Team:Bielefeld-CeBiTec/Results/unnatural base pair/biosynthesis

Biosynthesis

Short Summary

The plant L. croton tiglium was of great importance to our project due to its ability to produce isoguanosine. In order to to fully understand the production pathways of this nucleoside, we aimed at reproducing the biosynthesis of L. croton tiglium in vitro. First, we developed RNA libraries from all plant tissues. The expression libraries generated by trinity assembly were used to identify essential enzymes of purine metabolism existing in L. croton tiglium. After extracting the DNA of these proteins of interest from the library, we expressed them in E. coli and purified them. After estimating the concentration of the purified proteins, we proved their functionality in iso-GMP and isoguanosine formation assays. Finally, the identity of these reaction products was confirmed by liquid chromatographic analysis.

Usage of the Data Generated by the Trinity Assembly

Trinity assembly is a method for reconstruction of transcriptomes from RNA-sequencing data. The expression library of L. croton tiglium generated by this method revealed a lot of interesting genes and functions associated with iso-guanosine production. After we figured out their sequences by using different databases like uniprot, we used BLAST to identify them within all the trinity sequences. Out of these, we identified some that play an interesting role in the purine pathway and some others that are responsible for the carotenoid cleavage dioxygenases. These are of interest as they could be used as a combinatorical olfactorical reporter in combination with carotenoid-producing parts for future iGEM teams.

Identification of Candidate Genes

The synthesis of purine bases is a paramount aspect for the creation of bases. As already described and explained at unnatural base pairs, different reactions are needed for the creation of the final products (GMP and AMP). Moreover, there are also many reactions that are needed for the organism to work, as well as for the catalysis of side-products which are important for additional reactions. Further examination of the enzyme reactions of the purine metabolism allowed us to figure out some that might be of specific interest for the incorporation and creation of unnatural bases in L. croton tiglium.

Firstly, there is the guanosine monophosphate synthetases (GMPS), an enzyme from the class of ligases that form carbon-nitrogen-bonds with glutamine as an amido-N-donor. It is also known as Guanosine monophosphate synthetase and is abbreviated with guaA. GMPS is important for us since it is needed for the amination of XMP (xanthosine monophosphate) to create GMP and possibly iso-GMP. Besides , GMPS can be found in many organisms apart from L. Croton tiglium, including H. sapiens and E.coli. Comparing the found sequences of the GMPS with the trinity assembly allowed us to figure out two slightly different sequences for it. These sequences have the size of 314 amino acids and molecular mass of 59.46 kDa. To us, the GMPS is of special interest as it may be able to not only create GMP out of XMP but also iso-GMP.
Another interesting enzyme from the purine metabolism is the Inosine monophosphate-dehydrogenase (IMPDH) that matched with three trinity sequences. IMPDH is an enzyme from the class of the Oxydoreductases which are acting on CH-OH groups of donors with NAD+ or NADP+ as acceptors. IMPDH has a molecular mass of53-58 kDA depending on the exact sequence and a length of approximately 500 to 550 amino acids. In the purine metabolism, IMPDH is the catalysator of the synthetasis of XMP out of inosine monophosphate (IMP). For L. croton tiglium it means that it could possibly enable the creation of an iso-form of XMP that might then even be a creator of iso-GMP.
Further on, the cytidine deaminase seemed to be of immense potential. The CDA, which belongs to the class of hydrolases acting on carbon-nitrogen bonds different from peptide bonds is usually used to deaminate cytidine to uridine. However, there is also a risk of back reactions and a potential for further ones. For our purposes, we thought of a reaction from xanthosine to iso-GMP. The found amino acid sequence for cda from trinity assembly is 535 amino acids long and has a molecular mass of 33.95 kDa.
Aside from these enzymes, we thought of the adenylosuccinate synthetase as an interesting aspect of the purine pathway. The ADSS belonging to the class of ligases, which are forming carbon-nitrogen bonds, could only be found within one sequence of the trinity assembly. This has a molecular weight of 53.32 kDA and a size of 489 amino acids. In L. croton tiglium, it causes the reaction of IMP to adenylosuccinate that will then be further processed into AMP.
The final enzyme that we focussed on is xanthine dehydrogenase. With two different reactions as possible outcomes that cause the same products, the XDH will usually convert xanthine into urate that will be further processed afterwards. XDH is an enzyme from the class of oxidoreductases that is acting on CH or CH2 groups with NAD+ or NADH+ as an acceptor and could even be matched with six sequences of the trinity assembly. It has a molecular mass of 64.12 kDA and a size of 587 amino acids.

