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Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC
蓝细菌黑暗、厌氧培养及其分泌物有机酸的HPLC定量检测   

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Abstract

Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with a butyl rubber cap, following which N2 gas was introduced into the vial. The organic acids produced were quantified by high-performance liquid chromatography via post-labeling with bromothymol blue as a pH indicator. After separation by ion-exclusion chromatography, the organic acids were identified by comparing their retention time with that of standard solutions. These procedures allow researchers to quantify the organic acids produced by microorganisms, contributing to knowledge about the biology and biotechnology of cyanobacteria.

Keywords: Anaerobic condition(厌氧条件), Cyanobacteria(蓝细菌), HPLC(HPLC), Lactate(乳酸盐), Organic acids(有机酸), Succinate(琥珀酸), Synechocystis sp.(集胞藻属)

Background

Cyanobacteria are a group of bacteria that perform oxygenic photosynthesis. Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) is a non-nitrogen-fixing, unicellular cyanobacterium that is commonly used for basic and applied research studies. Synechocystis 6803 cells are able to excrete organic acids, such as succinate and lactate, under both dark and anaerobic conditions (Osanai et al., 2015). Genetic manipulation and modification of the incubation conditions, such as addition of potassium or NaHCO3, have succeeded in increasing the levels of organic acids excreted from Synechocystis 6803 cells (Osanai et al., 2015; Hasunuma et al., 2016; Iijima et al., 2016; Ueda et al., 2016). Since succinate and lactate are commodity chemicals used to make various materials, such as bioplastics, their bio-based production is a desirable way to reduce the environmental burden. Herein, we describe the methods of Synechocystis 6803 incubation under dark, anaerobic conditions, and quantification of the organic acids by HPLC via post-labeling with bromothymol blue.

Materials and Reagents

  1. 50 ml disposable polypropylene tubes
  2. Test tubes for cultivation (Iwaki, catalog number: TEST30NP )
  3. 20 ml headspace vials (GL Sciences, catalog number: 1030-46026 )
  4. 22 G x 1″ needle (Terumo Medical, catalog number: NN-2225R )
  5. 22 G x 1 ½″ needle (Terumo, catalog number: NN-2238S )
  6. Aluminum foil
  7. SupraPure hydrophilic PVDF syringe filters (Recenttec, catalog number: R7-PVDF033S022I )
  8. Target DP vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: C4000-1 )
  9. Screw caps (GL Sciences, catalog number: 1030-45261 )
  10. 300 µl target polyspring inserts (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: C4010-S630 )
  11. Disposable syringes (Terumo, catalog number: SS-10LZ )
  12. Open-top-style aluminum caps with butyl rubber septa (Systech, catalog number: 98552 )
  13. MF-Millipore membrane filters (Merck Millipore, catalog number: HAWP03700 )
  14. Cyanobacterium Synechocystis sp. PCC 6803
  15. Ammonium chloride (NH4Cl) (Wako Pure Chemical Industries, catalog number: 017-02995 )
  16. 60% perchloric acid (HClO4) (Wako Pure Chemical Industries, catalog number: 160-05755 )
  17. Trichloroacetic acid
  18. Succinate
  19. Lactate
  20. Citric acid (Nacalai Tesque, catalog number: 09109-85 )
  21. Acetate
  22. Ferric ammonium citrate
  23. Na2EDTA (Nacalai Tesque, catalog number: 15111-45 )
  24. Sodium nitrate (NaNO3) (Wako Pure Chemical Industries, catalog number: 195-02545 )
  25. Potassium phosphate dibasic (K2HPO4) (Wako Pure Chemical Industries, catalog number: 164-04295 )
  26. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 131-00405 )
  27. Calcium chloride dihydrate (CaCl2·2H2O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
  28. Sodium carbonate (Na2CO3) (Nacalai Tesque, catalog number: 31311-25 )
  29. Boric acid (H3BO3) (Wako Pure Chemical Industries, catalog number: 021-02195 )
  30. Manganese chloride tetrahydrate (MnCl2·4H2O) (Wako Pure Chemical Industries, catalog number: 139-00722 )
  31. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 264-00402 )
  32. Pentahydrate copper sulphate (CuSO4·5H2O) (Wako Pure Chemical Industries, catalog number: 039-04412 )
  33. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Wako Pure Chemical Industries, catalog number: 196-02472 )
  34. Sulfuric acid (H2SO4) (Wako Pure Chemical Industries, catalog number: 199-15995 )
  35. Cobalt dinitrate hexahydrate, Co(NO3)2·5H2O (Wako Pure Chemical Industries, catalog number: 031-03752 )
  36. HEPES (Dojindo Molecular Technologies, catalog number: 342-01375 )
  37. Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
  38. Bromothymol blue (BTB) (Wako Pure Chemical Industries, catalog number: 027-03052 )
  39. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00451 )
  40. Disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) (Wako Pure Chemical Industries, catalog number: 196-02835 )
  41. Succinic acid (Wako Pure Chemical Industries, catalog number: 190-04332 )
  42. DL-Lactic acid lithium salt (MP Biomedicals, catalog number: 100824 )
  43. Sodium acetate (Wako Pure Chemical Industries, catalog number: 190-01071 )
  44. Modified BG-11 liquid medium (see Recipes)
  45. Mobile phase (3 mM HClO4) (see Recipes)
  46. Reaction solution (0.2 mM BTB in 15 mM Na2HPO4) (see Recipes)
  47. 500 mM succinate solution (see Recipes)
  48. 500 mM lactate solution (see Recipes)
  49. 500 mM citrate solution (see Recipes)
  50. 500 mM acetate solution (see Recipes)
  51. 1 M acetate solution (see Recipes)

