Part:BBa_K3038004

Mlut_11700 N-term C-Myc Acyl CoA oxidase

Description

Mlut_11700 is an acyl-CoA oxidase. This enzyme 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.

Enzymatic digestion and ligation in pSB1C3

After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1C3. The insert (Mlut_11700) is then ligated into the plasmid.

Design of Mlut_11700/pSB1C3 with Geneious software.
This map shows the terminator corresponding to the pBAD, flanking the coding sequence of the Mlut_11700 protein. A tag is also present in N-ter. Finally, in the plasmid is present and chloramphenicol resistance cassette.

Cloning into thermocompetent cells JM109

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

Clones on a selective LB medium (+ chloramphenicol 25 µg/mL) following the transformation of E. coli thermocompetent cells with the Mlut_11700/pSB1C3 ligations.

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.

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