Expression of Vitreosicilla Hemoglobin Gene(vgb) In Pseudomonas denitrificans and the Central Carbon Metabolic Flux Analysis on Vitamin B12 Production

doi:10.13523/j.cb.20160903

China Biotechnology

2016, Vol. 36

Issue (9): 21-30 DOI: 10.13523/j.cb.20160903

Expression of Vitreosicilla Hemoglobin Gene(vgb) In Pseudomonas denitrificans and the Central Carbon Metabolic Flux Analysis on Vitamin B₁₂ Production

SHI Hui-lin¹, WANG Ze-jian¹, WU Jie-qun², GUO Mei-jin¹, CHU Ju¹, ZHUANG Ying-ping¹

1 State Key Laboratory of Bioreactor Engineering, National Engineering Research Center for Biotechnology, East China University of Science and Technology, Shanghai 200237, China;
2 Shanghai Insitiutute of Organic Chemistry Huzhou Center of Bio-synthetic Innovation, Huzhou 333000, China

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Abstract

Based on the establishment of the conditions of conjugational transfer of Pseudomonas denitrificans, vgb gene was successfully integrated into the chromosome of P. denitrificans via single crossover to obtain vgb recombinant strain Pvgb-16, and its effects on the carbon center metabolic flux and vitamin B₁₂ biosynthesis were illuminated through the ¹³C isotope labeling experiment. The results demonstrated that the recombinant strain with vgb gene had the higher specific growth rate and specific vitamin B₁₂ production rate, which was increased by 22% and 52% respectively, compared to the original strain under the same oxygen supply condition. In addition, cultivations were carried out with 20% or 20% glucose as substrates, the labeling patterns of the proteinogenic amino acids were used to accurately estimate the fluxes in the central carbon metabolism of P. denitrificans. The results showed that the glucose consumption rate of recombinant strain was enhanced for 14.2%, and the further carbon metabolic flux distribution analysis revealed that the improvement of the PP pathway for higher NADPH generation and the biosynthesis rate of glycine from betaine for higher synthesis of precursor aminolevulinic acid greatly enhanced vitamin B₁₂ biosynthesis rate and productivity in the vgb recombinant strain. It can be concluded that the expression of vgb gene in P. denitrificans can effectively promote the cell growth rate, the accumulation rate and yield of vitamin B₁₂ production, which would be of great significance for the further research and application in industrial fermentation.

Key words： Pseudomonas denitrificans Vitamin B₁₂ vgb gene Central carbon metabolic flux analysis

Received: 06 June 2016 Published: 25 September 2016

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SHI Hui-lin, WANG Ze-jian, WU Jie-qun, GUO Mei-jin, CHU Ju, ZHUANG Ying-ping. Expression of Vitreosicilla Hemoglobin Gene(vgb) In Pseudomonas denitrificans and the Central Carbon Metabolic Flux Analysis on Vitamin B₁₂ Production. China Biotechnology, 2016, 36(9): 21-30.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20160903 OR https://manu60.magtech.com.cn/biotech/Y2016/V36/I9/21

[1] 马蕙, 王丽丽, 张春晓, 等. 维生素B12的生物合成, 发酵生产与应用. 生物工程学报. 2008, 24(6):927-932. Ma H, Wang L L, Zhang C X, et al. The biosynthesis, fermentation and application of vitamin B₁₂. Chinese Journal of Biotechnology, 2008, 24(6):927-932.
[2] Li K T, Liu D H, Zhuang Y P, et al. Influence of Zn²⁺, Co²⁺ and dimethylbenzimidazole on vitamin B12 biosynthesis by Pseudomonas denitrificans. World Journal of Microbiology and Biotechnology, 2008, 24(7):2525-2530.
[3] Li K T, Liu D H, Chu J, et al. An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans. Bioprocess and Biosystems Engineering, 2008, 31(6):605-610.
[4] Wang Z J, Wang H Y, Li Y L, et al. Improved vitamin B12 production by step-wise reduction of oxygen uptake rate under dissolved oxygen limiting level during fermentation process. Bioresource Technology, 2010, 101(8):2845-2852.
[5] Pablos T E, Mora E M, Borgne S L, et al. Vitreoscilla hemoglobin expression in engineered Escherichia coli:improved performance in high cell-density batch cultivations. Biotechnolology Journal, 2011, 6(8):993-1002.
[6] Stark B C, Pagilla K R, Dikshit K L. Recent applications of Vitreosicilla hemoglobin technology in bioproduct synthesis and bioremediation. Applied Microbiology and Biotechnology, 2015, 99(4):1627-1636.
[7] Shen X W, Yang Y, Jian J, et al. Production and characterization of homopolymer poly (3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4. Bioresource Technology, 2009, 100(18):4296-4299.
[8] Zhu H, Sun S, Zhang S. Enhanced production of total flavones and exopolysaccharides via Vitreoscilla hemoglobin biosynthesis in Phellinus igniarius. Bioresource Technology, 2011, 102(2):1747-1751.
[9] Li X, Peng R H, Fan H Q. Vitreoscilla hemoglobin overexpression increases submergence tolerance in cabbage. Plant Cell Reports, 2005, 23(10):710-715.
[10] Pablos T E, Sigala J C, Borgne S L, et al. Aerobic expression of Vitreoscilla hemoglobin efficiently reduces overflow metabolism in Escherichia coli. Biotechnology Journal, 2014, 9(6):791-799.
[11] Ramachandran B, Dikshit K L, Dharmalingam K. Recombinant E. coil expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions:a proteome-level study. Journal of Biosciences, 2012, 37(4):617-633.
[12] 王泽建. 宏观和微观代谢分析相结合的系统生物过程研究-微生素B₁₂发酵过程优化. 上海:华东理工大学,生物工程学院,2010. Wang Z J. Optimization of Vitamin B₁₂ Fermentation Process by Integration of Microbial Macro and Micro Metabolic Analysis in the Context of Industrial Systems Bioprocess Engineering in Pseudomonas denitrificans. Shanghai:East China University of Science and Technology, School of Biological Engineering, 2010.
[13] Nanchen A, Fuhrer T, Sauer U. Determination of metabolic flux ratios from 13C-experiments and gas chromatography-mass spectrometry data:protocol and principles. Methods in Molecular Biology, 2007, 358(5):177-197.
[14] Wang Z J, Wang P, Liu Y W, et al. Metabolic flux analysis of the central carbon metabolism of the industrial vitamin B12 producing strain Pseudomonas denitrificans using 13C-labeled glucose. Journal of the Taiwan Institute of Chemical Engineers, 2012, 43(2):181-187.
[15] Wierckx N, Ruijssenaars H J, Winde J H D, et al. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12:verification and complementation of hypotheses derived from transcriptomics. Journal of Biotechnology, 2009, 143(2):124-129.

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