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The Korean Society of Phycology (한국조류학회(藻類))
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Insertional mutations exhibiting high cell-culture density HCD phenotypes are enriched through continuous subcultures in Chlamydomonas reinhardtii

  • Thung, Leena (Institute of Chemical Engineering and Sciences, A-STAR) ;
  • He, Jing (Ocean Research Centre of Zhoushan, Zhejiang University) ;
  • Zhu, Qingling (Ocean College, Zhejiang University) ;
  • Xu, Zhenyu (Ocean College, Zhejiang University) ;
  • Liu, Jianhua (Ocean Research Centre of Zhoushan, Zhejiang University) ;
  • Chow, Yvonne (Institute of Chemical Engineering and Sciences, A-STAR)
  • Received : 2017.08.30
  • Accepted : 2018.02.28
  • Published : 2018.03.15
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Abstract

Low efficiency in microalgal biomass production was largely attributed to the low density of algal cell cultures. Though mutations that reduced the level of chlorophyll or pigment content increased efficiency of photon usage and thus the cell-culture density under high-illumination growth conditions (e.g., >$500{\mu}mol\;photon\;m^{-2}\;s^{-1}$), it was unclear whether algae could increase cell-culture density under low-illumination conditions (e.g., ${\sim}50{\mu}mol\;photon\;m^{-2}\;s^{-1}$). To address this question, we performed forward genetic screening in Chlamydomonas reinhardtii. A pool of >1,000 insertional mutants was constructed and subjected to continuous subcultures in shaking flasks under low-illumination conditions. Complexity of restriction fragment length polymorphism (RFLP) pattern in cultures indicated the degree of heterogeneity of mutant populations. We showed that the levels of RFLP complexity decreased when cycles of subculture increased, suggesting that cultures were gradually populated by high cell-culture density (HCD) strains. Analysis of the 3 isolated HCD mutants after 30 cycles of subcultures confirmed that their maximal biomass production was 50-100% higher than that of wild type under low-illumination. Furthermore, levels of chlorophyll content in HCD mutant strains were similar to that of wild type. Inverse polymerase chain reaction analysis identified the locus of insertion in two of three HCD strains. Molecular and transcriptomic analyses suggested that two HCD mutants were a result of the gain-of-function phenotype, both linking to the abnormality of mitochondrial functions. Taken together, our results demonstrate that HCD strains can be obtained through continuous subcultures under low illumination conditions.

