Acyl ACP Thioesterase (TesA)

Description

TesA is an acyl-ACP thioesterase gene. This protein mainly ensures the conversion of long-chain carbonated CoA or ACP fatty acids for which it has a higher affinity.

Improvement of BBa_K1472601

Addition of a 6his tag at the C-terminal part of the coding sequence.

GenBank

TesA : GenBank: EG11542
https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3

Protein sequence

MADTLLILGDSLSAGYRMSA SAAWPALLNDKWQSKTSVVN ASISGDTSQQGLARLPALLK QHQPRWVLVELGGNDGLRGF QPQQTEQTLRQILQDVKAAN AEPLLMQIRLPANYGRRYNE AFSAIYPKLAKEFDVPLLPF FMEEVYLKPQWMQDDGIHPN RDAQPFIADWMAKQLQPLVN HDSHHHHHH

Reaction

The Acyl ACP thioesterase converts fatty acids ACP or fatty acids CoA into free fatty acids.

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.

Design

Thanks to Geneious software we have designed a gene with a promoter, and a tag. This part doesn’t have a terminator because it is produced to create a composite part with other gene involved in 2-nonanone synthesis. The promoter will therefore be associated with the design of the last gene of the composite part. 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. This part is already existing with number BBa_K1472601. But we decided to improve it by adding a 6-his tag. This allows to purify and detect the protein in the host strain by using Ni-NTA columns.

https://parts.igem.org/File:T--Poitiers--TesA_design-tab3.jpg

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. After amplification of the synthetic gene, the 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 (TesA) is then ligated into the plasmid.

The PCR product, as well as the digestion products, are deposited on 0.8 % agarose gel. In well 2, the TesA tagged with 6 his in C-ter amplified by PCR. The most intense band observed corresponds to the size expected for TesA around 900 pb. Another band, this time very weak, is visible below 400 pb. This band may be due to a specific pairing of the primers.

Electrophoresis gel photography following the deposit of TesA PCR products.
The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is NEB 1 kb Plus DNA Ladder

Enzymatic digestion

The products of digestion are also loaded on the gel. In well 2 we see the purified PCR TesA product. There is little DNA loss here, which is encouraging. Wells 3 and 4 respectively show the digestion of the plasmid and the TesA gene by the restriction enzymes EcoRI and PstI. This is to form cohesive ends between the two. We obtain bands at the expected sizes, about 2200 pb for the plasmid and 900 pb for the synthetic gene TesA.

Electrophoresis photography following loads on agarose gel 0.8% of enzymatic digestion products. The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder (left in the figure). Lane 1 corresponds to the marker, lane 2 to the purified PCR product, lane 3 to the digested pSB1A3 plasmid and lane 4 to the digested TesA synthetic gene.

However it is important to note that agarose gel migration does not verify the effectiveness of digestion. Indeed, since the restriction sites are at the end of the sequences, only a few base pairs have been removed on either side. The resolution of an agarose gel does not make it possible to observe the size of the fragments so precisely. This step makes it possible to ensure that we did not have a loss of DNA during experiments.

Ligation in pSB1A3

Design of TesA/pSB1A3 with Geneious software.
This map shows the pBAD promoter in amount of the coding sequence of the TesA protein. Also present the 6-His tag. Finally, in the plasmid is present and ampicillin resistance cassette.

Cloning into E. coli Thermocompetent cells JM109

The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained. Different volumes of transformed bacteria are spread on Petri dish with selective medium. The number of clones obtained is consistent with the proportion of bacteria spread on the Petri dishes.

Clones on a selective LB medium (+ ampicillin 100 µg/mL) following the transformation of E. coli thermocompetent cells with the TesA/pSB1A3 ligation.

PCR colony screening

After bacterial transformation, colony PCR is performed with the forward primer of the TesA gene and a reverse primer of the plasmid. 24 clones are tested. The PCR products are deposited on 0.8% agarose gel. Clones 1, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 19, 21, 23 and 24 have the right profile, an insert-vector fragment of 1100 pb. Wells 2 and 11 show nothing so they probably did not integrate the ligation products. Wells 10, 16, 20 and 22 seem to have incorporated the aspecific band obtained after PCR on the synthetic gene.

