Abstract
In the cultivation of microalgae for the production of useful compounds, cell disruption to extract the products of interest is a bottleneck process. To establish a cost-effective method to recover these cellular compounds, we developed a method to induce cell lysis via phosphate deficiency in the cyanobacterium Synechocystis sp. PCC 6803. In this system, the promoter from the phoA gene for alkaline phosphatase expressed bacteriophage genes encoding the lytic enzymes holin and endolysin, thus the cell lysis is induced under phosphate-deficient condition. We observed that 90% of the cells, introduced this bacteriophage genes, were lysed after 24 h of incubation under phosphate-deficient conditions. We also developed a method to induce cell lysis in highly concentrated cells for the efficient recovery of valuable cellular products and observed over 90% cell lysis after 16 h of incubation under these conditions. This inducible lysis system may contribute to decreased cell disruption costs in the algal biotechnology industry.
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References
Aiba H, Nagaya M, Mizuno T (1993) Sensor and regulator proteins from the cyanobacter ium Synechococcus species PCC7942 that belong to the bacterial signai-transduction protein families: implication in the adaptive response to phosphate limitation. Mol Microbiol 8:81–91
Barry A, Wolfe A, English C, Ruddick C, Lambert D (2016) National algal biofuels technology review. U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office
Berry J, Summer EJ, Struck DK, Young R (2008) The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes. Mol Microbiol 70:341–351
Berry J, Rajaure M, Pang T, Young R (2012) The spanin complex is essential for lambda lysis. J Bacteriol 194:5667–5674
Dassey AJ, Hall SG, Theegala CS (2014) An analysis of energy consumption for algal biodiesel production: comparing the literature with current estimates. Algal Res 4:89–95
Dollard MA, Billard P (2003) Whole-cell bacterial sensors for the monitoring of phosphate bioavailability. J Microbiol Methods 55:221–229
Farooq W, Suh WI, Park MS, Yang J (2015) Water use and its recycling in microalgae cultivation for biofuel application. Bioresour Technol 184:73–81
Gao Y, Feng X, Xian M, Wang Q, Zhao G (2013) Inducible cell lysis systems in microbial production of bio-based chemicals. Appl Microbiol Biotechnol 97:7121–7129
Gray J, Wardzala E, Yang M, Reinbothe S, Haller S, Pauli F (2004) A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers. Plant Mol Biol 54:39–54
Grigorieva G, Shestakov S (1982) Transformation in the cyanobacterium Synechocystis sp. 6803. FEMS Microbiol Lett 13:367–370
Grima EM, Belarbi EH, Fernández FGA, Medina AR, Chisti Y (2003) Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol Adv 20:491–515
Günerken E, Hondt ED, Eppink MHM, Garcia-Gonzalez L, Elst K, Wijffels RH (2015) Cell disruption for microalgae biorefineries. Biotechnol Adv 33:243–260
Hirani TA, Suzuki I, Murata N, Hayashi H, Eaton-Rye JJ (2001) Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp . PCC 6803. Plant Mol Biol 45:133–144
Hirose Y, Shimada T, Narikawa R, Katayama M, Ikeuchi M (2008) Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc Natl Acad Sci U S A 105:9528–9533
Holman WIM (1943) A new technique for the determination of phosphorus by the molybdenum blue method. Biochem J 37:256–259
Inaba M, Suzuki I, Szalontai B, Kanesaki Y, Los DA, Hayashi H, Murata N (2003) Gene-engineered rigidification of membrane lipids enhances the cold inducibility of gene expression in Synechocystis. J Biol Chem 278:12191–12198
Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3:109–136
Larena I, De Cal A, Melgarejo P (2004) Solid substrate production of Epicoccum nigrum conidia for biological control of brown rot on stone fruits. Int J Food Microbiol 94:161–167
Ling MM, Robinson BH (1997) Approaches to DNA mutagenesis: an overview. Anal Biochem 254:157–178
Liu X, Curtiss R III (2009) Nickel-inducible lysis system in Synechocystis sp. PCC 6803. Proc Natl Acad Sci U S A 106:21550–21554
Liu X, Fallon S, Sheng J, Curtiss R III (2011) CO2-limitation-inducible green recovery of fatty acids from cyanobacterial biomass. Proc Natl Acad Sci U S A 108:6905–6908