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References

  1. Bolger, A. M., Lohse, M. & Usadel, B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120. https://doi.org/10.1093/bioinformatics/btu170
  2. Cheng, X., Liu, G., Ke, W., Zhao, L., Lv, B., Ma, X., Xu, N., Xia, X., Deng, X., Zheng, C. & Huang, K. 2017. Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas. Plant Methods 13:36. https://doi.org/10.1186/s13007-017-0183-5
  3. Dent, R. M., Haglund, C. M., Chin, B. L., Kobayashi, M. C. & Niyogi, K. K. 2005. Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol. 137:545-556. https://doi.org/10.1104/pp.104.055244
  4. Engler-Blum, G., Meier, M., Frank, J. & Muller, G. A. 1993. Reduction of background problems in nonradioactive northern and southern blot analyses enables higher sensitivity than 32P-based hybridizations. Anal. Biochem. 210:235-244. https://doi.org/10.1006/abio.1993.1189
  5. Erjavec, N., Bayot, A., Gareil, M., Camougrand, N., Nystrom, T., Friguet, B. & Bulteau, A. L. 2013. Deletion of the mitochondrial Pim1/Lon protease in yeast results in accelerated aging and impairment of the proteasome. Free Radic. Biol. Med. 56:9-16. https://doi.org/10.1016/j.freeradbiomed.2012.11.019
  6. Folda, A., Citta, A., Scalcon, V., Calì, T., Zonta, F., Scutari, G., Bindoli, A. & Rigobello, M. P. 2016. Mitochondrial thioredoxin system as a modulator of cyclophilin D redox state. Sci. Rep. 6:23071. https://doi.org/10.1038/srep23071
  7. Gonzalez-Ballester, D., de Montaigu, A., Higuera, J. J., Galvan, A. & Fernandez, E. 2005. Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas. Plant Physiol. 137:522-533. https://doi.org/10.1104/pp.104.050914
  8. Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B. W., Nusbaum, C., Lindblad-Toh, K., Friedman, N. & Regev, A. 2011. Fulllength transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29:644-652. https://doi.org/10.1038/nbt.1883
  9. Harris, E. H. 1989. The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, San Diego, CA, 780 pp.
  10. Harris, E. H. 2001. Chlamydomonas as a model organism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:363-406. https://doi.org/10.1146/annurev.arplant.52.1.363
  11. Kang, S. G., Ortega, J., Singh, S. K., Wang, N., Huang, N. N., Steven, A. C. & Maurizi, M. R. 2002. Functional proteolytic complexes of the human mitochondrial ATPdependent protease, hClpXP. J. Biol. Chem. 277:21095-21102. https://doi.org/10.1074/jbc.M201642200
  12. Leon-Banares, R., Gonzalez-Ballester, D., Galvan, A. & Fernandez, E. 2004. Transgenic microalgae as green cellfactories. Trends Biotechnol. 22:45-52. https://doi.org/10.1016/j.tibtech.2003.11.003
  13. Lumbreras, V., Stevens, D. R. & Purton, S. 1998. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J. 14:441-447. https://doi.org/10.1046/j.1365-313X.1998.00145.x
  14. Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Maréchal-Drouard, L., Marshall, W. F., Qu, L. -H., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., Shapiro, H., Lucas, S. M., Grimwood, J., Schmutz, J., Cardol, P., Cerutti, H., Chanfreau, G., Chen, C. -L., Cognat, V., Croft, M. T., Dent, R., Dutcher, S., Fernandez, E., Ferris, P., Fukuzawa, H., Gonzalez-Ballester, D., Gonzalez-Halphen, D., Hallmann, A., Hanikenne, M., Hippler, M., Inwood, W., Jabbari, K., Kalanon, M., Kuras, R., Lefebvre, P. A., Lemaire, S. D., Lobanov, A. V., Lohr, M., Manuell, A., Meier, I., Mets, L., Mittag, M., Mittelmeier, T., Moroney, J. V., Moseley, J., Napoli, C., Nedelcu, A. M., Niyogi, K., Novoselov, S. V., Paulsen, I. T., Pazour, G., Purton, S., Ral, J. -P., Riano-Pachón, D. M., Riekhof, W., Rymarquis, L., Schroda, M., Stern, D., Umen, J., Willows, R., Wilson, N., Zimmer, S. L., Allmer, J., Balk, J., Bisova, K., Chen, C. -J., Elias, M., Gendler, K., Hauser, C., Lamb, M. R., Ledford, H., Long, J. C., Minagawa, J., Page, M. D., Pan, J., Pootakham, W., Roje, S., Rose, A., Stahlberg, E., Terauchi, A. M., Yang, P., Ball, S., Bowler, C., Dieckmann, C. L., Gladyshev, V. N., Green, P., Jorgensen, R., Mayfield, S., Mueller-Roeber, B., Rajamani, S., Sayre, R. T., Brokstein, P., Dubchak, I., Goodstein, D., Hornick, L., Huang, Y. W., Jhaveri, J., Luo, Y., Martinez, D., Ngau, W. C. A., Otillar, B., Poliakov, A., Porter, A., Szajkowski, L., Werner, G., Zhou, K., Grigoriev, I. V., Rokhsar, D. S. & Grossman, A. R. 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245-250. https://doi.org/10.1126/science.1143609
  15. Moellering, E. R. & Benning, C. 2010. RNA interference silencing of a major lipid droplet protein affects lipiddroplet size in Chlamydomonas reinhardtii. Eukaryot. Cell 9:97-106. https://doi.org/10.1128/EC.00203-09
  16. Ochman, H., Gerber, A. S. & Hartl, D. L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621-623.
  17. Polle, J. E. W., Kanakagiri, S. D. & Melis, A. 2003. tla1, a DNA insertional transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size. Planta 217:49-59.
  18. Pollock, S. V., Colombo, S. L., Prout, D. L. Jr., Godfrey, A. C. & Moroney, J. V. 2003. Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a $low-CO_2$ atmosphere. Plant Physiol. 133:1854-1861. https://doi.org/10.1104/pp.103.032078
  19. Pollock, S. V., Pootakham, W., Shibagaki, N., Moseley, J. L. & Grossman, A. R. 2005. Insights into the acclimation of Chlamydomonas reinhardtii to sulfur deprivation. Photosynth. Res. 86:475-489. https://doi.org/10.1007/s11120-005-4048-9
  20. Sartory, D. P. & Grobbelaar, J. U. 1984. Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis. Hydrobiologia 114:177-187. https://doi.org/10.1007/BF00031869
  21. Shimogawara, K., Fujiwara, S., Grossman, A. & Usuda, H. 1998. High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148:1821-1828.
  22. Sizova, I., Fuhrmann, M. & Hegemann, P. 2001. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Genetics 277:221-229.
  23. Sueoka, N. 1960. Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc. Natl. Acad. Sci. U. S. A. 46:83-91. https://doi.org/10.1073/pnas.46.1.83
  24. Tam, L. W. & Lefebvre, P. A. 1993. Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics 135:375-384.
  25. Trapnell, C., Pachter, L. & Salzberg, S. L. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105-1111. https://doi.org/10.1093/bioinformatics/btp120
  26. Wang, Z. T., Ullrich, N., Joo, S., Waffenschmidt, S. & Goodenough, U. 2009. Algal lipid bodies: stress induction,purification, and biochemical characterization in wildtype and starchless Chlamydomonas reinhardtii. Eukaryot.Cell 8:1856-1868. https://doi.org/10.1128/EC.00272-09
  27. Work, V. H., Radakovits, R., Jinkerson, R. E., Meuser, J. E., Elliott, L. G., Vinyard, D. J., Laurens, L. M. L., Dismukes, G. C. & Posewitz, M. C. 2010. Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains. Eukaryot. Cell 9:1251-1261. https://doi.org/10.1128/EC.00075-10
  28. Yoshioka, S., Taniguchi, F., Miura, K., Inoue, T., Yamano, T. & Fukuzawa, H. 2004. The novel Myb transcription factor LCR1 regulates the $CO_2-responsive$ gene Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Cell 16:1466-1477. https://doi.org/10.1105/tpc.021162