1.Effect of overexpression of nicotinic acid mononucleotide adenylyltransferase on succinic acid production in Escherichia coli NZN111.
Dongmei GOU ; Liya LIANG ; Rongming LIU ; Changqing ZHANG ; Mingke WU ; Jiangfeng MA ; Kequan CHEN ; Jianguo ZHU ; Min JIANG
Chinese Journal of Biotechnology 2012;28(9):1059-1069
Escherichia coli NZN111 is a promising strain with ldhA and pflB genes inactivated for the production of succinic acid. However, with these mutations, NAD+ could not be regenerated from NADH, and an unbalanced NADH/NAD+ ratio eliminated cell growth and glucose utilization under anaerobic conditions. Nicotinic acid mononucleotide adenylyltransferase (NAMNAT), encoded by the nadD gene, catalyzes the reaction from nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) during the synthetic pathway of NAD(H). Overexpression of the nadD gene could enhance the concentration of NAD(H) and maintain a suitable NADH/NAD+ ratio. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-nadD, and overexpressed NAMNAT with 1.0 mmol/L of IPTG under anaerobic conditions in sealed bottles. Compared to E. coli NZN111, the concentrations of NAD+ and NADH in the recombinant strain increased by 3.21-fold and 1.67-fold, respectively. The total concentration of NAD(H) was increased by 2.63-fold, and the ratio of NADH/NAD+ decreased from 0.64 to 0.42. The recombinant strain restored the cell growth and glucose utilization under anaerobic conditions. After 72 h, the recombinant strain could consume 14.0 g/L of glucose to produce 6.23 g/L of succinic acid, and the concentration of succinic acid was 19-fold higher than in E. coli NZN111.
Anaerobiosis
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Escherichia coli
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genetics
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metabolism
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Glucose
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metabolism
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Mutation
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NAD
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metabolism
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Nicotinamide-Nucleotide Adenylyltransferase
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genetics
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metabolism
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Recombinant Proteins
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genetics
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metabolism
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Succinic Acid
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metabolism
2.Nicotinamide mononucleotide adenylyltransferase 1 gene NMNAT1 regulates neuronal dendrite and axon morphogenesis in vitro.
Hong ZHAO ; Jing-Yu ZHANG ; Zi-Chao YANG ; Ming LIU ; Bao-Zhi GANG ; Qing-Jie ZHAO
Chinese Medical Journal 2011;124(20):3373-3377
BACKGROUNDWallerian degeneration is a self-destructive process of axonal degeneration that occurs after an axonal injury or during neurodegenerative disorders such as Parkinson's or Alzheimer's disease. Recent studies have found that the activity of the nicotinamide adenine dinucleotide (NAD) synthase enzyme, nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) can affect the rate of Wallerian degeneration in mice and drosophila. NMNAT1 protects neurons and axons from degeneration. However, the role of NMNAT1 in neurons of central nervous system is still not well understood.
METHODSWe set up the culture of primary mouse neurons in vitro and manipulated the expression level of NMNAT1 by RNA interference and gene overexpression methods. Using electroporation transfection we can up-regulate or down-regulate NMNAT1 in cultured mouse dendrites and axons and study the neuronal morphogenesis by immunocytochemistry. In all functional assays, FK-866 (CAS 658084-64-1), a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase was used as a pharmacological and positive control.
RESULTSOur results showed that knocking down NMNAT1 by RNA interference led to a marked decrease in dendrite outgrowth and branching and a significant decrease in axon growth and branching in developing cortical neurons in vitro.
CONCLUSIONSThese findings reveal a novel role for NMNAT1 in the morphogenesis of developing cortical neurons, which indicate that the loss of function of NMNAT1 may contribute to different neurodegenerative disorders in central nervous system.
Animals ; Axons ; metabolism ; Blotting, Western ; Cells, Cultured ; Dendrites ; metabolism ; Immunohistochemistry ; Mice ; Morphogenesis ; genetics ; physiology ; Neurons ; cytology ; metabolism ; Nicotinamide-Nucleotide Adenylyltransferase ; genetics ; metabolism
3.Construction of recombinant lentiviral vectors for mouse NMNAT1 gene expression and its interference RNA.
Hong ZHAO ; Zi-chao YANG ; Jing-yu ZHANG
Journal of Zhejiang University. Medical sciences 2011;40(6):622-629
OBJECTIVETo construct two recombinant lentiviral vectors carrying mouse NMNAT1 gene and RNAi targeting NMNAT1.
METHODSAccording to GenBank, the full-length cDNA sequence of mouse NMNAT1, an interfering sequence targeting NMNAT1 and a negative sequence were designed, synthesized and inserted into plasmid pLenti6 lentiviral vector. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. The virus titer was tested by qPCR methods. After infection of Hela cells with these lentiviruses, the expression of NMNAT1 was detected by qPCR and Western blot.
RESULTSAll the recombinant plasmids were confirmed by sequencing. The titer of virus was over 2 X10(8) TU/mL. Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1. The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence.
CONCLUSIONThe lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NMNAT1 have been successfully constructed.
Animals ; Gene Expression ; Genetic Vectors ; HeLa Cells ; Humans ; Lentivirus ; genetics ; Mice ; Nicotinamide-Nucleotide Adenylyltransferase ; genetics ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection
4.Wld(S), Nmnats and axon degeneration--progress in the past two decades.
Yan FENG ; Tingting YAN ; Zhigang HE ; Qiwei ZHAI
Protein & Cell 2010;1(3):237-245
A chimeric protein called Wallerian degeneration slow (Wld(S)) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld(S) attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld(S) is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld(S), indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.
Animals
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Axons
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physiology
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Humans
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Mice
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Mice, Mutant Strains
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Models, Neurological
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Mutant Proteins
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genetics
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physiology
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Mutation
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NAD
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biosynthesis
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Nerve Degeneration
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etiology
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genetics
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physiopathology
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Nerve Tissue Proteins
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genetics
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physiology
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Nicotinamide-Nucleotide Adenylyltransferase
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genetics
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physiology