pBAD/TesA/Mlut_11700/Terminator into psB1C3

Description

TesA (BBa_K3038002) and Mlut_11700 (BBa_K3038004) are two created biobricks associated to create a composite part.

GenBank

TesA : GenBank: EG11542
https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3
Mlut_11700 : GenBank: C5CBS9
https://www.uniprot.org/uniprot/C5CBS9

Protein Sequence

TesA:
MADTLLILGDSLSAGYRMSA SAAWPALLNDKWQSKTSVVN ASISGDTSQQGLARLPALLK QHQPRWVLVELGGNDGLRGF QPQQTEQTLRQILQDVKAAN AEPLLMQIRLPANYGRRYNE AFSAIYPKLAKEFDVPLLPF FMEEVYLKPQWMQDDGIHPN RDAQPFIADWMAKQLQPLVN HDSHHHHHH

Mlut_11700:
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

In order to produce the molecule of interest 2-nonanone, we worked with the Lawrence Berkeley National Laboratory, USA which is working on biofuels and modified E. coli strain and obtain a production of 2-nonanone. This production is possible using free fatty acids as substrate. Here we present the cloning of thioesterase I (TesA), an enzyme involved in the synthesis of free fatty acids in E. coli. We used an Acyl ACP thioesterase to reproduce a strain called EGS895, design by an American team. This strain produces methyl ketones and in particular 2-nonanone, which is one of our molecules of interest. We therefore want to reproduce this strain and optimize it later to increase production yields of 2-nonanone.

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 C-term tagged with a Flag 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 columns.

Manipulations

Enzymatic digestion and ligation in pSB1C3

After amplification of the synthetic gene, sample is purified, the amplicons are digested. TesA with EcorI and SpeI and Mlut_11700 with XbaI and PstI. The pSB1C3 plasmid is digested by EcoRI and PstI.

Design of TesA/Mlut_11700/pSB1C3 with Geneious software.
This map shows the pBAD promoter and its terminator flanking the coding sequence of the TesA and Mlut_11700 protein. A 6 his tag is present on TesA gene, and a c-myc tag is present on Mlut_11700 gene. 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 TesA/Mlut_11700/pSB1C3 ligations.

PCR colony screening

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

Reference

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