Extraction of Enzyme DNA out of the cDNA Library

After we had identified all interesting genes from the purine pathway, we had to extract them from the cDNA of the tissue samples. Thus, we designed primers for all sequences that could be found within the trinity assembly of L. croton tiglium. In total, we had 13 pairs of primers that we used in separate PCRs with all tissue samples. We could extract at least one gene possibility for each protein of interest from these PCRs. However, as there were two possible outcomes for the GMPS that could work differently, we codon-optimized the one we could not gain from the tissues and ordered it via gene synthesis prior to the protein purification experiments.
We further extracted the ccd gene out of L. croton tiglium tissue samples with these PCRs, cloning it into the Phytobrick-vector BBa_P10500 using the opportunity of the blue-white selection creating a new part.

Preparation of the protein purification

After the extraction, we had to figure out a way to gain the expressed proteins. Therefore, we used a modification of the NEB Impact® Kit. The Impact system itself works with the help of intein tags. Impact stands for “Intein Mediated Purification with Affinity Chitin-binding Tag”. In short, the target gene sequence of the protein is at first integrated into a vector designed for the system that contains an intein tag. After the expression of this vector within competent E.coli cells of the ER2566 strain, the protein will be bound to the intein. In this combination multiple washing steps can be done at once, followed by a cleavage between the intein and the protein, followed by a final elution step.

Since we did many tests with the enzyme needed, we did all of the following steps multiple times. Therefore, we firstly had to clone the sequences into a strain that adds an intein tag to the protein sequences and is usable for the impact protocol. For this purpose, we used the vector pTXB1. Since this was only available in small amounts as a plasmid containing another vector, we first had to integrate it into electro competent E.coli DH5alpha cells. Plates were incubated overnight (O/N) at 37°C. From these we made another plasmid isolation. We then used two different methods (PCR as well as restriction enzymes) to linearize the plasmid and to exclude the initially integrated insert. Next, we did Gibson Assemblies with all possible candidates for the enzymes we had previously extracted and integrated them into the previously linearized ptxb1 vectors.
Now we transformed the vectors into electro and chemo competent DH5-alpha E.coli so we could analyze which of those had worked well with a colony PCR and sequencing. All of the proteins of which we had positive clones were isolated and then integrated into electro competent ER2566 E.coli, since they have certain properties needed for the impact protocol. This strain contains a copy of the T7 RNA polymerase that is only expressed when IPTG is present. That way, the proteins cannot be expressed prior to further usage. In addition, there are no critical proteases within ER2566. From this step on, we followed the modified version of the protocol for impact, which can also be found here.
We first propagated the cells in 250 mL LB with supplemented antibiotics until they reached an _OD600 of at least 0.5. Then we added IPTG and let the proteins express overnight. On the next day we first pelleted our cells, resuspended them in lysis buffer and decomposed them with help of the French press. While we centrifuged to separate the cell extracts from the debris, we prepared the columns for the purification. For this, we used chitin beads that made up approximately 5 mL volume and equilibrated the columns.

Protein Purification

After everything was prepared we could start the purification of the proteins. Still following the protocol, we first poured the supernatant onto the columns taking care of a flow through that was less than half a mL per minute with special braces for columns. Afterwards, we washed the columns and induced the cleavage. Approximately 18 hours later, we eluted the columns and concentrated the proteins with protein filter columns falcon tubes and washed them with protein-wash- and storage buffer .

