Difference between revisions of "Part:BBa K200007"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K200007 short</partinfo>
 
<partinfo>BBa_K200007 short</partinfo>
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===Overview===
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This is the coding sequence for [http://en.wikipedia.org/wiki/Cellulase cellulase] production in E.coli. It codes for endo-b-1,4-glucanase E (CelE) cellulase, which is one of the three major proteins of the cellulosome of Clostridium cellulolyticum.<cite>1</cite>
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<br><br>
  
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This is a BioBrick for cellulase production in E.coli. CelE is one of the three major proteins of the cellulosome of Clostridium cellulolyticum.
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===More Details===
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Cellulase mainly catalyses the reactions that changes crystalline cellulose to cellobiose<cite>2</cite> and then finally to glucose. It also catalyses, to a small extent, the break down of [http://en.wikipedia.org/wiki/Carboxymethyl_cellulose carboxymethyl cellulose].
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This cellulase is protease resistant.
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<br><br>The gene was used as part of the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London <i>The E.ncapsulator</i>] project as one of the showcase proteins of the protein production module of the project.
  
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Cellulase catalyses the reactions, from crystal to cellulose to cellobiose/cellotetrose and to glucose. The cellulose is protease resistant.
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===Usage and Biology===
  
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It can be used in gram positive bacteria.
  
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As part of the Imperial 2009 iGEM E.ncapsulator project, E.coli was engineered to synthesise cellulase to a tunable threshold, and successfully coat and protect it through the stomach, until release in the small intestine.  
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The enzyme has a C-end cellulosome-binding domain (CBD), which is used to deliver its resident catalytic domain
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to the cellulosome. The cellulosome is multi-subunit complex that is involved in the hydrolysis of crystalline cellulose. [http://www.springerlink.com/content/lp8g657n13g76187/fulltext.pdf [3]]
  
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In Clostridum, CelE activity activates other cellulosomal enzymes synergistically.<cite>2</cite>
  
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K200007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K200007 SequenceAndFeatures</partinfo>
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[http://www.ncbi.nlm.nih.gov/nuccore/125972525?report=genbank&log$=seqview&from=961597&to=964041 Sequence from NCBI]
 
[http://www.ncbi.nlm.nih.gov/nuccore/125972525?report=genbank&log$=seqview&from=961597&to=964041 Sequence from NCBI]
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<biblio>
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#1 pmid=9055408
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#2 pmid=10714996
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</biblio>
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==Contribution ==
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*'''Group:''' [https://2020.igem.org/Team:SCU-WestChina SCU-WestChina iGEM team 2020]
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*'''Author:''' Xue Qin
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*'''Summary:''' function and structure addition
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1. Endo -β- 1,4-glucanase can be used to develop biocatalysts for the hydrolysis of hemicellulose to monosaccharides. Through the establishment of the library, we screened the mutants with random diversity and directional evolution, and obtained five best mutations which were 2-3 times higher than the wild-type enzyme activity, and evaluated the related sites.
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Table 1: Activity value of wild type rCelStrep and mutants, nucleotide and aminoacid substitutions in rCelStrep variants
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[[File:T--SCU-WestChina--improved_part6.png]]
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2. Homology models of the three-dimensional structures of the catalytic (Fig. 1) and binding (Fig. 2) modules of rCelStrep from Streptomyces sp. G12 were obtained. With a 37,000 Da molecular mass, this enzyme is composed of a signal peptide for secretion, of 37 aminoacids, a catalytic module belonging to the GH 12 family, of 222 aminoacids, bound through a small linker region to a carbohydrate binding module (CBM) belonging to the family CMB2 of 107 aminoacids.
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[[File:T--SCU-WestChina--improved_part7.png]]
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Fig 1: Homology model of the catalytic module of CelStrep. Surface view of the catalytic cleft of CelStrep with cellotriose modelled in the − 1, − 2 and − 3 subsites and cellobiose modelled in the + 1 and + 2 subsites. The two catalytic glutamate residues, E120 and E203, are coloured in red. The substituted labelled amino-acids are coloured according to the epCelStrep variant: epCS_2 (yellow), epCS_4 (blue), epCS_5 (green)
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[[File:T--SCU-WestChina--improved_part8.png]]
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Fig.2: Homology model of CBM structure of CelStrep overlaid to CBM of Cex from Cellulomonas fimi. Ribbon view of the structural overlay of the CBM of CelStrep (magenta) with the CBM of C. fini (cian). The three substrate binding tryptophan residues of C. fini are coloured in cian and the corresponding residues, W249, W286 and D304 of Streptomyces sp. GH12 in magenta. The substituted labelled amino-acids are coloured according to the epCelStrep variant: epCS_1 (pink), epCS_4 (blue), epCS_5 (green)
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*Reference
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Cecchini DA, Pepe O, Pennacchio A, Fagnano M, Faraco V. Directed evolution of the bacterial endo-β-1,4-glucanase from Streptomyces sp. G12 towards improved catalysts for lignocellulose conversion. AMB Express. 2018;8(1):74. Published 2018 May 5. doi:10.1186/s13568-018-0602-7

