Difference between revisions of "Part:BBa K3038004"

(→‎Mlut11700 Nter Cmyc)
(→‎Acyl CoA oxydase)
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===Mlut11700 Nter Cmyc_Acyl CoA oxydase===
 
===Mlut11700 Nter Cmyc_Acyl CoA oxydase===
  
−
===Acyl CoA oxydase===
+
==Description==
 +
 
 +
 
 +
Mlut_11700 is an acyl-CoA oxydase. This enzye is involved into the production of methyl ketones by the degradation pathway of fatty acids (β-oxidation)
 +
 
 +
For the competition, Mlut_11700 is tagged in N-term by a C-Myc tag. This tag allows the purification and detection of the protein in order to test the activity. This enzyme was interesting for us because of its implication into the degradation pathway of fatty acids, the β-oxidation. This degradation leads to a production of methyl ketones that are molecules of interest in our project.
 +
 
 +
===GenBank===
 +
 
 +
Mlut_11700 : GenBank: C5CBS9<br/>
 +
https://www.uniprot.org/uniprot/C5CBS9
 +
 
 +
===Protein Sequence ===   
 +
 
 +
Without the C-Myc-tag :<br/>
 +
 +
MTVHEKLAPQ SPTHSTEVPT DVAEIAPERP TPGSLDAAAL EEALLGRWAA
 +
 +
ERRESRELAK DPALWRDPLL GMDEHRARVL RQLGVLVERN AVHRAFPREF
 +
 
 +
GGEDNHGGNI SAFGDLVLAD PSLQIKAGVQ WGLFSSAILH LGTAEHHRRW
 +
 
 +
LPGAMDLSVP GAFAMTEIGH GSDVASIATT ATYDEATQEF VIHTPFKGAW
 +
 
 +
KDYLGNAALH GRAATVFAQL ITQGVNHGVH CFYVPIRDEK GAFLPGVGGE
 +
 
 +
DDGLKGGLNG IDNGRLHFTQ VRIPRTNLLN RYGDVAEDGT YSSPIASPGR
 +
 
 +
RFFTMLGTLV QGRVSLSLAA TTASFLGLHG ALAYAEQRRQ FNASDPQREE
 +
 
 +
VLLDYQNHQR RLIDRLARAY ADAFASNELV VKFDDVFSGR SDTDVDRQEL
 +
 
 +
ETLAAAVKPL TTWHALDTLQ EAREACGGAG FLAENRVTQM RADLDVYVTF
 +
 
 +
EGDNTVLLQL VGKRLLTDYS KEFGRLNVGA VSRYVVHQAS DAIHRAGLHK
 +
 
 +
AVQSVADGGS ERRSANWFKD PAVQHELLTE RVRAKTADVA GTLSGARGKG
 +
 
 +
QAAQAEAFNT RQHELIEAAR NHGELLQWEA FTRALEGITD ETTKTVLTWL
 +
 
 +
RDLFALRLIE DDLGWFVAHG RVSSQRARAL RGYVNRLAER LRPFALELVE
 +
 
 +
AFGLEPEHLR MAVATDAETQ RQEEAHAWFT ARRAAGEEPE DEKAVRAREK
 +
 
 +
AARGRRG
 +
 
 +
Molecular size : 78 kDa (from nucleotide sequence)
 +
 
 +
==Usage and biology==
  
 
Methyl ketones are formed by the hydrolysis of an acyl-ACP intermediate and the subsequent decarboxylation of the 3-keto acid. These volatile substances were first found in rue (Ruta graveolens) [250] but are widespread among plant, animal and microbial species [251]. Wild-type E. coli cells do not produce significant amounts of methyl ketones, but the ability can be established by metabolic engineering. In the first study small amounts of methyl ketones were obtained by overexpression of the genes shmks1 and shmks2 (methylketone synthases 1 and 2) from wild tomato (Solanum habrochaites) [252]. Park et al. [253] applied overexpression of these genes in an E. coli strain that was blocked in four pathways of the fermentation metabolism by deletion of the genes adhE, ldhA, poxB and pta. This strain procuced 450 mg l-1 methyl ketones. Shortly before, a methyl ketone titer of 380 mg l-1 was published upon overexpression of the genes fadB, fadM and Mlut11700 (an acyl-CoA oxidase of Micrococcus luteus) in an E. coli strain with deleted fadE and fadA genes [254]. The combination of the genes fadB, fadM and Mlut11700 was also sufficient for chemolithoautotrophic production of up to 180 mg l-1 methyl ketones in a strain of Ralstonia eutropha with both β-oxidation operons deleted [255].
 