Estimation of the Protein Concentration

After we had purificated the proteins, we had to estimate their total concentrations. For that we used a modification of the Bradford estimation (Bradford, M., (1976) Analy. Biochem. 72:248-254.), Roti®-Nanoquant

by by Roth. You can find an English version the protocol here. Depending on the protein, we reached concentrations from 1.35 up to 5.31 grams per liter. With the help of an SDS page we confirmed that all proteins had been extracted correctly. Regarding the heights of the bands, all proteins had been extracted correctly. We further checked whether all proteins could be extracted correctly with the help of an sds page. Regarding the heights of the bands, all proteins had been extracted correctly.

Enzyme activity assays

After we knew that all proteins had been extracted properly, our next step was to test their functionality. To do so, we used the device “Tecan infinite® 200” and the program “Tecan i-control , 1.10.4.0”. This device is a plate-reader that offers different opportunities to measure samples, originally designed for assays. It can also measure nearly all kinds of well plates including the possibility of repeated measurements at a given time interval. Since our main goal was to produce iso-GMP or iso-Guanosine out of the proteins of L. croton tiglium, we focused on the two main promising candidate enzymes, GMPS and CDA. First, we set up an enzyme activity assay for CDA with cytidine to ensure its activity following the protocol taken from (“Cytidine Deaminase from E.coli – Purificarion Properties and Inhibiton by the potential transition state analog 3,4,5,6-tetrahydrouridine” by Robert M. Cohen and Richard Wolfenden, published in “The Journal of Biological Chemistry” on December 25, 1971) that stated that the disappearance of cytidine can be measured in relation to the decrease of absorption at 282 nm. Therefore, we set up the following reaction mixture in a greater volume:

0.000167M cytidine
0.05M Tris-HCL-Buffer pH 7.5
Filled up with ddH₂O.

We created six identical measurement samples with 196 µL of the mixture and measured it for about 20 min (measurement all 30 sec) with the Tecan. We then paused the measurement program to add 4 µL of the previously extracted CDA or 4 µl of water to three samples each. Then, we immediately continued the measurement with the same program for about an hour.
After the general activity of the CDA was tested, we set up a possible reaction with xanthosine instead of cytidine, all other components being the same. However, since there was no real literature on this reaction, we first had to figure out the absorption rate at which xanthosine can be measured. This was done using a general spectrum analysis of different mixtures, three samples each:

without xanthosine, without cda
with xanthosine, without cda
without xanthosine , with cda
with xanthosine, with cda

We then calculated the mean values out of the absorption of the three samples each and compared them in different combinations, always calculating the positive difference between them:

1+3: difference between a reaction mixture with and without cda
1+2: difference between a reaction mixture with and without xanthosine
2+4: difference between no reaction and a possible reaction

We hereby could figure out the absorption rate at which xanthosine can be measured (B) as well as ensure that the peak was independent from the cda (A). Further on, we could identify the absorbance of cda at about 254-260 nnm (A and C). (Figure 1)

Figure (1): Results of the analysis of the absorbance of xanthosine at different nanometers.
The difference between a mixture with and without xanthosine(red) can clearly be made up at about 282 nnm.

Afterwards we set up new activity assays, using 196 µL of the reaction mixture in six of the well plates holes. After measuring the absorbance at 282 nm, we added 4 µL of either water or the enzyme to three biological duplicates each, continuing the (previous) measurements for about an hour. Then we set up the reaction mixture of the GMPS and the other form of the GMPS we ordered via gene synthesis (further referred to as “gene synthesis”). Again, we followed the protocol for the enzyme activity assay out of “The Effects of Removing the GAT Domain from E.coli GMP Synthetase“ (Abbott, J., Newell, J., Lightcap, C. et al. Protein J (2006) 25: 483. https://doi.org/10.1007/s10930-006-9032-5).We also regarded the original paper from 1985 (“GMP Synthetase " by Howard Zalkin, https://doi.org/10.1016/S0076-6879(85)13037-5) that stated the absorbance at 290 nm for the given amount of XMP within the mixture. In total, we set up the reaction mixture for 50 mL with the following concentrations:

60 mM HEPES
5mM ATP
0.2mM XMP
20mM MgCL2
200mM NH4CL
0.1mM DTT
0.8mM EDTA
Filled up with ddH₂O

However, since XMP and ATP are likely to denaturize, a 20 times higher solution of these (100mM ATP, 4mM XMP) was diluted each. In all following reactions, ATP and XMP were added at a 1:20 ratio. Further on, the reaction mixture including only MgCl2, NH4Cl, DTT, EDTA and HEPES was only filled up to 25 mL so that it could be used in a 1:2 ratio, still leaving room to add ATP and XMP to the complete mixture. For the measurements of the enzyme activity assay, we set up nine samples, all of them including:

100µL of the reaction mixture
10µL of the diluted ATP
10µL of the diluted XMP
76µL ddH₂O

Analytical Measurements of the Products of the Enzyme Reactions

After we had proven that the enzymes showed some activity, the last step was to show that the reactions brought out the desired products. For this purpose, we used the HPLC (high performance liquid chromatography) “LaChrom Ultra” from VWR in combination with the MicroTOFQ mass spectrometer from Bruker to determine the structure of the substances. The combination of these allowed us to separate the substances of the reaction mixtures, analyze their molecular weight and compare them with standards. Generally, in an HPLC measurement, substances (or sample mixtures) are pumped through a certain separation column containing a stationary phase that interacts with the analytes. The more interaction, the longer the analyte needs to flow through the complete column. The duration of this flow is measured by a detector so that conclusions about the analytes can be made.
In combination with the MicroTofQ system, a mass spectrometer, not only the duration of flow through can be measured but also the molecular mass of the substances can be estimated. In general, mass spectrometers transfer the analytes into their gas form and ionize them. Afterwards, they are accelerated and transferred to the analysis system that then separates them according to their masses. Combined, these two systems can give valuable statements about the substances included in a reaction mixture. For our purposes, we used parameters for the MicroTofQ like in (Ruwe et al 2017) with a measurement in negative mode were the masses would be measured substracting an H-atom. However, since we wanted to differentiate between different forms of substances with the same mass, we had to try additional measurement methods for the HPLC. Eventually, we used the “Zip-pHILIC” column with a length of 150 mm and a diameter of 2.1 mm from Merck.
For the mobile phase, we used Ammoniumbicarbonat (pH=9.3) as well as Acteonitril in a ratio of 27 % to 73 %. These were used in isocratic mode with a flow-through of 0.2ml/min. Injected out of the reaction mixture were 2 µL. The whole separation took place at 40 °C.

Final Discussion of the Enzyme Reactions

s it can be seen in Figure 2, the absorption of cytidine at 282 nm began to continuously decrease after the addition of the cytidine deaminase, whereas the absorption remained more or less constant when only water was added. With these results, the activity of our extracted cytidine deaminase could be proven.

Figure (2): Enzyme activity assay for the standard reaction of the cytidine deaminase
After the addition of water, the absorbance at 282 nm stayed the same whereas it decreased after the addition of the CDA. Thus, the enzyme activity is viewable.

The reaction of the cytidine deaminase with xanthosine showed diverse results (Figure 3). Here, also a slight decrease of the xanthosine concentration could be seen, which, however, was not significant.

Figure (3): Enzyme activity assay for the reaction of the cytidine deaminase with xanthosine as a substrate.
After adding CDA to the reaction mixture, a slight decrease in the absorbance at 282 nm was visible. However, as there is also a very small decrease for the addition of water, no significant statement can be made.