Latest revision as of 03:39, 28 October 2020

Cellulase

Overview

This is the coding sequence for [http://en.wikipedia.org/wiki/Cellulase cellulase] production in E.coli. It codes for endo-b-1,4-glucanase E (CelE) cellulase, which is one of the three major proteins of the cellulosome of Clostridium cellulolyticum.1

More Details

Cellulase mainly catalyses the reactions that changes crystalline cellulose to cellobiose2 and then finally to glucose. It also catalyses, to a small extent, the break down of [http://en.wikipedia.org/wiki/Carboxymethyl_cellulose carboxymethyl cellulose]. This cellulase is protease resistant.

The gene was used as part of the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London The E.ncapsulator] project as one of the showcase proteins of the protein production module of the project.

Usage and Biology

It can be used in gram positive bacteria.

The enzyme has a C-end cellulosome-binding domain (CBD), which is used to deliver its resident catalytic domain to the cellulosome. The cellulosome is multi-subunit complex that is involved in the hydrolysis of crystalline cellulose. [http://www.springerlink.com/content/lp8g657n13g76187/fulltext.pdf [3]]

In Clostridum, CelE activity activates other cellulosomal enzymes synergistically.2


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 199
    Illegal PstI site found at 700
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 700
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 287
    Illegal BamHI site found at 814
    Illegal XhoI site found at 524
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 199
    Illegal PstI site found at 700
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 199
    Illegal PstI site found at 700
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[http://www.ncbi.nlm.nih.gov/nuccore/125972525?report=genbank&log$=seqview&from=961597&to=964041 Sequence from NCBI] <biblio>

  1. 1 pmid=9055408
  2. 2 pmid=10714996

</biblio>


Contribution

1. Endo -β- 1,4-glucanase can be used to develop biocatalysts for the hydrolysis of hemicellulose to monosaccharides. Through the establishment of the library, we screened the mutants with random diversity and directional evolution, and obtained five best mutations which were 2-3 times higher than the wild-type enzyme activity, and evaluated the related sites. Table 1: Activity value of wild type rCelStrep and mutants, nucleotide and aminoacid substitutions in rCelStrep variants T--SCU-WestChina--improved part6.png

2. Homology models of the three-dimensional structures of the catalytic (Fig. 1) and binding (Fig. 2) modules of rCelStrep from Streptomyces sp. G12 were obtained. With a 37,000 Da molecular mass, this enzyme is composed of a signal peptide for secretion, of 37 aminoacids, a catalytic module belonging to the GH 12 family, of 222 aminoacids, bound through a small linker region to a carbohydrate binding module (CBM) belonging to the family CMB2 of 107 aminoacids. T--SCU-WestChina--improved part7.png

Fig 1: Homology model of the catalytic module of CelStrep. Surface view of the catalytic cleft of CelStrep with cellotriose modelled in the − 1, − 2 and − 3 subsites and cellobiose modelled in the + 1 and + 2 subsites. The two catalytic glutamate residues, E120 and E203, are coloured in red. The substituted labelled amino-acids are coloured according to the epCelStrep variant: epCS_2 (yellow), epCS_4 (blue), epCS_5 (green)

T--SCU-WestChina--improved part8.png

Fig.2: Homology model of CBM structure of CelStrep overlaid to CBM of Cex from Cellulomonas fimi. Ribbon view of the structural overlay of the CBM of CelStrep (magenta) with the CBM of C. fini (cian). The three substrate binding tryptophan residues of C. fini are coloured in cian and the corresponding residues, W249, W286 and D304 of Streptomyces sp. GH12 in magenta. The substituted labelled amino-acids are coloured according to the epCelStrep variant: epCS_1 (pink), epCS_4 (blue), epCS_5 (green)

  • Reference

Cecchini DA, Pepe O, Pennacchio A, Fagnano M, Faraco V. Directed evolution of the bacterial endo-β-1,4-glucanase from Streptomyces sp. G12 towards improved catalysts for lignocellulose conversion. AMB Express. 2018;8(1):74. Published 2018 May 5. doi:10.1186/s13568-018-0602-7