光学学报, 2016, 36 (4): 0417001, 网络出版: 2016-04-05   

氮源影响PHB 合成代谢的拉曼光谱分析

Raman Spectral Profiles of PHB Synthesis Influenced by Different Nitrogen Sources
作者单位
1 广西科学院生物物理实验室, 广西 南宁 530007
2 广西大学行健文理学院, 广西 南宁 530004
3 广西职业技术学院食品与生物技术系, 广西 南宁 530226
4 美国东卡罗来那大学物理系, 北卡罗来那州格林维尔 NC27858
摘要
应用拉曼光谱学结合奇异值分解方法在单细胞尺度上对杀虫贪铜菌(C. necator) H16 菌株在不同的氮源下合成聚β-羟基丁酸(PHB)的代谢动态进行分析。结果显示,硫酸铵是PHB 发酵的理想氮源,对应PHB 发酵速度、效率和产量在测试氮源中表现最好。奇异值分解结果显示,源自RNA、DNA、蛋白质和PHB 的拉曼峰是发酵的主要特征,随着发酵进程的延伸,菌体细胞差异、产物含量差别逐渐增大。分析PHB 产物快速合成过程,可见表征核酸、蛋白质和PHB 的782、1574、1660、1732 cm-1 等峰的强度变化活跃;不同氮源下,782 cm-1 峰与1660 cm-1 峰的强度呈正相关,而1660 cm-1 峰与1732 cm-1 峰的强度呈负相关。因此,不同氮源可能影响细胞的RNA 代谢和蛋白质代谢,从而间接影响PHB 的合成。拉曼光谱结合数据挖掘技术可以分析微生物发酵过程中的代谢信息,从分子光谱的角度为寻找最佳的发酵条件提供新的信息。
Abstract
Laser tweezers Raman spectroscopy and singular value decomposition are used to explore the synthesis dynamics of biodegradable plastic poly- β- hydroxybutyrate(PHB) in C. necator H16 strain cultured with different nitrogen sources at the single- cell level. The results show that ammonium sulfate performing best on fermentation speed, efficiency and productivity in all tested nitrogen sources, is demonstrated as an ideal nitrogen source for PHB fermentation. The singular value decomposition results reveal that the main fermentation features are the Raman peaks belonging to RNA, DNA, protein and PHB, and the difference in bacterial cells and product yield becomes more notable as the fermentation process works. The dynamics of Raman intensity peaks of nucleic acid, protein and PHB at 782, 1574, 1660, 1732 cm-1 are active to the fast PHB synthesis, and no matter what nitrogen sources are used, the intensity of peaks at 1660 cm-1 and 782 cm-1 is positively correlated, but the intensity of 1660 cm-1 and 1732 cm- 1 peaks is negatively correlated. The results suggest that nitrogen sources influence the RNA and the protein metabolism and then affect the synthesis of PHB indirectly. Raman spectroscopy combining with data analysis can provide the metabolism information of the microbial fermentation and offer a powerful experiment basis for optimizing fermentation process.
参考文献

[1] Grage K, Jahns A C, Parlane N, et al.. Bacterial polyhydroxyalkanoate granules: Biogenesis, structure, and potential use as nano-/microbeads in biotechnological and biomedical applications[J]. Biomacromolecules, 2009, 10(4): 660-669.

[2] Keshavarz T, Roy I. Polyhydroxyalkanoates: Bioplastics with a green agenda[J]. Current opinion in microbiology, 2010, 13(3): 321-326.

[3] Chen G Q. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry[J]. Chemical Society Reviews, 2009, 38(8): 2434- 2446.

[4] Cramm R. Genomic view of energy metabolism in Ralstonia eutropha H16[J]. Journal of Molecular Microbiology and Biotechnology, 2009, 16(1-2): 38-52.

[5] Peoples O P, Sinskey A J. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16, identification and characterization of the PHB polymerase gene (phbC)[J]. The Journal of Biological Chemistry, 1989, 264(26): 15298-15303.

[6] Peplinski K, Ehrenreich A, D ring C, et al.. Genome-wide transcriptome analyses of the ′Knallgas′ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism[J]. Microbiology, 2010, 156(Pt 7): 2136-2152.

[7] Yoshie N, Goto Y, Sakurai M, et al.. Biosynthesis and n.m.r. studies of deuterated poly (3-hydroxybutyrate) produced by Alcaligenes eutrophus H16[J]. International Journal of Biological Macromolecules, 1992, 14(2): 81-86.

