Abstract
Many freshwater cyanobacteria accumulate polyhydroxybutyrate (PHB) under nitrogen or phosphorus deprivation. While prior literature has shed lights on transcriptomic and metabolomic changes in the model cyanobacterium Synechocystis PCC 6803 cells, the quantitative contributions of the newly fixed carbon following nitrogen deprivation or the externally added acetate to PHB synthesis are not clear. Similarly, it is not clear how photomixotrophy affects precursor contributions. In this study, we show that (i) the pre-growth mode (photoautotrophic or photomixotrophic), while significantly impacting glycogen levels, does not have any significant effect on PHB levels, (ii) the carbon fixed following nitrogen deprivation contributes 26% of C for PHB synthesis in photoautotrophically pre-grown cells and its contribution to the PHB synthesis goes down with the addition of acetate at the resuspension phase or with photomixotrophic pre-growth, (iii) the acetate added at the start of nitrogen deprivation, doubles the intracellular PHB levels and contributes 44–48% to PHB synthesis and this value is not greatly affected by how the cells were pre-grown. Indirectly, the labeling studies also show that the intracellular C recycling is the most important source of precursors for PHB synthesis, contributing about 74–87% of the C for PHB synthesis in the absence of acetate. The addition of acetate significantly reduces its contribution. In photoautotrophic pre-growth followed by acetate addition under nitrogen starvation, the contribution of intracellular C reduces to about 34%. Thus, our study provides several novel quantitative insights on how prior nutritional status affects the precursor contributions for PHB synthesis.
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References
Anfelt J, Kaczmarzyk D, Shabestary K et al (2015) Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production. Microb Cell Fact. 14:167. https://doi.org/10.1186/s12934-015-0355-9
Ansari S, Fatma T (2016) Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and characterization. PloS ONE 11:e0158168. https://doi.org/10.1371/journal.pone.0158168
Asada Y, Miyake M, Miyake J et al (1999) Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria—the metabolism and potential for CO2 recycling. Int J Biol Macromol 25:37–42
Balaji S, Gopi K, Muthuvelan B (2013) A review on production of poly β hydroxybutyrates from cyanobacteria for the production of bio plastics. Algal Res 2:278–285. https://doi.org/10.1016/j.algal.2013.03.002
Bequette BJ, Sunny NE, El-Kadi SW, Owens SL (2006) Application of stable isotopes and mass isotopomer distribution analysis to the study of intermediary metabolism of nutrients. J Anim Sci 84(Suppl):E50–E59
Bhati R, Samantaray S, Sharma L, Mallick N (2010) Poly-beta-hydroxybutyrate accumulation in cyanobacteria under photoautotrophy. Biotechnol J 5:1181–1185. https://doi.org/10.1002/biot.201000252
Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(beta-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982
Carpine R, Du W, Olivieri G et al (2017) Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction. Algal Res 25:117–127. https://doi.org/10.1016/j.algal.2017.05.013
Damrow R, Maldener I, Zilliges Y (2016) The multiple functions of common microbial carbon polymers, glycogen and PHB, during stress responses in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803. Front Microbiol 7:966. https://doi.org/10.3389/fmicb.2016.00966
Depraetere O, Deschoenmaeker F, Badri H et al (2015) Trade-off between growth and carbohydrate accumulation in nutrient-limited Arthrospira sp. PCC 8005 studied by integrating transcriptomic and proteomic approaches. PLoS ONE 10:e0132461. https://doi.org/10.1371/journal.pone.0132461
Forchhammer K, Tandeau de Marsac N (1995) Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol 177:2033–2040
Hasunuma T, Kikuyama F, Matsuda M et al (2013) Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion. J Exp Bot 64:2943–2954. https://doi.org/10.1093/jxb/ert134
Hauf W, Schlebusch M, Huge J et al (2013) Metabolic changes in Synechocystis PCC6803 upon nitrogen-starvation: excess NADPH sustains polyhydroxybutyrate accumulation. Metabolites 3:101–118. https://doi.org/10.3390/metabo3010101
