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Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803   

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Original research article

A brief version of this protocol appeared in:
Frontiers in Microbiology
Jul 2016

Abstract

Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are massively accumulated in the progression of the general nitrogen stress response. Several different technologies have been established for in situ and in vitro PHB analysis from different microbial sources. In this protocol, a rapid and reliable spectrophotometric method is described for PHB quantification in the cyanobacterium Synechocystis sp. PCC 6803 upon nitrogen deprivation as described in (Damrow et al., 2016).

Keywords: Cyanobacteria, Synechocystis, PHB, Nitrogen deprivation

Background

Non-diazotrophic cyanobacteria such as Synechocystis sp. PCC 6803 respond to the lack of combined nitrogen sources by bleaching, a process known as chlorosis (Allen and Smith, 1969). This acclimation response is characterized by four major structural and morphological changes: (i) a massive accumulation of electron-dense glycogen inclusions (approx. 40 nm in diameter) between the thylakoid layers accompanied by (ii) the degradation of the phycobilisome antenna complexes, (iii) the disassembling of the thylakoid membrane layers including a reduction by number and packing density, and (iv) the formation of distinct electron-transparent PHB granules (approx. 400-500 nm in diameter) (Damrow et al., 2016). The physiological function of cyanobacterial PHB metabolism, synthesized just in a few species, is quite opaque due to the absence of both catabolic enzymes and evident phenotype of PHB-deficient mutants (Beck et al., 2012; van der Woude et al., 2014; Damrow et al., 2016; Namakoshi et al., 2016).