[8] Kobayashi T, Shiraki M, Abe T, et al.. Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly (3-hydroxybutyrate) depolymerase[J]. Journal of Bacteriology, 2003, 185(12): 3485-3490.

[9] Tian J, Sinskey A J, Stubbe J. Kinetic studies of polyhydroxybutyrate granuleformation in Wautersia eutropha H16 by transmission electron microscopy[J]. Journal of Bacteriology, 2005, 187(11): 3814-3824.

[10] Eggers J, Steinbuchel A. Poly (3-hydroxybutyrate) degradation in Ralstonia eutropha H16 is mediated stereoselectively to (S)-3- hydroxybutyryl coenzyme A (CoA) via crotonyl-CoA[J]. Journal of Bacteriology, 2013, 195(14): 3213-3223.

[11] Batcha A F M, Prasad D M R, Khan M R, et al.. Biosynthesis of poly (3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 from jatropha oil as carbon source[J]. Bioprocess and Biosystems Engineering, 2013, 37(5): 943-951.

[12] Sznajder A, Pfeiffer D, Jendrossek D. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16[J]. Applied and Environmental Microbiology, 2015, 81(5): 1847-1858.

[13] Jarute G, Kainz A, Schroll G, et al.. On-line determination of the intracellular poly (β-hydroxybutyric acid) content in transformed Escherichia coli and glucose during PHB production using stopped-flow attenuated total reflection FT-IR spectrometry[J]. Analytical Chemistry, 2004, 76(21): 6353-6358.

[14] Khanna S, Srivastava A K. On-line characterization of physiological state in poly (β-hydroxybutyrate) production by Wautersia eutropha [J]. Applied Biochemistry and Biotechnology, 2009, 157(2): 237-243.

[15] Brehm-Stecher B F, Johnson E A. Single-cell microbiology: Tools, technologies, and applications[J]. Microbiology and Molecular Biology Reviews, 2004, 68(3): 538-559.

[16] Peng L, Wang G, Liao W, et al.. Intracellular ethanol accumulation in yeast cells during aerobic fermentation: A Raman spectroscopic exploration[J]. Letters in Applied Microbiology, 2010, 51(6): 632-638.

[17] 李自达, 赖钧灼, 廖威, 等. 浓醪乙醇发酵的单细胞拉曼光谱表征[J]. 光学学报, 2012, 32(3): 0317001.

    Li Zida, Lai Junzhuo, Liao Wei, et al.. Raman spectroscopic profile of ethanol fermentation in high gravity cassava starch brewing[J]. Acta Optica Sinica, 2012, 32(3): 0317001.

[18] 覃赵军, 赖钧灼, 刘斌, 等. 不同初始pH 值的乙醇发酵过程拉曼光谱分析[J]. 中国激光, 2013, 40(2): 0215001.

    Qin Zhaojun, Lai Junzhuo, Liu Bin, et al.. Raman spectroscopic analysis of ethanol fermentation at various initial pH levels[J]. Chinese J Lasers, 2013, 40(2): 0215001.

[19] 覃赵军, 赖钧灼, 彭立新, 等. 拉曼光谱分析有机氮源促进乙醇发酵的机制[J]. 分析化学, 2014, 42(10): 1471-1477.

    Qin Zhaojun, Lai Junzhuo, Peng Lixin, et al.. Raman spectral profiles of promoting effects of organic nitrogen sources on ethanol fermentation using Saccharomyces cerevisiae[J]. Chinese Journal of Analytical Chemistry 2014, 42(10): 1471-1477.

[20] Chan J W. Recent advances in laser tweezers Raman spectroscopy (LTRS) for label-free analysis of single cells[J]. Journal of Biophotonics, 2013, 6(1): 36-48.

[21] Li M Q, Xu J, Romero-Gonzalez M E, et al.. Single cell Raman spectroscopy for cell sorting and imaging[J]. Current Opinion in Biotechnology, 2011, 23(1): 56-63.

[22] Gelder J D, Willemse-Erix D, Scholtes M J, et al.. Monitoring poly (3-hydroxybutyrate) production in Cupriavidus necator DSM 428 (H16) with Raman spectroscopy[J]. Analytical Chemistry, 2008, 80(6): 2155-2160.

[23] Hermelink A, St mmler M, Naumann D. Observation of content and heterogeneity of poly-β-hydroxybutyric acid (PHB) in Legionella bozemanii by vibrational spectroscopy[J]. Analyst, 2011, 136(6): 1129-1133.

[24] 覃赵军, 彭立新, 竺利波, 等. 碳源浓度影响微生物PHB合成代谢的单细胞拉曼光谱分析[J]. 中国激光, 2015, 42(3): 0315003.