Loessner MJ (2005) Bacteriophage endolysins — current state of research and applications. Curr Opin Microbiol 8:480–487
López-maury L, García-domínguez M, Florencio FJ, Reyes JC (2002) A two-component signal transduction system involved in nickel sensing in the cyanobacterium Synechocystis sp . PCC 6803. Mol Microbiol 43:247–256
Lübke C, Boidol W, Petri T (1995) Analysis and optimization of recombinant protein production in Escherichia coli using the inducible phoA promoter of the E. coli alkaline phosphatase. Enzym Microb Technol 17:923–928
Mironov KS, Sidorov RA, Trofimova MS, Bedbenov VS, Tsydendambaev VD, Allakhverdiev SI, Los DA (2012) Light-dependent cold-induced fatty acid unsaturation, changes in membrane fluidity, and alterations in gene expression in Synechocystis. Biochim Biophys Acta 1817:1352–1359
Miyake K, Abe K, Ferri S, Nakajima M, Nakamura M, Yoshida W, Kojima K, Ikebukuro K, Sode K (2014) A green-light inducible lytic system for cyanobacterial cells. Biotechnol Biofuels 7:56
Mizuno T, Kaneko T, Tabata S (1996) Compilation of all genes encoding bacterial two-component signal transducers in the genome of the cyanobacterium, Synechocystis sp. strain PCC 6803. DNA Res 3:407–414
Okamoto S, Ikeuchi M, Ohmori M (1999) Experimental analysis of recently transposed insertion sequences in the cyanobacterium Synechocystis sp. PCC 6803. DNA Res 6:265–273
Paithoonrangsarid K, Shoumskaya MA, Kanesaki Y, Satoh S, Tabata S, Los DA, Zinchenko VV, Hayashi H, Tanticharoen M, Suzuki I, Murata N (2004) Five histidine kinases perceive osmotic stress and regulate distinct sets of genes in Synechocystis. J Biol Chem 279:53078–53086
Passell H, Dhaliwal H, Reno M, Wu B, Ben Amotz A, Ivry E, Gay M, Czartoski T, Laurin L, Ayer N (2013) Algae biodiesel life cycle assessment using current commercial data. J Environ Manag 129:103–111
Schindler DW (1977) Evolution of phosphorus limitation in lakes. Science 195:260–262
Singh SK, Tiwari DN (2000) Control of alkaline phosphatase activity in Anabaena oryzae Fritsch. J Plant Physiol 157:467–472
Slabas AR, Suzuki I, Murata N, Simon WJ, Hall JJ (2006) Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase 34 gene. Proteomics 6:845–864
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35:171–205
Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69:183–215
Suzuki S, Ferjani A, Suzuki I, Murata N (2004) The SphS-SphR two component system is the exclusive sensor for the induction of gene expression in response to phosphate limitation in Synechocystis. J Biol Chem 279:13234–13240
Tandeau de Marsac N, Houmard J (1988) Complementary chromatic adaptation: physiological conditions and action spectra. Methods Enzymol 167:318–328
Vavilin D, Brune DC, Vermaas W (2005) 15N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems. Biochim Biophys Acta 1708:91–101
Wada H, Murata N (1989) Synechocystis PCC6803 mutants defective in desaturation of fatty acids. Plant Cell Physiol 30:971–978
Wang I, Smith DL, Young R (2000) Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54:799–825
Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol 167:766–778
Yeh K-C, Wu S-H, Murphy JT, Lagarias JC (1997) A cyanobacterial phytochrome two-component light sensory system. Science 277:1505–1508
Young R (2002) Bacteriophage holins: deadly diversity. J Mol Microbiol Biotechnol 4:21–36
Yuan J, Kendall A, Zhang Y (2015) Mass balance and life cycle assessment of biodiesel from microalgae incorporated with nutrient recycling options and technology uncertainties. GCB Bioenergy 7:1245–1259
Acknowledgments
We are grateful to the National Institute of Technology and Evaluation (NITE) for providing purified DNA of the S. enterica phage P22.
Funding
This work was financially supported by JSPS KAKENHI Grant no. JP24119501.
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Asada, R., Shiraiwa, Y. & Suzuki, I. A novel cell lysis system induced by phosphate deficiency in the cyanobacterium Synechocystis sp. PCC 6803. J Appl Phycol 31, 1069–1076 (2019). https://doi.org/10.1007/s10811-018-1652-6
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Keywords
- Cell disruption
- Endolysin
- Extraction
- Holin
- Phosphate sensor