Facing the world’s trash and global warming crisis, the demands for durable, recyclable, biodegradable, and synthetic-alternative plastics such as PHB is enormous and focus attention to cyanobacterial producers (Asada et al., 1999; Ansari and Fatma, 2016). Various different techniques are published for the analysis of PHB molecules (for updated review see [Godbole, 2016]). We are presenting a combination of hydrolytic degradation of PHB to 3-hydroxybutyrate (3-HB) in alkaline regime, and a coupled colorimetric enzymatic assay. Here the coupling with a phenazine methosulphate-p-iodonitrotetrazolium violet (PMS-INT) system directs the enzymatic redox reaction of both NADH oxidation and 3-HB reduction by the 3-hydroxybutyrate dehydrogenase (HBDH) and thus precludes an interfering backward reaction. This rapid spectrophotometric quantification of PHB just needs very simple lab equipment, is not much time-consuming, and is yet both reliable and reproducible.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml centrifuge tubes (Eppendorf® Safe-Lock microcentrifuge tubes) (Eppendorf, catalog number: 0030120086 )
  3. 15 ml centrifuge tubes (TubeSpin® Bioreactor 15) (TPP Techno Plastic Products, catalog number: 87015 )
  4. 50 ml centrifuge tubes (TubeSpin® Bioreactor 50) (TPP Techno Plastic Products, catalog number: 87050 )
  5. Synechocystis sp. PCC 6803 cells (Pasteur culture collection of cyanobacteria) (Institut Pasteur, catalog number: PCC 6803 )
  6. Crushed ice
  7. Bi-distilled water
  8. 0.5 N sodium hydroxide (NaOH) (Carl Roth, catalog number: 9356.1 )
  9. 1 N hydrochloric acid (HCl-ROTIPURAN® 37%) (Carl Roth, catalog number: X942.1 )
  10. 50 mM Tris-hydrochloride, pH 8.5 (PUFFERAN® ≥ 99%) (Carl Roth, catalog number: 9090.3 )
  11. 20 mM β-Nicotinamide adenine dinucleotide (NADH/H+), reduced disodium salt (Sigma-Aldrich, catalog number: N9785 )
    Note: This product has been discontinued.
  12. 1 mM β-Nicotinamide adenine dinucleotide (NAD+), reduced disodium salt (Sigma-Aldrich, catalog number: N0632 )
  13. 5 mM phenazine methosulfate (PMS) (Sigma-Aldrich, catalog number: P9625 )
  14. 5 mM p-Iodonitrotetrazolium violet (INT) (Sigma-Aldrich, catalog number: I8377 )
  15. 1 mM (R)-3-hydroxybutyric (R-3-HB) (Sigma-Aldrich, catalog number: 54920 ) with a molar mass of 104.10 g/mol
  16. 15 U/ml 3-hydroxybutyrate dehydrogenase (3-HBDH) grade II from Rhodobacter spheroides (Roche Diagnostics, catalog number: 10127833001 )
  17. BG11 stock solution ‘+N’; autoclaved (use 1:100) (see Recipes)
    1. Sodium nitrate (NaNO3 ≥ 99%, p.a., ACS, ISO) (Carl Roth, catalog number: A136.1 )
    2. Calcium chloride dihydrate (CaCl2·2H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 5239.1 )
    3. Citric acid (C6H8O7 ≥ 99.5%, p.a., ACS, anhydrous) (Carl Roth, catalog number: X863.1 )
    4. Magnesium sulfate heptahydrate (MgSO4·7H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: P027.1 )
    5. Ethylenediamine tetraacetic acid disodium salt dehydrate (EDTA ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 8043.2 )
  18. BG110 stock solution ‘-N’; autoclaved (use 1:100) (see Recipes)
    1. Calcium chloride (CaCl2·2H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 5239.1 )
    2. Citric acid (C6H8O7 ≥ 99.5%, p.a., ACS, anhydrous) (Carl Roth, catalog number: X863.1 )
    3. Magnesium sulfate (MgSO4·7H2O ≥ 99%, p.a., ACS) (Carl Roth, catalog number: P027.1 )
    4. Ethylenediamine tetraacetic acid disodium salt dehydrate (EDTA ≥ 99%, p.a., ACS) (Carl Roth, catalog number: 8043.2 )
  19. Trace Metal Mix for BG11; sterile filtrated (use 1:1,000) (see Recipes)
    1. Boric acid (H3BO3) (≥ 99.8%, p.a., ACS, ISO) (Carl Roth, catalog number: 6943.2 )
    2. Manganese(II) chloride tetrahydrate (MnCl2·4H2O ≥ 99%, p.a.) (Carl Roth, catalog number: T881.3 )
    3. Zinc sulfate heptahydrate (ZnSO4·7H2O ≥ 97%, extra pure) (Carl Roth, catalog number: 7316.1 )
    4. Sodium molybdate dihydrate (Na2MoO4·2H2O), ≥ 99.5% (Sigma-Aldrich, catalog number: M1651 )
    5. Cupric(II) sulfate pentahydrate (CuSO4·5H2O ≥ 99%, Ph.Eur., BP) (Carl Roth, catalog number: P025.1 )
    6. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Sigma-Aldrich, catalog number: 203106 )
  20. Extra solutions for BG11; sterile filtrated (use 1:1,000) (see Recipes)
    1. Ammonium iron(III) citrate (Carl Roth, catalog number: 9366.1 )
    2. Potassium phosphate dibasic (K2HPO4 ≥ 98%, Ph.Eur., BP) (Carl Roth, catalog number: T875.1 )
    3. Sodium carbonate carbonate monohydrate (Na2CO3·H2O ≥ 99.5%, p.a., ACS) (Carl Roth, catalog number: 2597.2 )
  21. Buffer solution for BG11; autoclaved (use 1:200) (see Recipes)
    1 M HEPES buffer (pH 8.0) (PUFFERAN® ≥ 99.5%) (Carl Roth, catalog number: 9105.4 )
  22. BG11 (‘+N’) medium (see Recipes)
  23. BG110 (‘-N’) medium (see Recipes)