The HPLC-MicroTofQ Measurements could only make up the xanthosine and various other substances. However, there were no significant masses and peaks for guanosine or iso-guanosine. (Figure 4)

Figure (4): HPLC-MicroTofQ measurement for the products of the reaction of CDA with xanthosine.
Even if many different masses could be found, none of these could be matched to guanosine or iso-guanosine. For these, a peak should be at about 282 g/mol.

So, with only a slight decrease of the absorbance and no viewable products in the HPLC, it seems reliable that there is only a very small amount of xanthosine converted to iso-guanosine, since the reaction is not specific to the CDA and thus rare. However, supplementary tests and experiments with different reaction mixtures would be needed to further analyze it.
The activity assays of the self-extracted GMPS and the gene synthesis both proved that the enzymes are working correctly, reducing the amount of XMP in the reaction mixture significantly. Therefore, the absorption at 290nm decreased a lot after adding the enzyme to the solution of the self-extracted form of GMPS, whereas the initial decrease was weaker for the gene synthesis. However, both decreased the amount of XMP about the same within the hour in which their reaction was measured. Thus, it can be said that both, the GMPS and the gene synthesis enzymes are working correctly. (See Figure 5 and Figure 6 for comparison)

Figure (5): Enzyme activity assay of the guanosine monophosphate synthetases ordere via gene synthesis.
A significant decrease in the absorption at 290 nm can be made up after the addition of the synthetized GMPS whereas the negative control with water stays at the same absorption.

Figure (6):Enzyme activity assay of the guanosine monophosphate synthetases extracted from Croton tiglium.
A significant decrease in the absorption at 290 nm can be made up after the addtion of the GMPS whereas the negative control with water stays at the same absorption.

As described earlier, it took some time to figure out the right requirements for the HPLC-MicroTofQ measurements, since iso-GMP and GMP have the exact same mass and are thus only separable by their structure. However, with the method chosen in the end, it was possible to identify analytes that seem to represent iso-GMP. Therefore, at first, the general substances within the reaction mix had to be figured out to ensure that only those representing GMP/iso-GMP will be included in the analyses. The general analysis of all substances included showed significant values for all the interesting substrates and products that should be within the reaction mix, including AMP, ADP and ATP, some remaining traces of XMP and of course GMP/iso-GMP (Figures 7 and 8).

Figure (7): HPLC-MicroTofQ measurement for the substances within the reaction mixture of the fully extracted GMPS.
Next to the substrates, ATP and XMP, also resulting substances like ATP and GMP can be found.

Figure (8): HPLC-MicroTofQ measurement for the substances within the reaction mxture of the GMPS with the synthetized sequence.
Next to the substrates, ATP and XMP, also resulting substances like ATP and GMP can be found.

We then compared the resulting form of GMP with a GMP-standard previously provided (10^-5 diluted solution) and the exact measurements of the HPLC. For both, the gene synthesis and the self-extracted form of GMPS the peaks of the substance’s flow-through found at the molecular mass of GMP and iso-GMP (approximately 363.22g/mol, in the graph at approximately 362 g/mol because of the missing H due to the measurement method) were significantly shifted to the right compared to the standard. Thus, the form of GMP that is created with the enzyme reactions of our GMPS and the gene synthesis has to be iso-GMP. (Figure 9)

Figure (9): HPLC-MicroTofQ measurement comparing the GMP standard and the reaction products’ flow-through.
In red the product of the gene synthesis. In blue the one found for the self-extracted GMPS, in green the standard. Even though the standard as well as the mixtures contained compounds that have the same molecular mass, they show different behaviors on the HPLC. The ordinary GMP was significantly faster than the one generated in the enzyme reactions. Thus, the form of GMP that results from the reactions is likely to be iso-GMP.

In conclusion, we did not only figure out the synthesis pathways in L. Croton tiglium but could even recreate some of them, showing that the enzymes expressed in L. Croton tiglium are more likely to generate a different form of GMP (presumably iso-GMP) than the standard one.