Hauf W, Watzer B, Roos N et al (2015) Photoautotrophic polyhydroxybutyrate granule formation is regulated by cyanobacterial Phasin PhaP in Synechocystis sp. strain PCC 6803. Appl Environ Microbiol 81:4411–4422. https://doi.org/10.1128/AEM.00604-15
Huang S, Chen L, Te R et al (2013) Complementary iTRAQ proteomics and RNA-seq transcriptomics reveal multiple levels of regulation in response to nitrogen starvation in Synechocystis sp. PCC 6803. Mol Biosyst 9:2565–2574. https://doi.org/10.1039/c3mb70188c
Kaewbai-Ngam A, Incharoensakdi A, Monshupanee T (2016) Increased accumulation of polyhydroxybutyrate in divergent cyanobacteria under nutrient-deprived photoautotrophy: an efficient conversion of solar energy and carbon dioxide to polyhydroxybutyrate by Calothrix scytonemicola TISTR 8095. Bioresour Technol 212:342–347. https://doi.org/10.1016/j.biortech.2016.04.035
Khetkorn W, Incharoensakdi A, Lindblad P, Jantaro S (2016) Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes. Bioresour Technol 214:761–768. https://doi.org/10.1016/j.biortech.2016.05.014
Koller M (2015) Cyanobacterial polyhydroxyalkanoate production: status Quo and Quo vadis? Curr Biotechnol 4:464–480
Koller M, Maršálek L, de Sousa Dias MM, Braunegg G (2017) Producing microbial polyhydroxyalkanoate (PHA) biopolyesters in a sustainable manner. New Biotechnol 37:24–38. https://doi.org/10.1016/j.nbt.2016.05.001
Krasikov V, Aguirre von Wobeser E, Dekker HL et al (2012) Time-series resolution of gradual nitrogen starvation and its impact on photosynthesis in the cyanobacterium Synechocystis PCC 6803. Physiol Plant 145:426–439. https://doi.org/10.1111/j.1399-3054.2012.01585.x
Lau N-S, Foong CP, Kurihara Y et al (2014) RNA-seq analysis provides insights for understanding photoautotrophic polyhydroxyalkanoate production in recombinant Synechocystis Sp. PLoS ONE 9:e86368. https://doi.org/10.1371/journal.pone.0086368
Lee SY (1996) Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 49:1–14. https://doi.org/10.1002/(SICI)1097-0290(19960105)49:1<1::AID-BIT1>3.0.CO;2-P
Lee WN, Byerley LO, Bergner EA, Edmond J (1991) Mass isotopomer analysis: theoretical and practical considerations. Biol Mass Spectrom 20:451–458. https://doi.org/10.1002/bms.1200200804
Lee WN, Byerley LO, Bassilian S et al (1995) Isotopomer study of lipogenesis in human hepatoma cells in culture: contribution of carbon and hydrogen atoms from glucose. Anal Biochem 226:100–112. https://doi.org/10.1006/abio.1995.1197
Matsuhashi A, Tahara H, Ito Y et al (2015) Slr2019, lipid A transporter homolog, is essential for acidic tolerance in Synechocystis sp. PCC6803. Photosynth Res 125:267–277. https://doi.org/10.1007/s11120-015-0129-6
Miyake M, Kataoka K, Shirai M, Asada Y (1997) Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria. J Bacteriol 179:5009–5013
Miyake M, Miyamoto C, Schnackenberg J et al (2000) Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate. Appl Biochem Biotechnol 84–86:1039–1044
Monshupanee T, Incharoensakdi A (2014) Enhanced accumulation of glycogen, lipids and polyhydroxybutyrate under optimal nutrients and light intensities in the cyanobacterium Synechocystis sp. PCC 6803. J Appl Microbiol 116:830–838. https://doi.org/10.1111/jam.12409
Monshupanee T, Nimdach P, Incharoensakdi A (2016) Two-stage (photoautotrophy and heterotrophy) cultivation enables efficient production of bioplastic poly-3-hydroxybutyrate in auto-sedimenting cyanobacterium. Sci Rep 6:37121. https://doi.org/10.1038/srep37121
Muro-Pastor MI, Reyes JC, Florencio FJ (2001) Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J Biol Chem 276:38320–38328. https://doi.org/10.1074/jbc.M105297200
Nakajima T, Kajihata S, Yoshikawa K et al (2014) Integrated metabolic flux and omics analysis of Synechocystis sp. PCC 6803 under mixotrophic and photoheterotrophic conditions. Plant Cell Physiol 55:1605–1612. https://doi.org/10.1093/pcp/pcu091
Nishioka M, Nishiuma H, Miyake M et al (2002) Metabolic flux analysis of a poly-β-hydroxybutyrate producing cyanobacterium,Synechococcus sp. MA19, grown under photoautotrophic conditions. Biotechnol Bioprocess Eng 7:295–302. https://doi.org/10.1007/BF02932839
Osanai T, Numata K, Oikawa A et al (2013) Increased bioplastic production with an RNA polymerase sigma factor SigE during nitrogen starvation in Synechocystis sp. PCC 6803. DNA Res Int J Rapid Publ Rep Genes Genomes 20:525–535. https://doi.org/10.1093/dnares/dst028