Equipment

  1. Wide-neck 500 ml Erlenmeyer flasks (Carl Roth, DURAN®, catalog number: C150.1 )
  2. Personal protective equipment (PPE)
    Note: These should be worn at all times when dealing with concentrated acids and alkali. See Note 3.
    1. Safety glasses (Sekuroka®-safety glasses EN166) (Carl Roth, catalog number: Y254.1 )
    2. Lab coat (Sekuroka®-lab coats) (Carl Roth, catalog number: T413.1 )
    3. Gloves (Rotiprotect®-nitrile evo) (Carl Roth, catalog number: CPX7.1 )
  3. Pipette set (Rainin Pipet-Lite LTS Starter Kit L-STARTXLS+) (Mettler-Toledo International, catalog number: 17014406 )
  4. Ice bucket
  5. Laminar flow bench (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II , catalog number: 51028225)
  6. Analytical balance (Mettler-Toledo International, model: XS105 )
  7. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM BiofugeTM Primo R , catalog number: 75005440)
  8. Light meter (LI-COR, model: LI-189 )
  9. Incubator (Infors, model: Multitron Pro , ‘Algae Special’)
  10. UV-Vis spectrophotometer (Analytic Jena, model: Specord® 50 PLUS )
  11. Bench pH/mV/°C meter (pH 1,000 L, pHenomenal®) (VWR, catalog number: 662-1422 )
  12. Thermomixer (Biometra, model: ThermoShaker TS1 , catalog number: 846-051-500)
  13. Vortex shaker (VWR, Peqlab, model: peqTWIST )
  14. Autoclave (Systec, model: Systec VX-150 )

Procedure

  1. Induction of PHB biosynthesis in Synechocystis sp. PCC 6803 upon nitrogen deprivation
    1. For nitrogen step down experiments as described in (Grundel et al., 2012; Damrow et al., 2016), an axenic pre-culture of Synechocystis sp. PCC 6803 (2 x 250 ml) grows in Erlenmeyer flasks to an OD750 of 0.5 at 28 °C under continuous illumination with white light of 80 µmol photons m-2 sec-1 in BG11 medium (see Recipes) containing 20 mM HEPES buffer (pH 8.0) and 17.6 mM sodium nitrate as the nitrogen source.
    2. For nitrogen deprivation, cells (500 ml) are pelleted by centrifugation at 4,000 x g for 10 min at room temperature and washed twice with 500 ml BG110 (see Recipes), which lacks sodium nitrate ‘-N’ (Stanier et al., 1971).
    3. The final cell suspension is split (2 x 250 ml) and both cultures ‘-N’ and ‘+N’ are supplemented with sodium acetate (final concentration 10 mM) as described by (Hein et al., 1998). Sodium nitrate is only added to the control ‘+N’ sample (final concentration 17.6 mM).
    4. The cultivation upon nitrogen deprivation is–likewise to the pre-culture cultivation–implemented in Erlenmeyer flasks at 28 °C under continuous shaking (80 rpm; Multitron Pro ‘Algae Special’) and continuous illumination using white light (80 µmol photons m-2 sec-1) for seven days.
  2. Quantification of dry weight
    1. 10 ml of each cell culture is pelleted by centrifugation (4,000 x g, 10 min), washed twice with 10 ml sterile water and finally concentrated up to 1 ml and transferred into a 1.5 ml reaction tube which has to be pre-weighted before.
    2. After another centrifugation step (10,000 x g, 3 min) the supernatant is entirely removed and the remaining cell pellet is dried completely at 80 °C in a thermomixer overnight (open cap).
    3. The dry weight of the cells (~5 mg) is determined by the weight of the ‘cell tubes’ subtracted from the dead weight of the tube (Table 1).