Electrophoresis photography following deposits on agarose gel 0.8% of colony PCR products.
The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder. Lane 1 to 10 corresponds to colony PCR performed on TesA/pSB1A3 ligation, lane 11 to 24 corresponds to colony PCR performed on TesA/pSB1A3.

Clones with the right profile are returned to liquid culture and minipreparations are performed. In order to avoid any risk of point mutation, sequencing is performed with the plasmid primer.

After sequencing, induction is performed on the thermocompetent E. coli bacteria JM109. The objective is to verify if the cloned gene leads to the production of a protein. The expected size of the TesA protein is 20 kDa.

Expression of TesA protein

After sequencing, the induction of the pBAD promotor is performed with arabinose on the thermocompetent bacteria JM109. The objective is to verify if the cloned gene leads to the production of the correct protein. The expected size of the TesA protein is 20 kDa. A little expression of the TesA protein is observed at the correct size when the pBAD promoter is induced with arabinose. The gene has therefore been correctly cloned into the strain and the protein is produced.

SDS Page 8% photography following the induction of JM109 with arabinose after 4 hours of culture.
Coloring with coomassie blue. The lane 1 to 4 correspond to induced or non-induced cultures transformed with TesA 6His tag/pSB1A3. Lane 6 to 8 correspond to induced or non-induced cultures transformed with ADR C-ter/pSB1A3. NI : Not induced I: Induced

M: Marker

The last step consist in evaluating the enzymatic activity of the protein in vitro.

Activity

The last step consists in evaluating the enzymatic activity of the TesA protein in vitro. An acid value (AV) was performed after a fatty acid extraction with hexane on bacterial culture media. Two conditions were used as our pBAD promoter is inducible by arabinose: induced and non induced samples. The volume of potassium hydroxide (KOH) in milliliters to neutralize the free acidity in our samples was measured thanks to an indicator dye: the phenolphthalein. The acid value, which represent here the quantity of KOH needed to neutralize the free fatty acids existent in the sample, was then calculated. It exhibited a difference between the induced sample and the non-one. The non-induced sample presented a smaller AV (0,03 mg) compared with the induced one (3,5 mg) which confirms that the induction leaded to an active TesA which increased the production of free fatty acids in the media.

Acidic value of TesA induced and non-induced fatty acids extracted samples.

Transformed bacteria with TesA gene and pBAD promoter were cultured until DO600nm = 0.5 then induced or not with 0.2% arabinose. Fatty acids were then extracted from our bacterial culture media with hexane and acidic value was then measured.

GC

The final step consists in evaluating the enzymatic activity of the TesA protein by checking the ability of the strain to produce octanoic and decanoic acid.

GC results after 24h of culture of JM109/pSB1A3’TesA strain and ethyl esterification of fatty acids.
X axis:Time (min). Y axis: Response (µV). A: Result of the GC analysis of pure hexane (peak on the left) and of 1g/l of octanoic and decanoic acid standards (peaks on the right). B: Result of the GC analysis of Non-induced and Induced JM109/pSB1A3’TesA at T+24h of culture after ethyl esterification of the FFAs an hexane extraction. The spectrum of the hexane, shown in A, is present in every spectrum. The spectrums of octanoic and decanoic acid standards present a response like a merge of two peaks. The retention time of the octanoic and decanoic acid standards only differ by 1 second. The spectrums of the samples are very similar to the spectrums of the standards.

As shown on this GC spectrums, the JM109/pSB1A3’TesA sample seem to present the same spectrum than the octanoic acid and decanoic acid standards, meaning that the JM109/pSB1A3’TesA might indeed produce one of these molecules. However, the retention time of the standards only differ by 1 second, preventing us from being able to differentiate the presence of one or the other of these molecules in the samples.

References

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