Osanai T, Oikawa A, Iijima H et al (2014a) Metabolomic analysis reveals rewiring of Synechocystis sp. PCC 6803 primary metabolism by ntcA overexpression. Environ Microbiol 16:3304–3317. https://doi.org/10.1111/1462-2920.12554
Osanai T, Oikawa A, Shirai T et al (2014b) Capillary electrophoresis-mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803. Environ Microbiol 16:512–524. https://doi.org/10.1111/1462-2920.12170
Panda B, Mallick N (2007) Enhanced poly-beta-hydroxybutyrate accumulation in a unicellular cyanobacterium, Synechocystis sp. PCC 6803. Lett Appl Microbiol 44:194–198. https://doi.org/10.1111/j.1472-765X.2006.02048.x
Panda B, Jain P, Sharma L, Mallick N (2006) Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803. Bioresour Technol 97:1296–1301. https://doi.org/10.1016/j.biortech.2005.05.013
Samantaray S, Nayak JK, Mallick N (2011) Wastewater utilization for poly-beta-hydroxybutyrate production by the cyanobacterium Aulosira fertilissima in a recirculatory aquaculture system. Appl Environ Microbiol 77:8735–8743. https://doi.org/10.1128/AEM.05275-11
Sauer J, Schreiber U, Schmid R et al (2001) Nitrogen starvation-induced chlorosis in Synechococcus PCC 7942. Low-level photosynthesis as a mechanism of long-term survival. Plant Physiol 126:233–243
Schlebusch M, Forchhammer K (2010) Requirement of the nitrogen starvation-induced protein Sll0783 for polyhydroxybutyrate accumulation in Synechocystis sp. strain PCC 6803. Appl Environ Microbiol 76:6101–6107. https://doi.org/10.1128/AEM.00484-10
Sudesh K, Taguchi K, Doi Y (2002) Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803. Int J Biol Macromol 30:97–104
Takahashi H, Uchimiya H, Hihara Y (2008) Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp. PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry. J Exp Bot 59:3009–3018. https://doi.org/10.1093/jxb/ern157
Thiel K, Vuorio E, Aro E-M, Kallio PT (2017) The effect of enhanced acetate influx on Synechocystis sp. PCC 6803 metabolism. Microb Cell Fact 16:21. https://doi.org/10.1186/s12934-017-0640-x
Tsang TK, Roberson RW, Vermaas WFJ (2013) Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction. Photosynth Res 118:37–49. https://doi.org/10.1007/s11120-013-9923-1
Tyo KE, Zhou H, Stephanopoulos GN (2006) High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and Synechocystis sp. strain PCC6803. Appl Environ Microbiol 72:3412–3417. https://doi.org/10.1128/AEM.72.5.3412-3417.2006
Tyo KEJ, Jin Y-S, Espinoza FA, Stephanopoulos G (2009) Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803. Biotechnol Prog 25:1236–1243. https://doi.org/10.1002/btpr.228
Varman AM, Xiao Y, Pakrasi HB, Tang YJ (2013) Metabolic engineering of Synechocystis sp. strain PCC 6803 for isobutanol production. Appl Environ Microbiol 79:908–914. https://doi.org/10.1128/AEM.02827-12
Wu GF, Wu QY, Shen ZY (2001) Accumulation of poly-beta-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803. Bioresour Technol 76:85–90
Xie J, Zhou J, Zhang H, Li Y (2011) [Increasing reductant NADPH content via metabolic engineering of PHB synthesis pathway in Synechocystis sp. PCC 6803]. Sheng Wu Gong Cheng Xue Bao Chin J Biotechnol 27:998–1004
Yang C, Hua Q, Shimizu K (2002) Metabolic flux analysis in Synechocystis using isotope distribution from 13C-labeled glucose. Metab Eng 4:202–216
Acknowledgements
We thank H.R. Girish for conducting the Mass Spectrometry analyses, Ms. Purnima Kumar for help with confocal microscopy, the Sophisticated Advanced Instrument Facility (SAIF) of All India Institute of Medical Sciences (AIIMS), New Delhi for the electron microscopy analyses, and Dr. S.C. Yadav, Department of Anatomy, AIIMS for providing Karnovsky’s fixative.
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VD is supported by Junior Research Fellowship by Council for Scientific and Industrial Research (CSIR).
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Dutt, V., Srivastava, S. Novel quantitative insights into carbon sources for synthesis of poly hydroxybutyrate in Synechocystis PCC 6803. Photosynth Res 136, 303–314 (2018). https://doi.org/10.1007/s11120-017-0464-x
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DOI: https://doi.org/10.1007/s11120-017-0464-x