      Table 1. Determined dry weights


  3. Extraction and de-polymerization of PHB to (R)-3-hydroxybutyric acid (Figure 1) by alkaline hydrolysis.


    Figure 1. Hydrolytic degradation of poly-β-hydroxybutyrate (PHB) to (R)-3-hydroxybutyric acid (R-3-HB)

    1. 0.5 N NaOH (< 5 mg DW→300 µl 0.5 N NaOH; > 5 mg DW→400 µl 0.5 N NaOH) are added to the dried cell pellet from step 2 (‘+N’ and ‘-N’, separately) and incubated at 85 °C for 1 h by continuously shaking in a thermomixer and vigorously vortexing from time to time both to break the cells and to hydrolyze PHB into its monomer (R)-3-hydroxybutyrate (R-3-HB) as described by (Yu and Marchessault, 2000; Cui et al., 2006).
    2. After cooling on ice, the samples (‘+N’ and ‘-N’, separately) are neutralized by the addition of 1 N HCl in a ratio of four volumes 0.5 N NaOH (e.g., 300 µl) to one volume 1 N HCl (e.g., 100 µl). The samples (‘+N’ and ‘-N’, separately) are thoroughly mixed by vortexing.
    3. Afterwards the R-3-HB containing fraction (supernatant) of each sample (‘+N’ and ‘-N’, separately) is separated from the cell debris (pellet) by centrifugation (1 min, 4,500 x g, room temperature). The R-3-HB containing fraction (supernatant) is transferred to a new 1.5 ml reaction tube for each sample (‘+N’ and ‘-N’, separately).
    4. For further quantitative analysis, the R-3-HB containing samples (‘+N’ and ‘-N’, separately) are diluted in ratio of 1:10 dilution with bi-distilled water.
  4. Spectrophotometric quantification of R-3-HB monomers
    1. The R-3-HB content in each sample (‘+N’ and ‘-N’, separately) is finally spectrophotometrically quantified via an enzymatic assay. In a redox reaction, R-3-HB is oxidized to acetoacetate while NAD+ is reduced to NADH/H+ catalyzed in a 1:1 stoichiometry by 3-hydroxybutyrate dehydrogenase (HBDH) (Figure 2).


      Figure 2. Coupled enzymatic assay of R-3-HB oxidation by the 3-hydroxybutyrate dehydrogenase and simultaneous reoxidation of NADH/H+ to NAD+ by the PMS-INT system forming a stable color precipitate

    2. In order to prevent a backward reaction (reduction of acetoacetate to R-3-HB; oxidation of NADH/H+ to NAD+), the NADH/H+ synthesis is directly coupled to a phenazinemethosulphate-p-iodonitrotetrazolium violet (PMS-INT) colorimetric assay as described by (Lim and Buttery, 1977; Hinman and Blass, 1981). Here, the electron is transferred in a 1:1 stoichiometry from NADH/H+ via PMS to INT. The reduced form of INT is a stable formazan, which absorbs light at a wavelength of 500 nm (or 505 nm) (Lim and Buttery, 1977) (Figure 2).
    3. The level of R-3-HB is proportional both to the level of synthesized NADH/H+ and of formazan (Figure 2).
      100 µl of the diluted sample from step 3 or 100 µl 1 mM standard ((R)-3-hydroxybutyric) is added into a cuvette (1 ml assay volume) containing (triplicates for each sample):
      730 µl 50 mM Tris-HCl buffer, pH 8.5 (optimum pH for oxidation reaction)
      50 µl 1 mM NAD+
      10 µl 0.5 mM PMS
      100 µl 0.05 mM INT
    4. The reaction is monitored at 25 °C as the change (∆E = E2 - E1) in A500 (or 505 nm) on spectrophotometer before (E1) and ten min after (E2) initiation with 10 µl 15 U/ml HBDH according to the protocol as described in (Hinman and Blass, 1981) (Table 2). Tris-HCl buffer, pH 8.5 (50 mM) is used as a blank.

