生物转化合成N-乙酰神经氨酸的关键因素
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摘要
在前期工作中已构建了一株能以外源N-乙酰氨基葡萄糖(GlcNAc)和胞内磷酸烯醇式丙酮酸(PEP)为底物,生物转化合成N-乙酰神经氨酸(Neu5Ac)的重组大肠杆菌,但其合成Neu5Ac的效率有待进一步提高。通过调整生物转化阶段培养基中氮源和碳源的组成,探寻Neu5Ac合成过程中的关键限制性因素。结果表明,增加唾液酸合成酶NeuB活性的措施没有提高Neu5Ac的产率,而底物之一胞内PEP的供给不足是限制Neu5Ac合成的关键性因素。通过限制转化培养基中的氮源,抑制重组大肠杆菌的生长,避免了PEP被用于细胞自身生长而更多地流向Neu5Ac合成方向;通过改用不依赖PTS跨膜转运系统的甘油作为碳源,在甘油/无氮转化培养基中Neu5Ac合成量最终达到(4.42±0.08) g/L,比初始合成条件高出10.3倍。
We constructed a recombinant Escherichia coli which could synthesize Neu5Ac with exogenous GlcNAc as sole substrate in previous work. Various nitrogen and carbon sources were used to investigate their effects on the production of Neu5Ac,and the key limiting factors in fluencing the whole process were revealed. It was shown that the increase of NeuB activity could not improve the Neu5Ac yield and the NeuB activity was not the limiting factor. The intracellular PEP was supposed to be the limiting factor for Neu5Ac production. The intracellular PEP was saved and shifted to the synthesis of Neu5Ac by adjusting the nitrogen source and restraining the cell growth. In addition,by using glycerol as sole carbon source which was PTS-independent,Neu5Ac synthesis in the glycerol/nitrogen-free medium reached(4.42 ±0.08) g/L,10.3 times higher than the initial synthesis conditions.
We constructed a recombinant Escherichia coli which could synthesize Neu5Ac with exogenous GlcNAc as sole substrate in previous work. Various nitrogen and carbon sources were used to investigate their effects on the production of Neu5Ac,and the key limiting factors in fluencing the whole process were revealed. It was shown that the increase of NeuB activity could not improve the Neu5Ac yield and the NeuB activity was not the limiting factor. The intracellular PEP was supposed to be the limiting factor for Neu5Ac production. The intracellular PEP was saved and shifted to the synthesis of Neu5Ac by adjusting the nitrogen source and restraining the cell growth. In addition,by using glycerol as sole carbon source which was PTS-independent,Neu5Ac synthesis in the glycerol/nitrogen-free medium reached(4.42 ±0.08) g/L,10.3 times higher than the initial synthesis conditions.
引文
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[2]VARKI A.Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins[J].Nature,2007,446(7139):1023-1029.
[3]ZHENG Zhiyong,ZHAN Xiaobei,WU Jianrong,et al.Structure analysis of polysialic acid in Escherichia coli[J].Journal of Food Science and Biotechnology,2005,24(5):38-41.(in Chinese)
[4]WANG B,BRAND-MILLER J.The role and potential of sialic acid in human nutrition[J].European Journal of Clinical Nutrition,2003,57(11):1351-1369.
[5]LEE Y C,WU H M,CHANG Y N,et al.The central cavity from the(Alpha/Alpha)6 barrel structure of Anabaena sp.CH1N-acetyl-d-glucosamine 2-epimerase contains two key histidine residues for reversible conversion[J].Journal of Molecular Biology,2007,367(3):895-908.
[6]LEE Y C,CHIEN H C R,HSU W H.Production of N-acetyl-d-neuraminic acid by recombinant whole cells expressing Anabaena sp.CH1 N-acetyl-d-glucosamine 2-epimerase and Escherichia coli N-acetyl-d-neuraminic acid lyase[J].Journal of Biotechnology,2007,129(3):453-460.
[7]LINTON D,KARLYSHEV A V,HITCHEN P G,et al.Multiple N-acetyl neuraminic acid synthetase(neuB)genes in Campylobacter jejuni:identification and characterization of the gene involved in sialylation of lipo-oligosaccharide[J].Molecular Microbiology,2000,35(5):1120-1134.
[8]SUNDARAM A K,PITTS L,MUHAMMAD K,et al.Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni[J].Biochemical Journal,2004,383:83-89.
[9]BARDFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[10]BENNETT B D,KIMBALL E H,GAO M,et al.Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli[J].Nature Chemical Biology,2009,5(8):593-599.
[11]UEHARA T,PARK J T.The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling[J].Journal of Bacteriology,2004,186(21):7273-7279.
[12]LI Jing,XU Min,ZHU Li,et al.Effect of dissolved oxygen level on the rheological properties of curdlan fermented by Agrobacterium sp.ATCC 31749[J].Journal of Food Science and Biotechnology,2013,32(12):1253-1260.(in Chinese)
[13]LEQUEUX G,BEAUPREZ J,MAERTENS J,et al.Dynamic metabolic flux analysis demonstrated on cultures where the limiting substrate is changed from carbon to nitrogen and vice versa[J].Journal of Biomedicine and Biotechnology,2010(10):103-105.
[14]JIANG L,LI S,HU Y,et al.Adaptive evolution for fast growth on glucose and the effects on the regulation of glucose transport system in Clostridium tyrobutyricum[J].Biotechnology and Bioengineering,2012,109(3):708-718.
[15]CLOMBURG J M,GONZALEZ R.Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol[J].Biotechnology and Bioengineering,2011,108(4):867-879.
[16]LIN B X,ZHANG Z J,LIU W F,et al.Enhanced production of N-acetyl-D-neuraminic acid by multi-approach whole-cell biocatalyst[J].Applied Microbiology and Biotechnology,2013,97(11):4775-4784.