研究报告

增强海藻糖胞内积累提高大肠杆菌耐受性与乙醇产率

  • 王惊春 ,
  • 田康明 ,
  • 苗佳 ,
  • 王彩喆 ,
  • 金鹏 ,
  • 王正祥
展开
  • 1 (天津科技大学 化工与材料学院,天津,300457)
    2 (天津科技大学 生物工程学院,天津,300457)
硕士研究生(王正祥教授为通讯作者,E-mail:zxwang0519@tust.edu.cn)。

收稿日期: 2019-04-02

  网络出版日期: 2019-11-15

基金资助

国家重点研发计划政府间国际科技创新合作重点专项(2018YFE0100400);天津市高等学校创新团队建设规划项目(TD12-5002)

Enhanced intracellular trehalose accumulation improves stress tolerance andethanol yield of Escherichia coli

  • WANG Jingchun ,
  • TIAN Kangming ,
  • MIAO Jia ,
  • WANG Caizhe ,
  • JIN Peng ,
  • WANG Zhengxiang
Expand
  • 1 (College of Chemical Engineering and Materials Science, Tianjin University of Science & Technology, TEDA, Tianjin 300457, China)
    2 (School of Biotechnology, Tianjin University of Science & Technology, TEDA, Tianjin 300457, China)

Received date: 2019-04-02

  Online published: 2019-11-15

摘要

为提高产乙醇大肠杆菌对发酵底物和乙醇的耐受性,并提高乙醇发酵性能,以大肠杆菌B0013-1031H为出发菌株,对其海藻糖代谢途径进行改造,获得了敲除海藻糖分解途径的突变株JC31和进一步加强海藻糖合成途径的突变株JC41。突变株JC31和JC41较出发菌株都具有更高的海藻糖合成与积累能力,其中JC41的胞内海藻糖含量可达出发菌株的12倍。与出发菌株相比,突变株JC31和JC41对葡萄糖和乙醇胁迫的耐受性显著提高。进一步引入乙醇合成途径,在葡萄糖质量浓度120 g/L的发酵条件下,菌株JC31-PA表现出最优的发酵性能,其最大乙醇产量为50.6 g/L,较对照菌株提高了5.42%;乙醇转化率为48.72 g/100 g葡萄糖,较对照提高了12.67%,达到理论转化率的95%。

本文引用格式

王惊春 , 田康明 , 苗佳 , 王彩喆 , 金鹏 , 王正祥 . 增强海藻糖胞内积累提高大肠杆菌耐受性与乙醇产率[J]. 食品与发酵工业, 2019 , 45(21) : 15 -21 . DOI: 10.13995/j.cnki.11-1802/ts.020729

Abstract

In order to improve the tolerance of ethanologenic Escherichia coli to substrate and ethanol and the performance of ethanol fermentation, the trehalose metabolic pathway of E. coli B0013-1031H was engineered and the mutants JC31 and JC41 were constructed. In strain JC31, the trehalose catabolism pathways were deleted, the trehalose synthesis pathway was further enhanced and resulted in strain JC41. The ability of trehalose synthesis and accumulation of JC31 and JC41 was higher than that of the original strain, in which, JC41 intracellularly accumulated trehalose up to 12-fold that of the original strain. The tolerance of JC31 and JC41 to glucose and ethanol stress was significantly improved. The ethanologenic recombinant E. coli JC31-PA obtained by engineering ethanol synthesis pathway fermented 120 g/L of glucose and yielded 50.6 g/L of ethanol, which was 5.42% higher than that of the control strain. The conversion rate of ethanol from glucose was 48.72 g/100 g glucose, which was 12.67% higher than that of the control.

