大肠杆菌产脂肪酶代谢通量分析
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摘要
考察了5 L发酵罐中大肠杆菌表达枯草芽孢杆菌脂肪酶LipA的发酵特性,诱导2 h后,脂肪酶比活达到最高值18. 11 U/mg。建立了E. coli代谢合成脂肪酶的反应网络,并通过代谢通量分析发现,经诱导产酶后,乳酸代谢通量增加,碳流通过糖酵解途径(embden-meyerhof-parnas pathway,EMP)生成乳酸,分流了部分合成生物量的碳流,降低了生物量合成的速率;缬氨酸通量的增加满足了脂肪酶合成的需求; ATP通量的减少,限制了脂肪酶的高效合成。该研究为进一步指导提高生产脂肪酶菌种的发酵条件优化和代谢途径改造提供了理论依据。
This study investigated the fermentation characteristics of lipase LipA of Bacillus subtilis expressed by Escherichia coli in 5 L fermentor. After 2 h of induction,the maximum extracellular activity of lipase reached 18. 11 U/mg. A reaction network for metabolic synthesis of lipase by E. coli was established. Through metabolic flux analysis,after inducing enzyme production,the metabolic flux of lactic acid increased. The carbon flux produced lactic acid through the embden-meyerhof-parnas(EMP) pathway,thus diverted the carbon flux of some synthetic biomass,and therefore reduced the biomass synthesis rate. The flux of proline increased to meet the needs of lipase synthesis.The reduction in the ATP flux limited efficient synthesis of lipase. This study provided a theoretical basis for further optimization of fermentation conditions and modification of metabolic pathways of lipase.
This study investigated the fermentation characteristics of lipase LipA of Bacillus subtilis expressed by Escherichia coli in 5 L fermentor. After 2 h of induction,the maximum extracellular activity of lipase reached 18. 11 U/mg. A reaction network for metabolic synthesis of lipase by E. coli was established. Through metabolic flux analysis,after inducing enzyme production,the metabolic flux of lactic acid increased. The carbon flux produced lactic acid through the embden-meyerhof-parnas(EMP) pathway,thus diverted the carbon flux of some synthetic biomass,and therefore reduced the biomass synthesis rate. The flux of proline increased to meet the needs of lipase synthesis.The reduction in the ATP flux limited efficient synthesis of lipase. This study provided a theoretical basis for further optimization of fermentation conditions and modification of metabolic pathways of lipase.
引文
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[25]沈雯.瘤胃p H下乳酸作为枯草芽孢杆菌可利用碳源的研究[D].北京:北京林业大学,2016
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[27] ANGCAJAS A B,HIRAI N,KANESHIRO K,et al. Diversity of amino acid signaling pathways on autophagy regulation:A novel pathway for arginine[J]. Biochemical&Biophysical Research Communications,2014,446(1):8-14
[28] NANOU K, ROUKAS T, PAPADAKIS E. Oxidative stress and morphological changes in Blakeslea trispora,induced by enhanced aeration during carotene production in a bubble column reactor[J]. Biochemical Engineering Journal,2011,54(3):172-177.
[2] BANEYX F,MUJACIC M. Recombinant protein folding and misfolding in Escherichia coli[J]. Nature Biotechnology,2004,22(11):1 399.
[3] ANTONIEWICZ M R. Methods and advances in metabolic flux analysis:a mini-review[J]. Journal of Industrial Microbiology&Biotechnology,2015,42(3):317-325.
[4] GEBRESELASSIE N A,ANTONIEWICZ M R.(13)Cmetabolic flux analysis of co-cultures:A novel approach[J]. Metabolic Engineering,2015,31:132.
[5] CALIK P,CALIK G,TAKAC S,et al. Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium:Effects of the oxygen transfer rate[J]. Biotechnology and Bioengineering.1999,64(2):151-167.
[6] SONG P,CHEN C,TIAN Q,et al. Two-stage oxygen supply strategy for enhanced lipase production by Bacillus subtilis,based on metabolic flux analysis[J]. Biochemical Engineering Journal,2013,71(1):1-10.
