Food and Fermentation Industries >

2020 , Vol. 46 >Issue 5: 31 - 37

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.022822

Fermentation condition optimization of recombinant lytic polysaccharidemonooxygenase extracellularly expressed in Escherichia coli

  • GUO Xiao ,
  • AN Yajing ,
  • CHAI Chengcheng ,
  • LU Fuping ,
  • LIU Fufeng
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  • (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education; Tianjin KeyLaboratory of Industrial Microbiology; National Engineering Laboratory for Industrial Enzymes;College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China)

Received date: 2019-10-08

  Online published: 2020-04-10

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Abstract

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent oxidases that are involved in the degradation of recalcitrant polysaccharides in biomass. Consequently, LPMOs have been widely used in the production of fuels and other value-added oligosaccharides from lignocellulose. In this study, a gene encoding LPMO from Myceliophthora thermophila C1 (MtC1LPMO) was expressed in Escherichia coli BL21(DE3). Firstly, the effect of signal peptides on the expression level of MtC1LPMO was studied. It was found that the fused MtC1LPMO with PelB signal peptide was secreted into the culture medium, increasing greatly the total content of soluble MtC1LPMO. To further improve the expression level of MtC1LPMO, the effects of induction time, inducer concentration, ethanol stress, induction incubation time and temperature on the productivity of the recombinant MtC1LPMO in E. coli BL21 were investigated. The highest yield of soluble MtC1LPMO was obtained by adding 0.5 mmol/L IPTG and 2% ethanol at a final concentration to the medium when OD600 reached 0.9 and further incubated at 30 ℃ for 16 h. The protein concentration of total soluble MtC1LPMO was 12.65 mg/L, which was 2.05 times of that before optimization. The extracellular ratio was 67.30% with the extracellular concentration of 8.51 mg/L. The specific activity of MtC1LPMO was 10.3 U/g using 2,6-dimethoxyphenol as the substrate at the standard condition.

Key words： lytic polysaccharide monooxygenase; Escherichia coli; signal peptide; secretory expression; fermentation optimization

Cite this article

GUO Xiao , AN Yajing , CHAI Chengcheng , LU Fuping , LIU Fufeng . Fermentation condition optimization of recombinant lytic polysaccharidemonooxygenase extracellularly expressed in Escherichia coli[J]. Food and Fermentation Industries, 2020 , 46(5) : 31 -37 . DOI: 10.13995/j.cnki.11-1802/ts.022822