Equipment

  1. Cultivation chamber (TOMY Digital Biology, model: CLE-303 )
  2. A gas mixer (KOFLOC, model: RK120XM )
  3. An air pump (Yasunaga, model: LP-30A )
  4. Spectrophotometer (Shimadzu, model: UV-2700 )
  5. High-speed refrigerated microcentrifuge (TOMY Digital Biology, model: MX-305 )
  6. N2 generator (Sanyo Denshi, model: SN4-4 )
  7. Rotary shaker (Nissin, model: NX-20D )
  8. Freeze dryer (Tokyo Rikaikai, EYELA, model: FDU-2200 )
  9. Ultrasonic cleaning machine (SND, model: US-108 )
  10. Autoclave
  11. The HPLC system (JASCO International, model: LC-2000Plus ) was composed of the following equipment:
    1. Photodiode array (PDA) detector (JASCO International, model: MD-2018Plus )
    2. Intelligent HPLC pump (JASCO International, model: PU-2080Plus )
    3. Line degasser (JASCO International, model: DG-2080-53 )
    4. Ternary gradient unit (JASCO International, model: LG-2080-02 )
    5. Intelligent column oven (JASCO International, model: CO-2065Plus )
    6. Intelligent autosampler (JASCO International, model: AS-2057Plus )
    7. Ion-exclusion column (300 x 8.0 mm) (GL Sciences, model: RSpak KC-811, catalog number: 5055-13509 ) and guard column (10 x 4.6 mm) (GL Sciences, model: GPC KF-G, catalog number: 5055-13150 )
    8. Reaction coil unit (JASCO International, model: RU-2080-51 )

Software

  1. ChromNAV software (Ver. 1.14)

Procedure

  1. Cultivation under light, aerobic (a) and incubation under dark, anaerobic conditions (b)
    1. The glucose-tolerant strain (designated as GT) of Synechocystis sp. PCC 6803, isolated by Williams (1988), is cultivated in modified BG-11 medium (see Recipes), consisting of BG-110 liquid medium (Rippka, 1988) supplemented with 5 mM NH4Cl (buffered with 20 mM HEPES-KOH, pH 7.8). In our study, the GT-I strain was used (Kanesaki et al., 2012). The GT cells are cultivated in liquid modified BG-11 medium under air containing 1% (v/v) CO2, using a gas mixer (RK120XM, KOFLOC) and an air pump (LP-30A, Yasunaga) at the flow rate of 30-50 ml/min, in a cultivation chamber (CLE-303, TOMY). The temperature is kept at 30 °C and white light is supplied continuously at 50-70 µmol photons·m-2 sec-1. Cell densities are measured at A730 using the Shimadzu UV-2700 spectrophotometer. Cells cultivated in 70 ml of modified BG-11 medium (started from A730 = 0.4) for 3 days under, light, aerobic condition. The light, aerobic cultivation affects the levels of succinate and lactate, and optimization of culture conditions is required for each laboratory.
    2. The cell are collected by centrifugation at 5,600 x g for 2 min and concentrated into 10 ml of HEPES buffer (20 mM HEPES-KOH, pH 7.8) or modified BG-11 to reach an A730 of 20 in a vial (Figure 1). The vial is sealed with open-top-style aluminum caps with butyl rubber septa, and two needles (Needle 22 G x 1″ and Needle 22 G x 1 ½″) are stabbed through a septum. The 22 G x 1 ½″ needle is connected to a N2 gas generator to introduce an N2 gas for 30-60 min in order to generate the anaerobic condition.
    3. After introduction of the N2 gas, the needles are removed and the vial is wrapped with aluminum foil. The cells are incubated at 30 °C with shaking on a rotary shaker at 160 rpm for 1-5 days. For 1 day, succinate and lactate were less excreted from the cells. Succinate and lactate levels increased during long incubation, however, the levels reached maximum at 3 or 4 days, and started to decrease at 5 days (Osanai et al., 2015).


      Figure 1. A 20 ml vial for anaerobic incubation. Cells were concentrated into 10 ml of buffer or medium to reach an A730 of 20, and the vial was then sealed with a butyl rubber cap. Two syringes were inserted into the cap, and N2 gas was introduced through one of two syringes for 30-60 min. After introduction of the N2 gas, the syringes were removed and the vial was wrapped in aluminum foil to maintain a dark condition.