[7]黄金,徐庆阳,温廷益,等.不同溶氧条件下L-苏氨酸生物合成菌株的代谢流量分析[J].微生物学报,2008,48(8):1 056-1 060.
[8]任婷月,周万里,张利群,等.一种检测葡萄糖氧化酶活力的新方法[J].食品与发酵工业,2015,41(1):212-215.
[9]阎博,赵林,谭欣,等.浊度法快速测定培养液中的生物量[J].天津化工,2003,17(1):45-46.
[10]宋萍,戚小灵,胡燚,等.响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基[J].中国生物工程杂志,2010,30(8):100-105.
[11]乔宇,丁宏标,闫俊艳,等.重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究[J].生物技术进展,2012,02(3):195-2009.
[12] ZUO S S,LUNDAHL P. A Micro-bradford membrane protein assay[J]. Analytical Biochemistry,2000,284(1):162.
[13] RAMESH K G,SUBAZINI T K,ASHOK S. ECO-MP:e coli-metabolic pathway-development of genome-scale metabolic pathway database for Escherichia coli[J]. Trends in Bioinformatics,2014,7(1):7-12.
[14]申铁,金杰军,乙引,等.大肠杆菌基因缺失菌株中碳中心代谢系统的通量平衡分析和通量变异分析[J].计算生物学,2013,3(3):15-19.
[15] MAHADEVAN I,GHOSH I. Analysis of E. coli promoter structures using neural networks[J]. Nucleic Acids Research,1994,22(11):2 158-2 165.
[16] BANEYX F AND MUJACIC M. Recombinant protein folding and misfolding in Escherichia coli[J]. Nature Biotechnology. 2004,22(11):1 399-1 408.
[17] KROEMER J O,WITTMANN C,SCHROEDER H,et al. Metabolic pathway analysis for rational design of Lmethionine production by Escherichia coli and Corynebacterium glutamicum[J]. Metabolic Engineering. 2006,8(4):353-369.
[18] LI M,HO P Y,YAO S J,et al. Effect of lpd A gene knockout on the metabolism in Escherichia coli based on enzyme activities,intracellular metabolite concentrations and metabolic flux analysis by C-13-labeling experiments[J]. Journal of Biotechnology,2006,122(2):254-266.
[19]张慧敏.酵母在不同培养环境下中间代谢途径代谢调控过程的研究[D].杭州:浙江大学,2005.
[20] KRMER J O,WITTMANN C,SCHRDER H,et al.Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum[J]. Metabolic Engineering,2006,8(4):353-369.
[21]陈飞,冯小海,吴波,等.丙酸杆菌的两种固定化细胞反应器发酵生产丙酸及其代谢通量分析[J].化工学报,2011,62(4):1 034-1 041.
[22] DOBSON C M. Review article protein folding and misfolding[J]. Nature,2003(6 968):884-890.
[23] GOLDBERG M E. The second translation of the genetic message:protein folding and assembly[J]. Trends in Biochemical Sciences,1985,10(10):388-391.
[24]赵树欣,梁慧珍,程丽娟,等.固定化丙酸菌发酵底物的研究[J].中国食品添加剂,2005(1):7-11.
[25]沈雯.瘤胃p H下乳酸作为枯草芽孢杆菌可利用碳源的研究[D].北京:北京林业大学,2016
[26]潘自皓.产转氨酶大肠杆菌高密度培养的研究[D].南京:南京工业大学,2006
[27] ANGCAJAS A B,HIRAI N,KANESHIRO K,et al. Diversity of amino acid signaling pathways on autophagy regulation:A novel pathway for arginine[J]. Biochemical&Biophysical Research Communications,2014,446(1):8-14
[28] NANOU K, ROUKAS T, PAPADAKIS E. Oxidative stress and morphological changes in Blakeslea trispora,induced by enhanced aeration during carotene production in a bubble column reactor[J]. Biochemical Engineering Journal,2011,54(3):172-177.