Decarboxylation

Why Decarboxylation?

Though there is great diversity between the many possible compounds termed “naphthenic acids” found in tailings ponds, all share the common chemical feature of a carboxylic acid group. This particular group is known to be the primary cause for the toxicity of these compounds, allowing them to enter through cell membranes and destroy cells (Frank et al, 2009). Naphthenic acids are a very problematic component of the tailings ponds, and are not easily degraded under regular circumstances. They are known to leak into local environments and cause many problems for wildlife (Clemente & Fedorak, 2005), as well as corrosion (Slavcheva et al, 1999). Methods for their degradation are obviously needed.

The main goal of OSCAR is to turn toxins like these into useable hydrocarbons. Removing the carboxylic acid group(s) from them is necessary for this to happen (Behar & Albrecht, 1984). Since naphthenic acids are a widely varying and unspecific mixture of compounds, an enzymatic process with very low substrate specificity is necessary, as particularly specific enzymes would not be able to universally decarboxylate all naphthenic acid substrates found in tailings ponds. Without a carboxylic acid group, an alkane suitable for use as fuel could potentially be obtained. The goal of this subproject was to find a suitable pathway to accomplish the decarboxylation of compounds such as naphthenic acids with the broadest specificity possible.

The PetroBrick

The 2011 Washington iGEM team developed the PetroBrick, a BioBrick consisting of two primary genes. These include acyl-ACP reductase (AAR), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (ADC), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain. Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that these fatty acids that the PetroBrick looks like, has a very similar structure to naphthenic acids.

Figure 1. A comparison of the structure of fatty acids and naphthenic acid

This led us to believe that the PetroBrick may also have the potential to turn naphthenic acids in to hydrocarbons! This could be a perfect solution to remeidating our naphthenic acids. First though, we had to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA form the registry and had to get the Washington team to send it to us. Initially the ability for the Petrobrick to produce alkanes from glucose was analyzed in order to verify that the Petrobrick was working as described by the 2011 Washington team. This was demonstrated in Figures 2 and 3.

Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an E.coli control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.

Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.

With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on naphthenic acids, so see if they could be selectively converted into alkanes! This experiment used commercially available naphthenic acid fractions including a large number of different complex naphthenic acid compounds.

Successful conversion of NA's into Hydrocarbons!

Figure 4: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.

Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 2, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.

The above graphs indicate that hydrocarbons were successfully produced from E.coli that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, E.coli containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of E.coli that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade naphthenic acids into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to!

Nocardia Carboxylic Acid Reductase (CAR)- Can we do better?

Despite our success, we wanted to improve on our results to see if we could get a higher yield or possibly target other compounds. One of our original fears in using the PetroBrick to decarboyxlate naphthenic acids was that the first enzyme AAR is thought to be highly specific for fatty acids bound to ACP. As a result, we had worried about its compatibility with naphthenic acids. We had sought another enzyme in the literature called carboxylic acid reductase (CAR) that was documented to perform a similar task as AAR, converting fatty acids to aldehydes, but with much lower specificity (He et al, 2004). This enzyme, from N. iowensis does not require covalent attachment to ACP so would likely be much broader in substrate specificity. It requires a second gene from N. iowensis, called Nocardia phosphopantetheinyl transferase (NPT) necessary to append a 4’-phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian et al, 2006).

Figure 6. Mechanism of action of CAR

Additionally, we had found a second alternative: olefin-forming fatty acid decarboxylase (OleT) from Jeotgalicoccus sp. ATCC 8456. This is a decarboxylase of the cytochrome P450 family that acts on fatty acids, but has also been documented to have low substrate specificity (Rude et al, 2011). What was attractive with this was that it was one single enzyme that go do the job of the PetroBrick! Now that we knew that our decarboxylation approach was valid, it was time to start testing and comparing this gene to the PetroBrick

Where are we at?

N. iowensis (NRRL 5646) was purchased from DSMZ and rehydrated, then grown in both solid and liquid brain heart infusion (BHI) media. CAR and NPT were successfully cloned out and amplified, and CAR was verified after ligation into the PET vector by a restriction digest. NPT was cloned into psb1c3 and similarly verified.

CAR was found to have six cut sites deemed illegal for BioBrick construction: one XbaI site, two EcoRI sites, and three NotI sites. These were first to be addressed by a multi-site mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more time-consuming series of conventional single-site mutagenesis procedure was favoured, using the KAPPA amplification system. A PET vector was chosen as an alternate vector to pSB1C3 to ligate with CAR for the duration of mutagenesis, while NPT was still ligated with pSB1C3. The XpaI and EcoRI sites were eliminated first, and following these steps and subsequent verification of their success, the mutated CAR+PET plasmid was successfully amplified with BioBrick primers after multiple attempts in preparation for the final construct. NPT was ligated in a construct following part J13002 from the iGEM parts registry, containing a ribosomal binding site and a TetR promoter.

How do the PetroBrick and CAR+NPT work together?

Our team decided to created a BioBrick construction containing three major parts. These included: the PetroBrick (as received from Washington), the CAR gene, and the NPT gene. This construction can be seen below.

Both CAR and NPT use the part JI3002, which consists of pTetR (a constitutive promotor), as well as an RBS (ribosomal binding site). This constitutive promoter allows the gene to always be expressed, so that decarboxylation may consistently occur. The construction also includes Washington’s PetroBrick, which contributes the genes ADC and AAR. While AAR is useful to help convert some carboxylic acids and fatty acids to aldehydes, it is not known to be highly specific like the CAR enzyme. We are primarily interested in using the PetroBrick for its ADC gene, which is responsible for producing the only enzyme in our system that is capable of converting aldehydes into hydrocarbons. Meanwhile, both AAR and CAR in our construct will work to convert the initial carboxylic acids into the aldehydes required to produce hydrocarbons.

OleT was successfully amplified from the Jeotgalicoccus sp. ATCC 8456. Like CAR, OleT was inserted in a PET vector before placing it into a BioBrick, as two illegal cut sites adjacent to one another needed to be mutagenized. The final construct for OleT is very simple, containing the J13002 promotor/RBS, and the OleT gene. This simple construct should be able to convert carboxylic acids to terminal alkenes on its own.