  2. Sample preparation for HPLC analysis
    1. After incubation, the aluminum cap with butyl rubber is removed with an opener. The cell cultures are centrifuged at 5,600 x g for 2 min. Using a SupraPure sterile syringe filter, the supernatants are filtered to remove debris.
    2. Then, 1 ml of supernatant is transferred into a 2 ml plastic tube. The supernatants are placed in a flask and dried at -80 °C with an FDU-2200 freeze dryer for 1 day. The freeze-dried samples are resolved with 100 µl of 3 mM perchloric acid (HClO4) (since HClO4 is highly corrosive, it should be handled with care) and centrifuged at 5,600 x g for 2 min.
    3. Then, transfer 100 µl of the supernatant into a Target DP vial using a glass insert. Set it in the autosampler for HPLC analysis.
    4. In case of samples containing large amount of proteins, freeze-dried samples should be resolved in 97 µl of 3 mM HClO4 and 3 µl of 100% trichloroacetic acid. Mix thoroughly and then centrifuge the samples at 20,400 x g for 10 min. Use the supernatant for HPLC analysis.
    5. To prepare standards, mix 100 μl of 500 mM succinate solution, 160 μl of 500 mM lactate solution, 100 μl of 500 mM citrate solution, 200 μl of 1 M acetate solution (see Recipes), and 440 μl of dH2O to make up a total 1 ml of organic acids standard. This solution was named STD1. The final concentration of each organic acid in STD1 is 80 mM lactate, 50 mM succinate, 50 mM citrate, and 200 mM acetate.
    6. Dilute STD1 to make a range of concentrations for drawing the analytical curves.
  3. HPLC analysis
    Quantification of the organic acids is performed by HPLC using post-labeling with BTB (Figure 2). Use 3 mM HClO4 solution as the mobile phase and 0.2 mM BTB in 15 mM Na2HPO4 solution as the reaction solution. The mobile phase and reaction solution are degassed for approximately 20 min using the ultrasonic cleaning machine and the aspirator. HPLC measurement is performed with the following conditions:
    1. Intelligent HPLC pump (Mobile phase): the flow rate is set at 0.7 ml/min. The pump is connected to the line degasser and the ternary gradient unit, and further connected to the intelligent column oven. Before sample injection, equilibrate the columns with mobile phase at same flow rate for one hour.
    2. Intelligent HPLC pump (Reaction solution): the flow rate is set at 1.2 ml/min.
    3. Intelligent column oven: the column temperature is 60 °C.
    4. Intelligent autosampler: the sampler temperature is 4 °C and the injection volume is 20 µl. To clean up the injection needle, use HClO4 solution. Flush three times with HClO4 before sample injection.
    5. Reaction coil unit was used for mixing mobile phase including samples and BTB solution.
    6. PDA: single beam photometer with deuterium (D2) and halogen (W) lamps; single data analysis is performed for 42 min using scans in the wavelength range of 200-900 nm. Before starting the HPLC analysis, wait for approximately 60 min until the flow is stable (baseline without drift and noise).


      Figure 2. Schematic diagram of HPLC analysis with bromothymol blue post-labeling. Two HPLC pumps are used for mobile phase and BTB solution, respectively. Samples are injected with autosampler and divided by size exclusion columns, followed by mixed with BTB solution. Organic acids are detected by PDA detector and analyzed by ChromNAV software.

Data analysis

Typical chromatograms measured by HPLC with BTB post-labeling are shown in Figure 3. Organic acid peaks are quantified using the absorbance at 445 nm. Use the ChromNAV program to quantify the peak areas of each organic acid. Draw standard curves for each organic acid by marking with a dot of known concentration samples. Calculate the concentrations of organic acids in the samples by using the following standard curves (Figure 4).


Figure 3. Typical chromatograms measured by HPLC with bromothymol blue post-labeling. The peak intensity was calculated from the absorbance at wavelength 445 nm. The retention times for each organic acid were as follows: citrate 17.55 min, succinate 22.36 min, lactate 23.80 min, and acetate 27.34 min.


Figure 4. Standard curves for succinate (orange) and lactate (blue). The intensity values were calculated from the chromatograms by integrating the peak areas of each organic acid.

Notes

In case of samples containing large amount of proteins, freeze-dried samples should be resolved in 97 µl of 3 mM HClO4 and 3 µl of 100% trichloroacetic acid. Mix thoroughly and then centrifuge the samples at 20,400 x g for 10 min. Use the supernatant for HPLC analysis.

