基于比速率及代谢流的黑曲霉突变株和野生株分析

doi:10.13523/j.cb.20140806

中国生物工程杂志

2014, Vol. 34

Issue (8): 35-40 DOI: 10.13523/j.cb.20140806

研究报告

基于比速率及代谢流的黑曲霉突变株和野生株分析

陈香粉, 鲁洪中, 唐文俊, 唐寅, 储炬, 庄英萍, 张嗣良

华东理工大学生物反应器工程国家重点实验室上海 200237

Comparison of Two Aspergillus niger Mutant and Wild Strains Based on q-rate and Flux Balance Analysis

CHEN Xiang-fen, LU Hong-zhong, TANG Wen-jun, TANG Yin, CHU Ju, ZHUANG Ying-pin, ZHANG Si-liang

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

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摘要：

黑曲霉具备优异的外源蛋白表达和分泌能力，从而被广泛应用于工业酶制剂的生产。通过研究黑曲霉突变株和野生株在相同培养条件下生理参数和代谢流的差异，确定了黑曲霉合成糖化酶过程中的限制性因素。宏观动力学分析发现，较之野生株，突变株具有较高的最大比生长速率，并且副产物得率降低了90%，底物利用率提高了近30%，表明突变株与野生株在碳源分配和产物转化率上具有明显的差异。利用流平衡分析（FBA）计算胞内代谢通量分布，发现还原力和核糖的供应水平是限制菌体合成的主要因素，而前体氨基酸是合成糖化酶最主要的限制性因素。这些研究结果为后续发酵工艺优化和菌株基因改造提供了有益的思路。

关键词： 黑曲霉; 糖化酶; 代谢通量分析; 副产物

Abstract:

Aspergillus niger is widely used in industrial enzyme production for its excellent protein expression and secretion capacity. The differences of physiological behaviors and metabolic flux distribution between Aspergillus niger mutant and wild strains under same cultivation conditions are investigated, so as to determine limited factors in glucoamylase production. Based on kinetic analysis, it is confirmed that the mutant strain gets a higher maximum specific growth rate (+30%), a lower by-product productivity (－90%) and a higher substrate uptake efficiency (+30%), which implies significant differences in carbon distribution and substrate usage efficiency between these two strains. By applying Flux Balance Analysis (FBA), it is found that supplies of reducing power and ribose are main factors which effect cell growth. What's more, precursor amino acids is confirmed to be the main limited factor in glucoamylase production. These conclusions provide significances for subsequent bioprocess optimization and strain gene modification.

Key words: Aspergillus niger Glucoamylase Flux balance analysis By-product

收稿日期: 2014-05-05 出版日期: 2014-08-25

ZTFLH:

Q936

基金资助:

国家高技术研究与发展计划资助项目（2014AA021705）

通讯作者: 唐寅，E-mail：tangyin@ecust.edu.cn E-mail: tangyin@ecust.edu.cn

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引用本文:

陈香粉, 鲁洪中, 唐文俊, 唐寅, 储炬, 庄英萍, 张嗣良. 基于比速率及代谢流的黑曲霉突变株和野生株分析[J]. 中国生物工程杂志, 2014, 34(8): 35-40.

CHEN Xiang-fen, LU Hong-zhong, TANG Wen-jun, TANG Yin, CHU Ju, ZHUANG Ying-pin, ZHANG Si-liang. Comparison of Two Aspergillus niger Mutant and Wild Strains Based on q-rate and Flux Balance Analysis. China Biotechnology, 2014, 34(8): 35-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140806 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I8/35

[1] Galbraith J C, Smith J E. Sporulation of Aspergillus niger in submerged liquid culture. Journal of General Microbiology, 1969, 59(1): 31-45.
[2] David H, Zçelik I S, Hofmann G, et al. Analysis of Aspergillus nidulans metabolism at the genome-scale. Bmc Genomics, 2008, 9(1): 163.
[3] Alper H, Jin Y-S, Moxley J, et al. Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metabolic Engineering, 2005, 7(3): 155-164.
[4] Mukhopadhyay A, Redding A M, Rutherford B J, et al. Importance of systems biology in engineering microbes for biofuel production. Current Opinion in Biotechnology, 2008, 19(3): 228-234.
[5] Pluschkell S, Hellmuth K, Rinas U. Kinetics of glucose oxidase excretion by recombinant Aspergillus niger. Biotechnology and Bioengineering, 1996, 51(2): 215-220.
[6] Nielsen J, Jorgensen H S. Metabolic control analysis of the Penicillin biosynthetic pathway in a high-yielding strain of Penicillium chrysogenum. Biotechnology Progress, 1995, 11(3): 299-305.
[7] Borodina I, Siebring J, Zhang J, et al. Antibiotic overproduction in streptomyces coelicolor A3 (2) mediated by phosphofructokinase deletion. Journal of Biological Chemistry, 2008, 283(37): 25186-25199.
[8] Gunnarsson N, Eliasson A, Nielsen J. Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics. Molecular Biotechnolgy of Fungal beta-Lactam Antibiotics and Related Peptide Synthetases, 2004, 88(5):137-178.
[9] Gao H J, Du G C, Chen J. Analysis of metabolic fluxes for hyaluronic acid (Ha) production by Streptococcus zooepidemicus. World Journal of Microbiology and Biotechnology, 2006, 22(4): 399-408.
[10] Sauer U, Eikmanns B J. The peppyruvateoxaloacetate node as the switch point for carbon flux distribution in bacteria. Fems Microbiology Reviews, 2005, 29(4): 765-794.
[11] Elik E, alik P, Oliver S G. Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate. Biotechnology and Bioengineering, 2010, 105(2): 317-329.
[12] Lasse P, Hansen K, Nielsen J, et al. Industrial glucoamylase fed-batch benefits from oxygen limitation and high osmolarity. Biotechnology and Bioengineering, 2012, 109(1): 116-124.
[13] Melzer G, Dalpiaz A, Grote A, et al. Metabolic flux analysis using stoichiometric models for Aspergillus niger: Comparison under glucoamylase-producing and non-producing conditions. Journal of Biotechnology, 2007, 132(4): 405-417.
[14] Niklas J, Schneider K, Heinzle E. Metabolic flux analysis in Eukaryotes. Current Opinion in Biotechnology, 2010, 21(1): 63-69.
[15] Dromms R, Styczynski M. Systematic applications of metabolomics in metabolic engineering. Metabolites, 2012, 2(4): 1090-1122.
[16] Young J D. Inca: A computational platform for isotopically non-stationary metabolic flux analysis. Bioinformatics, 2014, 30(9): 1333-1335.
[17] Antoniewicz M R, Kelleher J K, Stephanopoulos G. Elementary metabolite units (Emu): A novel framework for modeling isotopic distributions. Metabolic Engineering, 2007, 9(1): 68-86.
[18] Weitzel M, Nöh K, Dalman T, et al. ¹³C flux2—high-performance software suite for ¹³C-metabolic flux analysis. Bioinformatics, 2013, 29(1): 143-145.

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