      Table 2. Absorption change at 500 nm (or 505 nm) for nitrogen-deprived samples


    5. The R-3-HB (and thus PHB) level is calculated via the calibration series of six different NADH/H+ concentrations ranging from 0, (0.1), 0.25, 0.5, 0.75, and 1.0 mM (triplicates for each concentration) in a PMS-INT formazan reaction. Tris-HCl buffer, pH 8.5 (50 mM) is used as a blank. Here, the ∆E (Table 3) is determined at 25 °C as the change in A500 (or 505 nm) on spectrophotometer before (E1) and three min after (E2) addition of 100 µl respective NADH/H+ standard solution to a cuvette containing (triplicates for each sample):
      730 µl 50 mM Tris-HCl buffer, pH 8.5
      60 µl bi-distilled water
      10 µl 0.5 mM PMS
      100 µl 0.05 mM INT

      Table 3. Absorption change at 500 nm (or 505 nm) for NADH calibration series

Data analysis

  1. NADH/H+ standard curve
    The calibration curve is plotted as a function of respective absorbance (∆E value) versus known concentration of NADH/H+ [mM]. The fitting curve and parameters are obtained by linear regression analysis. The linear regression coefficient should be in the range of 0.97 ≤ R2 ≤ 1 (Figure 3).


    Figure 3. NADH/H+ calibration curve

  2. R-3-HB standard control reaction
    Assuming a complete chemical conversion (1:1 stoichiometry), the respective absorbance of control reaction (0.1 mM R-3-HB) corresponds to 0.1 mM NADH/H+ (see NADH/H+ standard curve, Figure 3).
  3. PHB level quantification related to the dry weight
    The converted NADH/H+ and thus R-3-HB concentrations are calculated from the slope function of the linear regression analysis for each absorbance (∆E value). Including all dilution and concentration factors into the equations, the average R-3-HB amount of all three replicates is related to the determined dry weight (Table 4). Whereas in the control samples ‘+N’ no R-3-HB is detectable, the average R-3-HB (and thus PHB) content of the nitrogen-deprived cells of Synechocystis sp. PCC 6803 corresponds to 6-10% of the dry weight (Damrow et al., 2016).

    Table 4. Determination of PHB content per dry weight

Notes

  1. All cell passages and media are implemented under sterile conditions using a laminar flow bench and an autoclave. The intensity of photosynthetically active radiation (PAR) is measured in µE m-2 sec-1 (μmol photons per m2 and sec) using a LI-189 light meter.
  2. The dried cell pellets are stored at 4 °C for at least three weeks.
  3. For safety reasons, adjust to protective measures and tight-closing tubes when handling acids and bases. For safety reasons, assure that the hot samples are cooled down before HCl addition. The supernatant containing R-3-HB monomers could be stored at -20 °C for at least three weeks.
  4. The PMS-INT reaction is light-sensitive. The samples are handled in darkness.

Recipes

Note: Unless otherwise indicated, bi-distilled water was used as a solvent in solutions.

  1. BG11 stock solution ‘+N’; autoclaved (use 1:100)
    17.65 M NaNO3
    0.18 M CaCl2·2H2O
    0.0031 M citric acid
    0.304 M MgSO4·7H2O
    0.0034 M EDTA
  2. BG11 stock solution ‘-N’; autoclaved (use 1:100)
    0.18 M CaCl2·2H2O
    0.0031 M citric acid
    0.304 M MgSO4·7H2O
    0.0034 M EDTA
  3. Trace Metal Mix for BG11; sterile filtrated (use 1:1,000)
    1.43 g/L H3BO3
    0.09 g/L MnCl2·4H2O
    0.11 g/L ZnSO4·7H2O
    0.195 g/L Na2MoO4·2H2O
    0.0395 g/L CuSO4·5H2O
    0.0247 g/L Co(NO3)2·6H2O
  4. Extra solutions for BG11; sterile filtrated (use 1:1,000)
    23 mM ferric ammonium citrate
    172 mM K2HPO4
    188 mM Na2CO3·H2O
  5. Buffer solution for BG11; autoclaved (use 1:200)
    1 M HEPES buffer (pH 8.0)
  6. BG11 (‘+N’)
    10 ml 100x BG11 (+N) stock solution
    1 ml Trace Metal Mix
    1 ml 23 mM ferric ammonium citrate
    1 ml 172 mM potassium phosphate dibasic
    1 ml 188 mM sodium carbonate
    5 ml 1 M HEPES buffer (pH 8.0)
    Add up to 1 L with deionized water
  7. BG110 (‘-N’)
    10 ml 100x BG11 (-N) stock solution diminished by sodium nitrate
    1 ml Trace Metal Mix
    1 ml 23 mM ferric ammonium citrate
    1 ml 172 mM potassium phosphate dibasic
    1 ml 188 mM sodium carbonate
    5 ml 1 M HEPES buffer (pH 8.0)
    Add up to 1 L with deionized water