    Qin Zhaojun, Peng Lixin, Zhu Libo, et al.. Raman spectral profiles of PHB synthesis by Cupriavidus necator H16 at different fructose levels[J]. Chinese J Lasers, 2015, 42(3): 0315003.

[25] Moritz T J, Taylor D S, Polage C R, et al.. Effect of cefazolin treatment on the nonresonant Raman signatures of the metabolic state of individual Escherichia coli cells[J]. Analytical Chemistry, 2010, 82(7): 2703-2710.

[26] De Gelder J, De Gussem K, Vandenabeele P, et al.. Methods for extracting biochemical information from bacterial Raman spectra: Focus on a group of structurally similar biomolecules--fatty acids[J]. Analytica Chimica Acta, 2007, 603(2): 167-175.

[27] De Gelder J, De Gussem K, Vandenabeele P, et al.. Methods for extracting biochemical information from bacterial Raman spectra: An explorative study on Cupriavidus metallidurans[J]. Analytica Chimica Acta, 2007, 585(2): 234-240.

[28] Hall E K, Singer G A, P lzlM, et al.. Looking inside the box: Using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time[J]. ISME Journal, 2011, 5(2): 196-208.

[29] 赵海霞, 袁丁, 何毓敏, 等. 3,5-二硝基水杨酸比色法测定竹节参中多糖的含量[J]. 食品工业科技, 2010, 31(6): 327-329.

    Zhao Haixia, Yuan Ding, He Yumin, et al.. Quantitative determination of Panax japonicus polysaccharide by DNS method[J]. Science and Technology of Food Industry, 2010, 31(6): 327-329.

[30] Xie C G, Dinno M A, Li Y Q. Near-infrared Raman spectroscopy of single optically trapped biological cells[J]. Optics Letters, 2002, 27 (27): 249-251.

[31] 黄庶识, 卢明倩, 李冰, 等. 重组大肠杆菌表达可溶性蛋白和包涵体过程的拉曼光谱实时分析[J]. 中国激光, 2014, 41(12): 1215003.

    Huang Shushi, Lu Mingqian, Li Bing, et al.. Real-time detection on the expression of soluble protein and inclusion body in the recombinant Escherichia coli with laser tweezers Raman spectroscopy[J]. Chinese J Lasers, 2014, 41(12): 1215003.

[32] De Gelder J, De Gussem K, Vandenabeele P, et al.. Reference database of Raman spectra of biological molecules[J]. Journal of Raman Spectroscopy, 2007, 38(9): 1133-1147.

[33] Movasaghi Z, Rehman S, Rehman I U. Raman spectroscopy of biological tissues[J]. Applied Spectroscopy Reviews, 2007, 42(5): 493-541.

[34] Furukawa T, Sato H, Murakami R, et al.. Raman microspectroscopy study of structure, dispersibility, and crystallinity of poly (hydroxybutyrate) /poly (l-lactic acid) blends[J]. Polymer, 2006, 47(9): 3132-3140.

[35] Izumi C M S, Temperini M L A. FT- Raman investigation of biodegradable polymers: Poly (3- hydroxybutyrate) and poly (3- hydroxybutyrate-co-3-hydroxyvalerate)[J]. Vibrational Spectroscopy, 2010, 54(2): 127-132.

[36] Maguire A, Vega Carrascal I, White L, et al.. Competitive evaluation of data mining algorithms for use in classification of leukocyte subtypes with Raman microspectroscopy[J]. Analyst, 2014, 140(7): 2473-2481.

[37] Nejadgholi I, Caytak H, Bolic M, et al.. Preprocessing and parameterizing bioimpedance spectroscopy measurements by singular value decomposition[J]. Physiological Measurement, 2015, 36(5): 983-999.

[38] Khmaladze A, Jasensky J, Price E, et al.. Hyperspectral imaging and characterization of live cells by broadband coherent anti-Stokes Raman scattering (CARS) microscopy with singular value decomposition (SVD) analysis[J]. Applied Spectroscopy, 2014, 68(10): 1116- 1122.

覃赵军, 陶站华, 廖威, 陈真英, 黎永青, 王桂文. 氮源影响PHB 合成代谢的拉曼光谱分析[J]. 光学学报, 2016, 36(4): 0417001. Qin Zhaojun, Tao Zhanhua, Liao Wei, Chen Zhenying, Li Yongqing, Wang Guiwen. Raman Spectral Profiles of PHB Synthesis Influenced by Different Nitrogen Sources[J]. Acta Optica Sinica, 2016, 36(4): 0417001.

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