Team:Poitiers/Design
Design
Our strategy is to produce attractive and repellent molecules for the Asian hornet to protect hives and control Asian hornet population on the territory.
For the production of molecules of interest, we wanted to achieve cloning, for this, a model of design was established and followed for all biobricks that we generated during the project.
A tag like C-myc, 6-His, HA are added. The latter is positioned between the promoter and the coding sequence or between the coding sequence and the terminator as a function of the active sites of the protein to not disturb its activity. Restriction enzymes sites of EcoRI, XbaI, SpeI and PstI enzymes are added.
Restriction enzymes allow to insert biobricks into linearized vector as explained after. Then, the promoter pBAD has been chosen because it’s inducible to arabinose which is a cheap molecule compared to IPTG (IsoPropyl β-D-1-ThioGalactopyranoside). This allows to control the production of protein and to not have a concentration of this protein which could disturb the bacteria. Tags are used and they serve to verify if proteins are produce or not into the final host. Tag makes possible the protein detection but also its purification. Gene sequence is then sent to Integrated DNA Technology (IDT) which synthesized the ordered sequences and sent them back to us in 20 µl tubes with 20 ng/µl.
An arabinose inducible promoter pBAD and its terminator (TER) was built according to the same design. This allows, thanks to a biobrick system, to add the promoter and the terminator in the plasmids so that they become expression plasmids. This makes possible to have functional translation transcription machinery even when the design of the gene of interest did not include a promoter and terminator.
Indeed, the bottom up approach consists in adding DNA cassettes with known biological functions to modify microorganisms according to our own desires. The generated cassettes are called Biobricks and they are built to be reusable from one organism to another.
Sequence A is digested with EcoRI and SpeI, sequence B is digested with XbaI and PstI. SpeI and XbaI are compatible. This then form a scar and allows to associate the Biobricks in the same vector by regenerating the four restriction sites.
Production of 2-nonanone molecule
A molecule identified in the alarm pheromone of the Asian hornet appears to show, at a high concentration, a repellent effect. For that, we have to look for a way to produce it. Indeed, the method of production in the Asian hornet itself is still unknown. After a state of the art, we found a team from Lawrence Berkeley National Laboratory working on biofuel production. In their research, they seek to optimize the production of methyl ketones in DH1 E. coli strain [2].
Green boxes indicate overexpressed genes and red boxes indicate chromosomal deletions. The blue box indicates the putative substrate for FadM (producing free keto acids), and the purple box indicates the final methyl ketone product (putatively generated by spontaneous decarboxylation of keto acids). The TesA = thioesterase used for fatty acid overproduction is not depicted in this figure.
In all the methyl ketones produced by this modified strain, 1% were 2-nonanone. We have therefore tried to recreate this strain by adding improvements. For example, tags on the coding sequences of overexpressed or added proteins.
An inducible pBAD promoter have been added as well as a 6 His tag to purify and detect the protein in the strain.
No terminators have been added to the TesA gene. Indeed, we wish to reproduce the constructions as made by the American team.
On the left, the plasmid comprised of the coding sequences of TesA, FadB and Mlut. This plasmid also contains a chloramphenicol resistance cassette. On the right, the plasmid comprised of the coding sequence of fadM. This plasmid also contains a kanamycin resistance cassette.
Production of 4OOA and 4ODA molecules
Production of FFA and HFA
Two molecules from the Asian hornet sex pheromone have been identified as having attractive effect on males. This is the 4-oxo octanoic acid (4OOA) and the 4-oxo decanoic acid (4ODA). Since these molecules are part of the family of oxidized fatty acids, we have tried to establish a potential metabolic pathway for their production. Since the enzymatic pathway for producing these molecules is still unknown in the asian hornet. Indeed, sequencing of the whole genome of this organism is still ongoing.
From this knowledge, we wanted to establish a synthetic pathway with fatty acids as substrate. We then select a potential host for the production of these molecules of interest. Following a bibliographic work, we became aware of an E. coli BL21 strain modified by a team from Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. These modifications allow an increase in the production of free fatty acids in the cell but also of hydroxylated fatty acids (HFA).
Four important changes in the strain have been made: first, the fadD gene, encoding an acyl-CoA synthetase, has been deleted to stop the degradation pathway of free fatty acids (FFA). Then, acetyl CoA carboxylase (ACCase) and thioesterase (‘TesA), which are two proteins involved in the FFA synthesis, have been cloned in plasmids and overexpressed in the strain upon IPTG addition. Finally, the cytochrome P450 BM3 from Bacillus megaterium (CYP102A1) has been cloned to convert FFA to HFA [1]. The strain called BL21/ΔfadD/pE-A1’tesA&pA-acc has shown interesting yields of HFA (550 mg/L in batch fermentation).
Blocking of the bêta-oxidation pathway and stimulation of the free fatty acids production pathway. Cloning of cytochrome CYP102A1 BM3 of Bacillus megaterium for conversion of free fatty acids into hydroxylated fatty acids. Hydroxylated fatty acids can be produced with longer or shorter carbon chains and the hydroxyl function can be placed from position 2 to X carbon.
In order to perform the overexpression of the TesA and ACCase protein as well as the cloning of the gene coding for the cytochrome, the chinese team built vectors which were then inserted into the strain.
pA-acc: This first plasmid contains the two subunits of acetyl CoA carboxylase enzyme as well as a chloramphenicol resistance cassette.
pE-A1’tesA: this second plasmid contains the coding sequence of the thioesterase and the coding sequence of the CYP102A1 from Bacillus megaterium. This plasmid contains a kanamycin resistance cassette.
Production of octanoic acid (C8)
After putting in place our production strategy, we looked for solutions that would potentially allow an increase in the yield of the different productions. Still using bibliographic research, we found a team working on the production of octanoic acid by a bacterial strain. The Chemical and Biological Engineering laboratory in Iowa State University developed a strain able to produce 1g/L of octanoic acid [3].We wish after the final modification of the E. coli BL21 strain, supplement its culture medium in octanoic or decanoic acids to try to increase the yield of 4OOA and 4OOA. Indeed, if the available free fatty acids are mainly C8 or C10, the conversion to hydroxylated fatty acids and then oxidized fatty acids will be mainly carried out on this fatty acids.
In the strain MG1655 (TE10) of Iowa State University, overexpression of the fabZ gene was performed as well as 3 knock out on fadE, fumAC and ackA genes in order to produce a high concentration of octanoic acid.