the amount of energy conservation in M -3 ZFeL does 369 not seem significant.

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The M -3 ZFeL characterization results suggest that the mutant is defective in the 372 components related to flagellum motion and/or formation that are not critical for survival and 373 proliferation, at least under controlled laboratory conditions. The production of mutant strains by 374 mutagenesis using high-LET irradiations, such as Ar-ion or Fe-ion irradiation, is advantageous 375 because it causes a small number of large deletions in the genome with few side mutations 376 (Hirano et al. 2015;Kazama et al. 2017). Although we could not identify the genetic cause of the 377 phenotype in M -3 ZFeL, the process of breeding these strains ensures minimal side mutations. 378 This will enable the immediate industrial use of the strain by reducing the possibility of showing 379 unexpected and undesirable traits, which may not be observed in the laboratory but appear under 380 harsh and unstable outdoor-culture conditions.

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The M -3 ZFeL mutant of E. gracilis produced in this study is highly promising for the 383 industrial production of food ingredients and chemical substances because it evidenced fast 384 sedimentation, high paramylon storage, and a growth speed comparable to that of the wild type, 385 at least under autotrophic conditions. In particular, the fast sedimentation of the mutant strain 386 will save time and energy during the harvesting processes, which will contribute to the use of E. 387 gracilis as a feedstock for biofuel by improving the balance between the energy invested to 388 produce it and the energy output. In practical cultivation to produce paramylon and lipid under 389 autotrophic conditions, processes to accumulate respective ingredients are added. In particular, 390 cultured cells are subjected to nitrogen-restricted conditions to accumulate paramylon; the 391 culture is then condensed and hypoxically conditioned to ferment the paramylon to wax ester 392 (Suzuki 2017). Our results showed that M -3 ZFeL can competently accumulate larger amounts of 393 paramylon under heterotrophic culture conditions than under autotrophic conditions; therefore, 394 we suggest that they also show sufficient paramylon accumulation by nitrogen restriction (Briand