References

[1] HEMSWORTH G R,JOHNSTON E M,DAVIES G J,et al. Lytic Polysaccharide Monooxygenases in Biomass Conversion[J]. Trends Biotechnol, 2015, 33(12): 747-761.
[2] LIU Z L. Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates[J]. Appl Microbiol Biotechnol, 2011, 90(3): 809-825.
[3] GRONENBERG L S,MARCHESCHI R J,LIAO J C. Next generation biofuel engineering in prokaryotes[J]. Curr Opin Chem Biol, 2013, 17(3): 462-471.
[4] PERCIVAL ZHANG Y H,HIMMEL M E,MIELENZ J R. Outlook for cellulase improvement: screening and selection strategies[J]. Biotechnol Adv, 2006, 24(5): 452-481.
[5] VIIKARI L,ALAPURANEN M,PURANEN T,et al. Thermostable Enzymes in Lignocellulose Hydrolysis[J]. Advances in Biochemical Engineering/biotechnology, 2007, 108(108): 121.
[6] MERINO S T,CHERRY J. Progress and challenges in enzyme development for biomass utilization[J]. Adv Biochem Eng Biotechnol, 2007, 108: 95-120.
[7] KUMAR R,SINGH S,SINGH O V. Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives[J]. J Ind Microbiol Biotechnol, 2008, 35(5): 377-391.
[8] JARVIS M. Chemistry - Cellulose stacks up[J]. Nature, 2003, 426(6 967): 611-612.
[9] VAAJE-KOLSTAD G,WESTERENG B,HORN S J,et al. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[J]. Science, 2010, 330(6 001): 219-222.
[10] HARRIS P V,WELNER D,MCFARLAND K C,et al. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family[J]. Biochemistry, 2010, 49(15): 3 305-3 316.
[11] LI X,BEESON W T T,PHILLIPS C M,et al. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases[J]. Structure, 2012, 20(6): 1 051-1 061.
[12] FORSBERG Z,VAAJE-KOLSTAD G,WESTERENG B,et al. Cleavage of cellulose by a CBM33 protein[J]. Protein Sci, 2011, 20(9): 1 479-1 483.
[13] HORN S J,VAAJE-KOLSTAD G,WESTERENG B,et al. Novel enzymes for the degradation of cellulose[J]. Biotechnology for Biofuels, 2012, 5(1): 45.
[14] ANTHONY LEVASSEUR E D, VINCENT LOMBARD, PEDRO M COUTINHO, BERNARD HENRISSAT. Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes[J]. Biotechnology for Biofuels, 2013, 6: 41.
[15] HEMSWORTH G R,HENRISSAT B,DAVIES G J,et al. Discovery and characterization of a new family of lytic polysaccharide monooxygenases[J]. Nature Chemical Biology, 2013, 10(2): 122-126.
[16] LO L L,SIMMONS T J,POULSEN J C,et al. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase[J]. Nat Commun, 2015, 6: 5 961.
[17] VOSHOL G P,VIJGENBOOM E,PUNT P J. The discovery of novel LPMO families with a new Hidden Markov model[J]. BMC Res Notes, 2017, 10(1): 105.
[18] SABBADIN F,HEMSWORTH G R,CIANO L,et al. An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion[J]. Nat Commun, 2018, 9(1): 756.
[19] FILIATRAULT-CHASTEL C,NAVARRO D,HAON M,et al. AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes[J]. Biotechnology for Biofuels, 2019, 12: 55.
[20] MONCLARO A V,FILHO E X F. Fungal lytic polysaccharide monooxygenases from family AA9: Recent developments and application in lignocelullose breakdown[J]. Int J Biol Macromol, 2017, 102: 771-778.
[21] FROMMHAGEN M,SFORZA S,WESTPHAL A H,et al. Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase[J]. Biotechnol Biofuels, 2015, 8(1): 101.
[22] VOGEL J. Unique aspects of the grass cell wall[J]. Curr Opin Plant Biol, 2008, 11(3): 301-307.
[23] TERPE K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems[J]. Applied Microbiology and Biotechnology, 2006, 72(2): 211-222.
[24] CHEN R. Bacterial expression systems for recombinant protein production: E. coli and beyond[J]. Biotechnology Advances, 2012, 30(5): 1 102-1 107.
[25] BANEYX F,MUJACIC M. Recombinant protein folding and misfolding in Escherichia coli[J]. Nat Biotechnol, 2004, 22(11): 1 399-1 408.
[26] CHOI J H,LEE S Y. Secretory and extracellular production of recombinant proteins using Escherichia coli[J]. Applied Microbiology and Biotechnology, 2004, 64(5): 625-635.
[27] VENTURA S,VILLAVERDE A. Protein quality in bacterial inclusion bodies[J]. Trends in Biotechnology, 2006, 24(4): 179-185.
[28] LOW K O,MUHAMMAD MAHADI N,MD ILLIAS R. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts[J]. Appl Microbiol Biotechnol, 2013, 97(9): 3 811-3 826.
[29] ZHANG H,ZHAO Y,CAO H,et al. Expression and characterization of a lytic polysaccharide monooxygenase from Bacillus thuringiensis[J]. Int J Biol Macromol, 2015, 79: 72-75.
[30] YANG Y,LI J,LIU X,et al. Improving extracellular production of Serratia marcescens lytic polysaccharide monooxygenase CBP21 and Aeromonas veronii B565 chitinase Chi92 in Escherichia coli and their synergism[J]. AMB Express, 2017, 7(1): 170.
[31] LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227(5 259): 680-685.
[32] BRESLMAYR E,HANZEK M,HANRAHAN A,et al. A fast and sensitive activity assay for lytic polysaccharide monooxygenase[J]. Biotechnol. Biofuels, 2018, 11: 79.
[33] PETERSEN T N,BRUNAK S,VON HEIJNE G,et al. SignalP 4.0: discriminating signal peptides from transmembrane regions[J]. Nat Methods, 2011, 8(10): 785-786.
[34] MAKRIDES S C. Strategies for achieving high-level expression of genes in Escherichia coli[J]. Microbiol Rev, 1996, 60(3): 512-538.
[35] DVORAK P,CHRAST L,NIKEL P I,et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway[J]. Microb Cell Fact, 2015, 14: 201.
[36] GLICK B R. Metabolic load and heterologous gene expression[J]. Biotechnology Advances, 1995, 13(2): 247-261.
[37] CHHETRI G,KALITA P,TRIPATHI T. An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli[J]. MethodsX, 2015, 2: 385-391.
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