大肠杆菌K235 能量代谢扰动影响聚唾液酸合成的研究

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.030

摘要/Abstract

摘要： 通过不同装液量、不同氧化还原状态的碳源和添加NAD⁺的前体烟酸来调控大肠杆菌胞内的能荷、还原力水平，研究胞内核苷酸和聚唾液酸合成之间的关系。结果表明，聚唾液酸合成量越高，胞内UTP含量越低、UMP含量越高；同时，合成UTP、CTP消耗大量ATP使胞内能荷水平较低。与其他碳源的发酵相比，以山梨醇为碳源时，由于胞内NADH水平的提高使菌体合成的聚唾液酸量最高。添加烟酸可以提高胞内的NAD⁺水平，导致胞内的氧化水平提高，从而使胞内还原产物如聚唾液酸、琥珀酸、乳酸的产量降低甚至消失。

关键词: 大肠杆菌, 聚唾液酸, 能荷, 核苷酸, 氧化还原势

Abstract: Different medium volumes, carbon sources with varied redox states and adding nicotinic acid, and the precursor of NAD⁺ were used to adjust the intracellular energy charge and reduction of Escherichia coli in order to investigate the relationships between biosynthesis of polysialic acid and intracellular nucleotides. The results showed that the higher the production of polysialic acid was, the lower UTP was, but higher UMP was. Meanwhile, energy charge level was lower because the synthesis of UTP and CTP consumed a lot of ATP. Using sorbitol as carbon source, more polysialic acid was produced than those of fermentation with glucose, xylose and sodium gluconate due to the increase of NADH. Addition of nicotinic acid to the fermentation medium elevated intracellular NAD⁺, which led to the raise of intracellular oxidative stress, sequentially the decrease of the reducing metabolites such as polysialic acid, succinate and lactate, and even the vanish of them.

Key words: Escherichia coli, polysialic acid, energy charge, nucleotides, redox potential

闫霞, 吴剑荣, 郑志永, 詹晓北. 大肠杆菌K235 能量代谢扰动影响聚唾液酸合成的研究[J]. 生物技术通报, 2015, 31(9): 209-217.

Yan Xia, Wu Jianrong, Zheng Zhiyong, Zhan Xiaobei. Influence of Perturbation of Energy Metabolism in Escherichia coli K235 on Biosynthesis of Polysialic Acid[J]. Biotechnology Bulletin, 2015, 31(9): 209-217.