Methyl ketones are formed by the hydrolysis of an acyl-ACP intermediate and the subsequent decarboxylation of the 3-keto acid. These volatile substances were first found in rue (Ruta graveolens) [250] but are widespread among plant, animal and microbial species [251]. Wild-type E. coli cells do not produce significant amounts of methyl ketones, but the ability can be established by metabolic engineering. In the first study small amounts of methyl ketones were obtained by overexpression of the genes shmks1 and shmks2 (methylketone synthases 1 and 2) from wild tomato (Solanum habrochaites) [252]. Park et al. [253] applied overexpression of these genes in an E. coli strain that was blocked in four pathways of the fermentation metabolism by deletion of the genes adhE, ldhA, poxB and pta. This strain procuced 450 mg l-1 methyl ketones. Shortly before, a methyl ketone titer of 380 mg l-1 was published upon overexpression of the genes fadB, fadM and Mlut11700 (an acyl-CoA oxidase of Micrococcus luteus) in an E. coli strain with deleted fadE and fadA genes [254]. The combination of the genes fadB, fadM and Mlut11700 was also sufficient for chemolithoautotrophic production of up to 180 mg l-1 methyl ketones in a strain of Ralstonia eutropha with both β-oxidation operons deleted [255].
  
 +
==Design==
 +
 +
Thanks to Geneious software we have designed a gene with a promoter and a N-term tagged with a C-Myc tag, and finally a terminater. The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. The tag allows to purify and detect the protein in the host strain by using specific antibodies.
 +
 +
==Manipulations==
 +
===PCR amplification===
 +
Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.
 +
 +
===Cloning design in pSB1A3===
 +
 +
===Cloning into pSB1A3===
 +
 +
After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1A3 according to the protocol described above. The insert is then ligated into the plasmid.
 +
 +
 +
===Cloning into thermocompetent cells JM109===
 +
 +
The thermocompetent <i>E. coli</i> JM109 bacteria are then transformed and clones are obtained.
 +
 +
===PCR colony screening===
 +
 +
After bacterial transformation, colony PCR is performed with the forward and reverse primer hybridizing into the plasmid. The PCR products are loaded on 0.8% agarose gel.
 +
 +
===Expression of the recombinant protein===
 +
NI : Not induced
 +
I: Induced
 +
M: Marker
 +
The last step consist in evaluating the enzymatic activity of the protein in vitro.
  
 
===References===
 
===References===

Revision as of 09:16, 19 October 2019

Mlut11700 Nter Cmyc_Acyl CoA oxydase

Description

Mlut_11700 is an acyl-CoA oxydase. This enzye is involved into the production of methyl ketones by the degradation pathway of fatty acids (β-oxidation)

For the competition, Mlut_11700 is tagged in N-term by a C-Myc tag. This tag allows the purification and detection of the protein in order to test the activity. This enzyme was interesting for us because of its implication into the degradation pathway of fatty acids, the β-oxidation. This degradation leads to a production of methyl ketones that are molecules of interest in our project.

GenBank

Mlut_11700 : GenBank: C5CBS9
https://www.uniprot.org/uniprot/C5CBS9

Protein Sequence

Without the C-Myc-tag :