Acknowledgments

We particularly acknowledge Wolfgang Lockau for his commitment and valuable discussions on this topic and for his sustained support of our research. We gratefully thank Gisa Baumert (Humboldt-Universität zu Berlin, Germany) for her excellent technical assistance. We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (to YZ) in the framework of the Collaborative Research Center 1078 on ‘Protonation Dynamics in Protein Function’ (SFB1078, project A4/Holger Dau).

References

  1. Allen, M. M. and Smith, A. J. (1969). Nitrogen chlorosis in blue-green algae. Arch Mikrobiol 69(2): 114-120.
  2. Ansari, S. and Fatma, T. (2016). Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and characterization. PLoS One 11(6): e0158168.
  3. Asada, Y., Miyake, M., Miyake, J., Kurane, R. and Tokiwa, Y. (1999). Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling. Int J Biol Macromol 25(1-3): 37-42.
  4. Beck, C., Knoop, H., Axmann, I. M. and Steuer, R. (2012). The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms. BMC Genomics 13: 56.
  5. Cui, Y., Barford, J. P. and Renneberg, R. (2006). Determination of poly(3-hydroxybutyrate) using a combination of enzyme-based biosensor and alkaline hydrolysis. Anal Sci 22(10): 1323-1326.
  6. Damrow, R., Maldener, I. and 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.
  7. Godbole, S. (2016). Methods for identification, quantification and characterization of polyhydroxyalkanoates. Int J Bioassays 5(04).
  8. Grundel, M., Scheunemann, R., Lockau, W. and Zilliges, Y. (2012). Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology 158(Pt 12): 3032-3043.
  9. Hein, S., Tran, H. and Steinbuchel, A. (1998). Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria. Arch Microbiol 170(3): 162-170.
  10. Hinman, L. M. and Blass, J. P. (1981). An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates. J Biol Chem 256(13): 6583-6586.
  11. Lim, H. H. and Buttery, J. E. (1977). Determination of ethanol in serum by an enzymatic PMS-INT colorimetric method. Clin Chim Acta 75(1): 9-12.
  12. Namakoshi, K., Nakajima, T., Yoshikawa, K., Toya, Y. and Shimizu, H. (2016). Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803. J Biotechnol 239: 13-19.
  13. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35(2): 171-205.
  14. van der Woude, A. D., Angermayr, S. A., Puthan Veetil, V., Osnato, A. and Hellingwerf, K. J. (2014). Carbon sink removal: Increased photosynthetic production of lactic acid by Synechocystis sp. PCC6803 in a glycogen storage mutant. J Biotechnol 184: 100-102.
  15. Yu, G-e. and Marchessault, R. H. (2000). Characterization of low molecular weight poly(β-hydroxybutyrate)s from alkaline and acid hydrolysis. Polymer 41(3): 1087-1098.
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zilliges, Y. and Damrow, R. (2017). Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803. Bio-protocol 7(14): e2402. DOI: 10.21769/BioProtoc.2402.
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