参考文献

[1] Rodriguez-Aparicio LB, Reglero A, Ortiz AI, et al. Effect of physical and chemical conditions on the production of colominic acid by Escherchia coli in a defined medium[J]. Applied Microbiology and Biotechnology, 1988, 27(5-6)：474-483.
[2] Barker SA, Jones RG, Somers PJ. Improvements in the production and isolation of colominic acid[J]. Carbohydrate Research, 1967, 3(3)：369-376.
[3] Inoue S, Inoue Y. Development profile of neural cell adhesion molecule glycoforms with a varying degree of polymerization of polysialic acid chains[J]. The Journal of Biological Chemistry, 2001, 276(34)：31863-31870.
[4] Nakata D, Troy FA. Degree of polymerization(DP)of polysialic acid(polySia)on neural cell adhesion molecules(N-CAMS)：development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS[J]. The Journal of Biological Chemistry, 2005, 280(46)：38305-38316.
[5] Haile Y, Berski S, Drager G, et al. The effect of modified polysialic acid based hydrogels on the adhesion and viability of primary neurons and glial cells[J]. Biomaterials, 2008, 29(12)：1880-1891.
[6] Chen C, Constantinou A, Chester KA, et al. Glycoengineering approach to half-Life extension of recombinant biotherapeutics[J]. Bioconjugate Chemistry, 2012, 23(8)：1524-1533.
[7] Camino CC, Maria LJ. High production of polysialic acid by Escherichia coli K-92 grow in a chemically defined medium regulated by temperature[J]. Biological Chemistry Hoppe-Seyler, 1990, 371(11)：1101-1106.
[8] 郭良栋, 钱世钧, 叶军, 等. 产多聚唾液酸的菌种筛选及产酸条件[J]. 微生物学报, 1998, 38(2)：103-107.
[9] 贾薇. 聚唾液酸生产菌的诱变育种及发酵研究[D]. 无锡：无锡轻工大学, 1999.
[10]Rode B, Endres C, Ran C, et al. Large-scale production and homogenous purification of long chain polysialic acids from E. coli K1[J]. Journal of Bacteriology, 2008, 135(2)：202-209.
[11]于军华. 聚唾液酸生产菌种的选育及其发酵工艺的研究[D]. 无锡：无锡轻工大学, 2000.
[12]张琦. 聚唾液酸发酵和分离纯化工艺的研究[D]. 无锡：江南大学, 2009.
[13]Berrios-Rivera SJ, San KY, Bennett GN. The effect of NAPRTase overexpression on the total levels of NAD, the NADH/NAD(+)ratio, and the distribution of metabolites in Escherichia coli[J]. Metabolic Engineering, 2002, 4(3)：238-247.
[14] Heuser F, Schroer K, Lutz S, et al. Enhancement of the NAD(P)(H)pool in Escherichia coli for biotransformation[J]. Engineering in Life Sciences, 2007, 7(4)：343-353.
[15]Geertman JM, van Maris AJ, van Dijken JP, et al. Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production[J]. Metabolic Engineering, 2006, 8(6)：532-542.
[16]Zhang YP, Li Y, Du CY, et al. Inactivation of aldehyde dehydrogenase：A key factor for engineering 1, 3-propanediol production by Klebsiella pneumoniae[J]. Metabolic Engineering, 2006, 8(6)：578-586.
[17]Hou J, Lages NF, Oldiges M, et al. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2009, 11(4-5)：253-261.
[18]Zhang Y, Huang Z, Du C, et al. Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol[J]. Metabolic Engineering, 2009, 11(2)：101-106.
[19]de Graef MR, Alexeeva S, Snoep JL, et al. The steady-state internal redox state(NADH/NAD+)reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli[J]. Journal of Bacteriology, 1999, 181(8)：2351-2357.
[20]Liu LM, Li Y, Shi ZP, et al. Enhancement of pyruvate productivity in Torulopsis glabrata：Increase of NAD⁽⁺⁾availability[J]. Journal of Bacteriology, 2006, 126(2)：173-185.
[21]Lin H, Bennett GN, San KY. Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli[J]. Journal of Industrial Microbiology and Biotechnology, 2005, 32(3)：87-93.
[22]Svennerholm L. Quantitative estimation of sialic acids[J]. Biochimica et Biophysica Acta, 1957, 24(4)：604-611.
[23]Hong SH, Lee SY. Importance of redox balance on the production of succinic acid by metabolically engineered Escherichia coli[J]. Applied Microbiology and Biotechnology, 2002, 58：286-290.
[24]San KY, Bennett GN, Berrios-Rivera SJ, et al. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli[J]. Metabolic Engineering, 2002, 4：182-192.
[25]Liu XL, Lin J, Hu HF, et al. Metabolic engineering of Escherichia coli to enhance shikimic acid production from sorbitol[J]. World Journal of Microbiology and Biotechnology, 2014, 30(9)：2543-2550.
[26]Wimpenn, JWT, Firth A. Levels of nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide in facultative bacteria and the effect of oxygen[J]. Journal of Bacteriology, 1972, 111(1)：24-32.
[27]Matin A, Gottschal JC. Influence of dilution rate on NAD(P)and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture[J]. Journal of General Microbiology, 1976, 94(2)：333-341.
[28]Berr?os-Rivera SJ, Bennett GN, San KY, et al. Metabolic engineering of Escherichia coli：increase of NADH availability by overexpressing an NAD⁺-dependent formate dehydrogenase[J]. Metabolic Engineering, 2002, 4(3)：217-229.