Recipes

  1. Modified BG-11 liquid medium (store at room temperature)
    1. Solution 1 (store at 4 °C)
      Weigh 0.6 g citric acid, 0.6 g ferric ammonium citrate, and 0.1 g Na2EDTA, and add distilled water (dH2O) to 200 ml
    2. Solution 2 (store at 4 °C)
      Weigh 30 g NaNO3, 0.78 g K2HPO4, and 1.5 g MgSO4·7H2O, and add dH2O to 1 L
    3. Solution 3 (store at 4 °C)
      Weigh 3.8 g CaCl2·2H2O and add dH2O to 200 ml
    4. Solution 4 (store at 4 °C)
      Weigh 4 g Na2CO3 and add dH2O to 200 ml
    5. Solution 5 (store at 4 °C)
      Weigh 2.86 g H3BO3, 1.81 g MnCl2·4H2O, 0.22 g ZnSO4·7H2O, 0.08 g CuSO4·5H2O, 0.021 g Na2MoO4, and 50 μl H2SO4, and add dH2O to 1 L (1*)
      Weigh 0.0494 g Co(NO3)2·5H2O and add to (1*)
    6. 1 M HEPES-KOH, pH 7.8 (store at 4 °C)
      Weigh 119.1 g HEPES and add dH2O to approximately 400 ml
      Adjust pH to 7.8 with 6 N KOH and add dH2O to 500 ml
      Autoclave (121 °C for 15 min) and store at 4 °C
    7. K2HPO4 solution (store at 4 °C)
      Weigh 0.39 g K2HPO4 and add dH2O to 200 ml
    8. MgSO4·7H2O solution (store at 4 °C)
      Weigh 0.75 g MgSO4·7H2O and add dH2O to 200 ml
      Mix 20 ml 1 M HEPES-KOH (pH 7.8), 50 ml Solution 2, 2 ml Solution 3, 1 ml Solution 4, and 1 ml Solution 5, and add dH2O to 1 L
      Autoclave the mixture and store at room temperature
      Before use, add 2 ml Solution 1 to 1 L mixture
  2. Mobile phase (3 mM HClO4)
    Weigh 0.5 g 60% HClO4 and add dH2O to 1 L
    Filter with the MF-Millipore membrane filter
  3. Reaction solution (0.2 mM BTB in 15 mM Na2HPO4)
    Weigh 125 mg BTB and add to 10 ml ethanol (1)
    Weigh 5.3 g Na2HPO4·12H2O and add to dH2O (2)
    Mix (1) and (2), and add dH2O to 1 L
    Filter the mixture with the MF-Millipore membrane filter
  4. 500 mM succinate solution
    Weigh 590.45 mg succinate and add to 10 ml dH2O using a measuring flask
  5. 500 mM lactate solution
    Weigh 480.05 mg DL-lactate lithium salt and add to 10 ml dH2O using a measuring flask
  6. 500 mM citrate solution
    Weigh 960.6 mg citrate and add dH2O to 10 ml using a measuring flask
  7. 1 M acetate solution
    Weigh 820.3 mg sodium acetate and add dH2O to 10 ml using a measuring flask

Acknowledgments

This protocol was adapted from our previous studies published by Ueda et al. (2016) and Iijima et al. (2016). This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology, Japan, by a grant to T.O. from ALCA (Project name ‘Production of cyanobacterial succinate by the genetic engineering of transcriptional regulators and circadian clocks’) from the Japan Science and Technology Agency, and by JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (Grant No. 16H06559).

References

  1. Hasunuma, T., Matsuda, M. and Kondo, A. (2016). Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism. Metab Eng Commun 3: 130-141.
  2. Iijima, H., Shirai, T., Okamoto, M., Pinto, F., Tamagnini, P., Hasunuma, T., Kondo, A., Hirai, M., T. and Osanai, T. (2016). Metabolomics-based analysis revealing the alteration of primary carbon metabolism by the genetic manipulation of a hydrogenase HoxH in Synechocystis sp. PCC 6803. Algal Res 18: 305-313.
  3. Kanesaki, Y., Shiwa, Y., Tajima, N., Suzuki, M., Watanabe, S., Sato, N., Ikeuchi, M. and Yoshikawa, H. (2012). Identification of substrain-specific mutations by massively parallel whole-genome resequencing of Synechocystis sp. PCC 6803. DNA Res 19(1): 67-79.
  4. Osanai, T., Shirai, T., Iijima, H., Nakaya, Y., Okamoto, M., Kondo, A. and Hirai, M. Y. (2015). Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium. Front Microbiol 6: 1064.
  5. Rippka, R. (1988). Isolation and purification of cyanobacteria. Methods Enzymol 167: 3-27.
  6. Ueda, S., Kawamura, Y., Iijima, H., Nakajima, M., Shirai, T., Okamoto, M., Kondo, A., Hirai, M. Y. and Osanai, T. (2016). Anionic metabolite biosynthesis enhanced by potassium under dark, anaerobic conditions in cyanobacteria. Sci Rep 6: 32354.
  7. Williams, J. G. K. (1988). Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol. 167: 766-778.