MTVHEKLAPQ SPTHSTEVPT DVAEIAPERP TPGSLDAAAL EEALLGRWAA

ERRESRELAK DPALWRDPLL GMDEHRARVL RQLGVLVERN AVHRAFPREF

GGEDNHGGNI SAFGDLVLAD PSLQIKAGVQ WGLFSSAILH LGTAEHHRRW

LPGAMDLSVP GAFAMTEIGH GSDVASIATT ATYDEATQEF VIHTPFKGAW

KDYLGNAALH GRAATVFAQL ITQGVNHGVH CFYVPIRDEK GAFLPGVGGE

DDGLKGGLNG IDNGRLHFTQ VRIPRTNLLN RYGDVAEDGT YSSPIASPGR

RFFTMLGTLV QGRVSLSLAA TTASFLGLHG ALAYAEQRRQ FNASDPQREE

VLLDYQNHQR RLIDRLARAY ADAFASNELV VKFDDVFSGR SDTDVDRQEL

ETLAAAVKPL TTWHALDTLQ EAREACGGAG FLAENRVTQM RADLDVYVTF

EGDNTVLLQL VGKRLLTDYS KEFGRLNVGA VSRYVVHQAS DAIHRAGLHK

AVQSVADGGS ERRSANWFKD PAVQHELLTE RVRAKTADVA GTLSGARGKG

QAAQAEAFNT RQHELIEAAR NHGELLQWEA FTRALEGITD ETTKTVLTWL

RDLFALRLIE DDLGWFVAHG RVSSQRARAL RGYVNRLAER LRPFALELVE

AFGLEPEHLR MAVATDAETQ RQEEAHAWFT ARRAAGEEPE DEKAVRAREK

AARGRRG

Molecular size : 78 kDa (from nucleotide sequence)

Usage and biology

Methyl ketones are formed by the hydrolysis of an acyl-ACP intermediate and the subsequent decarboxylation of the 3-keto acid. These volatile substances were first found in rue (Ruta graveolens) [250] but are widespread among plant, animal and microbial species [251]. Wild-type E. coli cells do not produce significant amounts of methyl ketones, but the ability can be established by metabolic engineering. In the first study small amounts of methyl ketones were obtained by overexpression of the genes shmks1 and shmks2 (methylketone synthases 1 and 2) from wild tomato (Solanum habrochaites) [252]. Park et al. [253] applied overexpression of these genes in an E. coli strain that was blocked in four pathways of the fermentation metabolism by deletion of the genes adhE, ldhA, poxB and pta. This strain procuced 450 mg l-1 methyl ketones. Shortly before, a methyl ketone titer of 380 mg l-1 was published upon overexpression of the genes fadB, fadM and Mlut11700 (an acyl-CoA oxidase of Micrococcus luteus) in an E. coli strain with deleted fadE and fadA genes [254]. The combination of the genes fadB, fadM and Mlut11700 was also sufficient for chemolithoautotrophic production of up to 180 mg l-1 methyl ketones in a strain of Ralstonia eutropha with both β-oxidation operons deleted [255].

Design

Thanks to Geneious software we have designed a gene with a promoter and a N-term tagged with a C-Myc tag, and finally a terminater. The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. The tag allows to purify and detect the protein in the host strain by using specific antibodies.

Manipulations

PCR amplification

Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.

Cloning design in pSB1A3

Cloning into pSB1A3

After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1A3 according to the protocol described above. The insert is then ligated into the plasmid.


Cloning into thermocompetent cells JM109

The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained.

PCR colony screening

After bacterial transformation, colony PCR is performed with the forward and reverse primer hybridizing into the plasmid. The PCR products are loaded on 0.8% agarose gel.

Expression of the recombinant protein

NI : Not induced I: Induced M: Marker The last step consist in evaluating the enzymatic activity of the protein in vitro.

References

Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels Helge Jans Janßen1 and Alexander Steinbüchelcorresponding author1,2. Biotechnol Biofuels. 2014; 7: 7. doi: 10.1186/1754-6834-7-7. PMCID: PMC3896788. PMID: 24405789


Engineering of Bacterial Methyl Ketone Synthesis for Biofuels. Ee-Been Goh,a,c Edward E. K. Baidoo,a,c Jay D. Keasling,a,c,d and Harry R. Beller. Appl Environ Microbiol. 2012 Jan; 78(1): 70–80.doi: 10.1128/AEM.06785-11. PMCID: PMC3255637. PMID: 22038610


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1682
    Illegal XhoI site found at 1925
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1816
    Illegal BsaI.rc site found at 2047
    Illegal SapI site found at 172