参考文献

[1] ZABED H,SAHU J N,SUELY A,et al.Bioethanol production from renewable sources:Current perspectives and technological progress[J].Renewable and Sustainable Energy Reviews,2017,71:475-501.
[2] INGRAM L O,CONWAY T,CLARK D P,et al.Genetic engineering of ethanol production in Escherichia coli[J].Applied and Environmental Microbiology,1987,53(10):2 420-2 425.
[3] 孙金凤,徐敏,张峰,等.利用木糖和葡萄糖合成乙醇的新型重组大肠杆菌的研究[J].微生物学报,2004,44(5):600-604.
[4] ASGHARI A,BOTHAST R J,DORAN J B,et al.Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11[J].Journal of Industrial Microbiology,1996,16(1):42-47.
[5] INGRAM L O,LAI X,MONIRUZZAMAN M,et al.Fuel ethanol production from lignocellulose using genetically engineered bacteria[J].Acs Symposium,1997,666:57-73.
[6] ALSAKER K V,PAREDES C,PAPOUTSAKIS E T.Metabolite stress and tolerance in the production of biofuels and chemicals:Gene-expression-based systems analysis of butanol,butyrate,and acetate stresses in the anaerobe Clostridium acetobutylicum[J].Biotechnology and Bioengineering,2010,105(6):1 131-1 147.
[7] PINKART H C,WHITE D C.Phospholipid biosynthesis and solvent tolerance in Pseudomonas putida strains[J].Journal of Bacteriology,1997,179(13):4 219-4 226.
[8] HEIPIEPER H J,BONT J A M D.Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes[J].Applied and Environmental Microbiology,1994,60(12):4 440-4 444.
[9] SANDOVAL N R,KIM J Y H,GLEBES T Y,et al.Strategy for directing combinatorial genome engineering in Escherichia coli[J].Proceedings of the National Academy of Sciences,2012,109(26):10 540-10 545.
[10] ZALDIVAR J,MARTINEZ A,INGRAM L O.Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli[J].Biotechnology and Bioengineering, 1999, 65(1):24-33.
[11] PIPER P W.The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap[J].FEMS Microbiology Letters,1995,134(2-3):0-127.
[12] ZINGARO K A,ELEFTHERIOS T P.Toward a semisynthetic stress response system to engineer microbial solvent tolerance[J].MBIO,2012,3(5):429-493.
[13] SHIMA J,HINO A,YAMADAIYO C,et al.Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial baker’s yeast[J].Applied and Environmental Microbiology,1999,65(7):2 841-2 846.
[14] 戴秀玉,王忆琴,周坚,等.大肠杆菌海藻糖的代谢调控[J].中国生物工程杂志,2000,20(6):26-29.
[15] GIAEVER H M,STYRVOLD O B,KAASEN I,et al.Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli[J].Journal of Bacteriology,1988,170(6):2 841-2 849.
[16] WHITEZIEGLER C A,UM S,PÉREZ N M,et al.Low temperature (23 ℃) increases expression of biofilm-,cold-shock- and RpoS-dependent genes in Escherichia coli K-12[J].Microbiology,2008,154(Pt 1):148.
[17] BOOS W,EHMANN U,BREMER E,et al.Trehalase of Escherichia coli.Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions[J].Journal of Biological Chemistry,1987,262(27):13 212-13 218.
[18] REPOILA F,GUTIERREZ C.Osmotic induction of the periplasmic trehalase in Escherichia coli K12:characterization of the treA gene promoter[J].Molecular Microbiology,2010,5(3):747-755.
[19] HORLACHER R,UHLAND K,KLEIN W,et al.Characterization of a cytoplasmic trehalase of Escherichia coli[J].Journal of Bacteriology,1996,78(21):6 250-6 257.
[20] KLEIN W,HORLACHER R,BOOS W.Molecular analysis of treB encoding the Escherichia coli enzyme II specific for trehalose[J].Journal of Bacteriology,1995,177(14):4 043-4 052.
[21] PURVIS J E,YOMANO L P,INGRAM L O.Enhanced trehalose production improves growth of Escherichia coli under osmotic stress[J].Applied and Environmental Microbiology,2005,71(7):3 761-3 769.
[22] 孙金凤,田康明,沈微,等.底物选择性大肠杆菌共发酵葡萄糖和木糖产生乙醇[J].食品与发酵工业,2018,44(12):1-7.
[23] 周丽,牛丹丹,李宁,等.基于Red重组系统和Xer重组系统的大肠杆菌多基因删除方法[J].微生物学通报,2010, 37(6):923-928.
[24] ZHOU Li,ZUO Zhirui,CHEN Xianzhong,et al.Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli[J].Current Microbiology,2011,62:981-989.
[25] TANAKA K,ISHII Y,OGAWA J,et al.Enhancement of acetic acid tolerance in Saccharomyces cerevisiae by overexpression of the HAA1 gene,encoding a transcriptional activator[J].Applied and Environmental Microbiology,2012,78(22):8 161-8 163.
[26] 章克昌.酒精与蒸馏酒工艺学[M].北京:中国轻工业出版社, 1995.
文章导航

/