简介

琥珀酸盐和乳酸盐是用于生产生物塑料的商品化学品。最近发现这样的有机酸从单细胞蓝细菌集胞藻的细胞中排出。 PCC 6803在黑暗,无氧条件下。为了进行黑暗的厌氧培养,将细胞浓缩在小瓶内,然后用丁基橡胶帽密封,随后将N 2气体引入小瓶中。通过用溴百里酚蓝作为pH指示剂的后标记,通过高效液相色谱定量产生的有机酸。通过离子排阻色谱分离后,通过将其保留时间与标准溶液的保留时间进行比较来鉴定有机酸。这些程序允许研究人员量化微生物产生的有机酸,有助于蓝藻生物学和生物技术的知识。

背景 蓝细菌是一组进行氧合光合作用的细菌。集胞藻 sp。 PCC 6803(以下称为集胞藻6803)是非固氮,单细胞蓝细菌,通常用于基础和应用研究。集胞藻6803细胞能够在黑暗和厌氧条件下分泌有机酸,如琥珀酸盐和乳酸盐(Osanai等,2015)。培养条件的遗传操作和修饰,例如添加钾或NaHCO 3,成功地提高了从集胞藻6803细胞排出的有机酸的水平(Osanai >等人,2015; Hasunuma等人,2016; Iijima等人,2016; Ueda等人。 ,2016)。由于琥珀酸和乳酸盐是用于制造各种材料(如生物塑料)的商品化学品,因此其生物基生产是减少环境负担的理想方式。在这里,我们描述了在黑暗,厌氧条件下孵育的集胞藻6803的方法,并通过用溴百里酚蓝后标记通过HPLC定量有机酸。

关键字:厌氧条件, 蓝细菌, HPLC, 乳酸盐, 有机酸, 琥珀酸, 集胞藻属

材料和试剂

  1. 50ml一次性聚丙烯管
  2. 试管(Iwaki,目录号:TEST30NP)
  3. 20 ml顶空瓶(GL Sciences,目录号:1030-46026)
  4. 22 G x 1"针(Terumo Medical,目录号:NN-2225R)
  5. 22 G x 1½"针(Terumo,目录号:NN-2238S)
  6. 铝箔
  7. SupraPure亲水性PVDF注射器过滤器(Recenttec,目录号:R7-PVDF033S022I)
  8. 目标DP小瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:C4000-1)
  9. 螺旋盖(GL Sciences,目录号:1030-45261)
  10. 300μl目标聚合物插入物(Thermo Fisher Scientific,Thermo Scientific TM,目录号:C4010-S630)
  11. 一次性注射器(Terumo,目录号:SS-10LZ)
  12. 开口式铝合金帽,带丁基橡胶隔垫(Systech,目录号:98552)
  13. MF-Millipore膜过滤器(Merck Millipore,目录号:HAWP03700)
  14. 蓝藻属集胞藻PCC 6803
  15. 氯化铵(NH 4 Cl)(Wako Pure Chemical Industries,目录号:017-02995)
  16. 60%高氯酸(HClO 4)(Wako Pure Chemical Industries,目录号:160-05755)
  17. 三氯乙酸
  18. 琥珀酸盐
  19. 乳酸盐
  20. 柠檬酸(Nacalai Tesque,目录号:09109-85)
  21. 醋酸盐
  22. 柠檬酸铁铵
  23. Na 2 EDTA(Nacalai Tesque,目录号:15111-45)
  24. 硝酸钠(NaNO 3)(Wako Pure Chemical Industries,目录号:195-02545)
  25. 磷酸氢二钾(K 2 N 2 HPO 4)(和光纯药工业公司,目录号:164-04295)
  26. 硫酸镁七水合物(MgSO 4·7H 2 O)(和光纯药,目录号:131-00405)
  27. 氯化钙脱水(CaCl 2·2H 2 O)(Wako Pure Chemical Industries,目录号:031-00435)
  28. 碳酸钠(Na 2 CO 3)(Nacalai Tesque,目录号:31311-25)
  29. 硼酸(H 3 BO 3/3)(和光纯药工业公司,目录号:021-02195)
  30. 四水合氯化锰(MnCl 2·4H 2 O)(Wako Pure Chemical Industries,目录号:139-00722)
  31. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Wako Pure Chemical Industries,目录号:264-00402)
  32. 五水合硫酸铜(CuSO 4·5H 2 O)(和光纯药,目录号:039-04412)
  33. 钼酸钠脱水(Na 2 MoO 4·2H 2 O)(和光纯药工业株式会社,目录号:196-02472) >
  34. (H 2 SO 3 SO 4)(和光纯药工业公司,目录号:199-15995)
  35. 硝酸钴六水合物,Co(NO 3 3)2·5H 2 O(和光纯药工业公司,目录号:031-03752)
  36. HEPES(Dojindo Melecular Technologies,目录号:342-01375)
  37. 氢氧化钾(KOH)(Wako Pure Chemical Industries,目录号:168-21815)
  38. 溴百里酚蓝(BTB)(Wako Pure Chemical Industries,目录号:027-03052)
  39. 乙醇(Wako Pure Chemical Industries,目录号:057-00451)
  40. 磷酸氢二钠十二水合物(Na 2 HPO 4·12H 2 O)(和光纯药工业公司,目录号:196-02835)
  41. 琥珀酸(和光纯药,目录号:190-04332)
  42. DL-乳酸锂盐(MP Biomedicals,目录号:100824)
  43. 乙酸钠(和光纯药,目录号:190-01071)
  44. 改良的BG-11液体介质(见配方)
  45. 流动相(3mM HClO 4)(参见食谱)
  46. 反应溶液(15mM Na 2 HPO 4中的0.2mM BTB)(参见食谱)
  47. 500mM琥珀酸溶液(参见食谱)
  48. 500 mM乳酸盐溶液(见配方)
  49. 500 mM柠檬酸溶液(见配方)
  50. 500 mM醋酸溶液(见食谱)
  51. 1 M醋酸溶液(见配方)

设备

  1. 栽培室(TOMY数字生物学,型号:CLE-303)
  2. 气体混合器(KOFLOC,型号:RK120XM)
  3. 空气泵(Yasunaga,型号:LP-30A)
  4. 分光光度计(Shimadzu,型号:UV-2700)
  5. 高速冷冻微量离心机(TOMY数字生物学,型号:MX-305)
  6. N 2发电机(Sanyo Denshi,型号:SN4-4)
  7. 旋转振动筛(Nissin,型号:NX-20D)
  8. 冷冻干燥机(东京Rikaikai,EYELA,型号:FDU-2200)
  9. 超声波清洗机(SND,型号:US-108)
  10. 高压釜
  11. HPLC系统(JASCO International,型号:LC-2000Plus)由以下设备组成:
    1. 光电二极管阵列(PDA)检测器(JASCO International,型号:MD-2018Plus)
    2. 智能型HPLC泵(JASCO International,型号:PU-2080Plus)
    3. 线式脱气机(JASCO International,型号:DG-2080-53)
    4. 三元梯度单位(JASCO International,型号:LG-2080-02)
    5. 智能立柱炉(JASCO International,型号:CO-2065Plus)
    6. 智能自动进样器(JASCO International,型号:AS-2057Plus)
    7. 离子排除柱(300×8.0mm)(GL Sciences,型号:RSpak KC-811,目录号:5055-13509)和保护柱(10×4.6mm)(GL Sciences,型号:GPC KF-G,目录号:5055-13150)
    8. 反应线圈单元(JASCO International,型号:RU-2080-51)

软件

  1. ChromNAV软件(Ver。1.14)

程序

  1. 光照培养,需氧(a)和黑暗培养,厌氧条件(b)
    1. 集胞藻的葡萄糖耐量菌株(称为GT)由Williams(1988)分离的PCC 6803在补充有5mM NH 3的BG-11 液体培养基(Rippka,1988)的改良的BG-11培养基(参见食谱)中培养> 4 Cl(用20mM HEPES-KOH缓冲,pH7.8)。在我们的研究中,使用了GT-I株(Kanesaki等人,2012)。使用气体混合器(RK120XM,KOFLOC)和空气泵(LP-30A)将GT细胞在含有1%(v/v)CO 2的空气中的液体改性BG-11培养基中培养, Yasunaga)以30-50ml/min的流速,在培养室(CLE-303,TOMY)中。将温度保持在30℃,在50-70μmol光子·m -2 sec -1连续供给白光。使用Shimadzu UV-2700分光光度计在 730 测量细胞密度。细胞培养在70ml改良的BG-11培养基(起始于730)中,在轻,有氧条件下培养3天。轻,有氧培养影响琥珀酸和乳酸的含量,每个实验室都需要优化培养条件。
    2. 通过以5,600×g离心离心收集细胞2分钟,并浓缩成10ml HEPES缓冲液(20mM HEPES-KOH,pH7.8)或修饰的BG-11以达到A 730 (图1)。将小瓶用带有丁基橡胶隔片的开放式铝盖密封,并通过隔膜刺穿两针(针22 G x 1"和针22 G x 1 1/2")。 22G×1 1/2"针连接到N 2气体发生器以引入N 2气体30-60分钟以产生厌氧条件。
    3. 在引入N 2气体后,取出针,并用铝箔包裹小瓶。将细胞在30℃下在旋转振荡器上以160rpm摇动孵育1-5天。 1天,琥珀酸盐和乳酸盐从细胞中排泄较少。在长时间的孵育期间,琥珀酸和乳酸水平升高,但是在3或4天达到最大值,并在5天开始下降(Osanai等,2015)。


      图1.用于厌氧培养的20ml小瓶。将细胞浓缩至10ml缓冲液或培养基中以达到730℃/ 20,然后用丁基橡胶帽密封小瓶。将两个注射器插入帽中,并通过两个注射器之一引入N 2气体30-60分钟。在引入N 2气体后,取出注射器,将小瓶包裹在铝箔中以保持黑暗状态。

  2. HPLC分析样品制备
    1. 孵育后,用开瓶器除去带有丁基橡胶的铝盖。细胞培养物以5,600×g离心2分钟。使用SupraPure无菌注射器过滤器,过滤上清液以除去碎屑
    2. 然后将1ml上清液转移到2ml塑料管中。将上清液置于烧瓶中,并在-80℃下用FDU-2200冷冻干燥器干燥1天。冷冻干燥的样品用100μl3mM高氯酸(HClO 4)分解(由于HClO 4高度腐蚀性,应小心处理)并离心在5,600×g下进行2分钟。
    3. 然后,使用玻璃插入物将100μl上清液转移到Target DP小瓶中。将其置于自动进样器进行HPLC分析。
    4. 在含有大量蛋白质的样品的情况下,冷冻干燥的样品应在97μl3mM HClO 4和3μl100%三氯乙酸中分解。充分混合,然后以20,400 x g离心样品10分钟。使用上清液进行HPLC分析
    5. 为了制备标准品,混合100μl500mM琥珀酸盐溶液,160μl500mM乳酸盐溶液,100μl500mM柠檬酸盐溶液,200μl1M乙酸盐溶液(参见Recipes)和440μldH 2 O组成总共1ml有机酸标准品。该解决方案命名为STD1。 STD1中每种有机酸的最终浓度为80mM乳酸盐,50mM琥珀酸盐,50mM柠檬酸盐和200mM乙酸盐。
    6. 稀释STD1以绘制分析曲线的浓度范围。
  3. HPLC分析
    有机酸的定量通过使用用BTB进行后标记的HPLC进行(图2)。使用3mM HClO 4溶液作为流动相和0.2mM BTB在15mM Na 2 HPO 4·4溶液中作为反应溶液。使用超声波清洗机和吸气器将流动相和反应溶液脱气约20分钟。 HPLC测量在以下条件下进行:
    1. 智能型HPLC泵(流动相):流量设定为0.7ml/min。该泵连接线路脱气器和三元梯度单元,并进一步连接到智能柱箱。样品注射前,用流动相平衡柱,流速相同,流速为1小时
    2. 智能型HPLC泵(反应溶液):流量设定为1.2ml/min
    3. 智能柱箱:柱温60°C
    4. 智能自动进样器:取样器温度为4°C,进样体积为20μl。要清理注射针,请使用HClO 4溶液。在样品注入前用HClO 4冲洗三次。
    5. 反应线圈单元用于混合流动相,包括样品和BTB溶液
    6. PDA:具有氘(D 2子)和卤素(W)灯的单光束光度计;使用200-900nm波长范围内的扫描,进行42分钟的单次数据分析。开始HPLC分析之前,请等待大约60分钟,直到流量稳定(基线没有漂移和噪音)

      图2.使用溴百里酚蓝标记后的HPLC分析示意图。
      两种HPLC泵分别用于流动相和BTB溶液。样品注射自动进样器并除以尺寸排阻柱,然后与BTB溶液混合。有机酸通过PDA检测器检测,并通过ChromNAV软件进行分析。

数据分析

通过HPLC测定的经BTB标记后的典型色谱图如图3所示。有机酸峰使用445nm处的吸光度进行定量。使用ChromNAV程序来量化每种有机酸的峰面积。通过用已知浓度样品的点标记来绘制每种有机酸的标准曲线。使用以下标准曲线计算样品中有机酸的浓度(图4)

图3.用标记后的溴百里酚蓝色的HPLC测量的典型色谱图。从波长445nm处的吸光度计算峰强度。每种有机酸的保留时间如下:柠檬酸盐17.55分钟,琥珀酸盐22.36分钟,乳酸盐23.80分钟,乙酸盐27.34分钟。


图4.琥珀酸(橙色)和乳酸(蓝色)的标准曲线。
通过整合每种有机酸的峰面积,从色谱图计算强度值。

笔记

在含有大量蛋白质的样品的情况下,冷冻干燥的样品应在97μl3mM HClO 4和3μl100%三氯乙酸中分解。充分混合,然后以20,400 x g离心样品10分钟。使用上清液进行HPLC分析。

食谱

  1. 改良的BG-11液体介质(室温下储存)
    1. 解决方案1(存储在4°C)
      称量0.6g柠檬酸,0.6g柠檬酸铁铵和0.1g Na 2 EDTA,并加入蒸馏水(dH 2 O 2)至200ml
    2. 解决方案2(存储在4°C)
      称取30g NaNO 3,0.78g K 2 HPO 4,和1.5g MgSO 4·7H 2 O,并将dH 2 O添加到1L
    3. 解决方案3(存储在4°C)
      称取3.8g CaCl 2·2H 2 O,并加入dH 2 O至200ml
    4. 解决方案4(存储在4°C)
      称取4g Na 2 CO 3,并加入dH 2 O至200ml
    5. 解决方案5(存储在4°C)
      称取2.86g H 3 BO 3,1.81g MnCl 2·4H 2 O,0.22g ZnSO 3 > 4 2 O,0.08g CuSO 4·5H 2 O,0.021g Na 2, sub> MoO 4,和50μlH 2 SO 4,并加入dH 2 O至1L( 1 *)
      称量0.0494g Co(NO 3 3)2< 2< 2> O并加入(1 *)
    6. 1 M HEPES-KOH,pH 7.8(储存于4°C)
      称量119.1 g HEPES并加入dH 2 O至约400 ml
      用6N KOH将pH调节至7.8,并加入dH 2 O至500ml
      高压灭菌器(121°C 15分钟),并保存在4°C
    7. K 2 2 HPO 4溶液(在4℃下储存)
      称取0.39g K 2 H 2 HPO 4,并加入dH 2 O 2至200ml
    8. MgSO 4·7H 2 O溶液(4℃保存)
      称量0.75g MgSO 4·7H 2 O,并加入dH 2 O至200ml
      混合20 ml 1M HEPES-KOH(pH 7.8),50ml溶液2,2ml溶液3,1ml溶液4和1ml溶液5,并加入dH 2 O至1L < br /> 高压灭菌混合物并在室温下储存 使用前,加入2ml溶液1至1L混合物
  2. 流动相(3mM HClO 4 )
    称量0.5g 60%HClO 4,并加入dH 2 O至1L
    用MF-Millipore膜过滤器过滤
  3. 反应溶液(15mM Na 2 HPO 4中的0.2mM BTB)
    称量125 mg BTB,加入10ml乙醇(1) 称取5.3g Na 2 HPO 4·12H 2 O,并加入到dH 2 O(2)
    混合(1)和(2),并将dH <2> O添加到1L
    用MF-Millipore膜过滤器过滤混合物
  4. 500mM琥珀酸溶液
    称量590.45mg琥珀酸盐并加入10ml dH 2 O,使用量瓶
  5. 500 mM乳酸盐溶液
    称量480.05mg DL乳酸锂盐,并使用量瓶将其加至10ml dH 2 O 3
  6. 500 mM柠檬酸溶液
    称量960.6mg柠檬酸盐并加入dH 2 O至10ml,使用测量瓶
  7. 1 M醋酸溶液
    称量820.3mg乙酸钠,并用量杯加入dH 2 O至10ml

致谢

这个协议是从以前的Ueda等人发表的研究中改编的。 (2016)和Iijima等人。 (2016)。这项工作得到日本教育,文化,体育,科技部的支持。来自日本科学技术厅(JSPS KAKENHI,创新地区科学研究资助项目名称("转基因调节剂和昼夜节律钟基因工程遗传工程蓝藻琥珀酸盐生产项目名称")(授权号16H06559 )。

参考

  1. Hasunuma,T.,Matsuda,M。和Kondo,A.(2016)。通过集胞藻生产提高无糖的琥珀酸生物 PCC 6803鉴定糖原分解代谢中的限制步骤。 Metab Eng Commun 3:130-141。
  2. Iijima,H.,Shirai,T.,Okamoto,M.,Pinto,F.,Tamagnini,P.,Hasunuma,T.,Kondo,A.,Hirai,M.,T.and Osanai,T。(2016) 。基于代谢组学的分析揭示了原代碳代谢的改变通过在集胞藻中的氢化酶HoxH的遗传操作。 PCC 6803. Algal Res 18:305-313。
  3. Kanesaki,Y.,Shiwa,Y.,Tajima,N.,Suzuki,M.,Watanabe,S.,Sato,N.,Ikeuchi,M.and Yoshikawa,H。(2012)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22193367"target ="_ blank">通过大规模平行的全基因组重排序鉴定亚型特异性突变 sp。 PCC 6803。 DNA Res 19(1):67-79。
  4. Osanai,T.,Shirai,T.,Iijima,H.,Nakaya,Y.,Okamoto,M.,Kondo,A.and Hirai,MY(2015)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/26500619"target ="_ blank">代谢酶和转录调节物的遗传操作增加了单细胞蓝细菌的琥珀酸盐排泄。微生物 6:1064。
  5. Rippka,R。(1988)。分离和纯化蓝细菌。 方法Enzymol 167:3-27。
  6. Ueda,S.,Kawamura,Y.,Iijima,H.,Nakajima,M.,Shirai,T.,Okamoto,M.,Kondo,A.,Hirai,MY and Osanai,T。(2016)一个class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27576448"target ="_ blank">阴离子代谢物生物合成在黑暗条件下增强,蓝藻中的厌氧条件。/a> 6:32354.
  7. Williams,JGK(1988)。  构建特定突变光合系统II光合反应中心通过遗传工程方法在集胞藻6803中。方法Enzymol 。 167:766-778。
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引用:Yasuda, C., Iijima, H., Sukigara, H. and Osanai, T. (2017). Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC. Bio-protocol 7(9): e2257. DOI: 10.21769/BioProtoc.2257.
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