Difference between revisions of "Team:Bielefeld-CeBiTec/Results/unnatural base pair/biosynthesis"

The plant Croton tiglium is of great importance to our project due to its ability to produce isoguanosine. In order to utilize the production pathways of this novel DNA component, we aimed at rebuilding this biosynthesis pathway of C. tiglium in E.coli. Therefore, we collected samples from different tissues of this plant. First, we isolated RNA from all these plant tissues. One normalized library as well as five tissue specific libraries were constructed for RNA-Seq analysis. A trinity assembly revealed unique enzymes of the purine metabolism in C. tiglium. The DNA of the enzymes of interest was extracted from the library or ordered via Gene synthesis for expression in E. coli to purify these enzymes via IMPACT® Kit(New England Biolabs). Functionality of the produced enzymes was confirmed in iso-GMP and isoguanosine formation assays. For further validation of the reaction products, we applied high performance liquid chromatographic (HPLC) analysis followed by mass spectrometry (MS) analysis. Parts encoding these enzymes were submitted to facilitate the use of these novel DNA components in the iGEM community.

Croton tiglium samples were kindly provided by the botanic garden of the Philipps University in Marburg. Total RNA was extracted from frozen tissue samples of young leaves, stem, inflorescence, seeds and roots. Mortar and pestle were used to grind the material in liquid nitrogen. Spectrum Plant Total RNA Kit was used for the RNA extracting according to the suppliers’ instructions. mRNAs were enriched based on their polyA tail via oligo-dT beads. DropSense16 (trinean) was used for quality control.

Enriched mRNA samples from different tissues were used for the construction of a normalized library ( vertis biotechnology ag ). In parallel, tissue specific samples were submitted to fragmentation prior to reverse transcription into cDNA via ProtoScriptII (NEB) based on suppliers’ recommendations. The Illumina TruSeq Stranded mRNA Sample Preparation Guide was used for the generation of five tissue specific libraries with an average insert size of 400 bp. Those libraries represent young leaves, stem, inflorescence, seed, and root.

Reads from tissue specific data sets were mapped to the initial transcriptome assembly via STAR {Dobin, 2013} with 90 % length and 95 % identity. A customized python script was developed to generate a matching annotation file for the assembly. featureCounts {Liao, 2014} was applied to quantify the expression of all sequences in the assembly. Since most transcripts are represented by multiple contigs representing different splice variants, we decided to include multi-mapped reads. Dedicated python scripts were used for further investigation of the expression data as well as for the generation of expression heatmaps via the seaborn module. VENN diagram generation was performed at Bioinformatics.psb.

In total, 45.5 million 2x250 nt paired-end reads were assembled into the 431.8 Mbp transcriptome comprising 388,181 contigs. The high continuity of the assembled contigs can be described by the E90N50 of 2,246 bp and the E90N90 of 452 bp. The completeness check indicated a sufficient amount of sequencing data were generated. BUSCO analysis revealed the presence of 94 % complete BUSCOs. In addition, 3 % are present in fragmented form and only 3 % are missing in this transcriptome assembly.

Known sequences for specific enzymes from related species were subjected to a uniprot, we used BLAST search against the transcriptome assembly. We identified several putative pathways for the isoG biosynthesis based on existing databases like uniprot, we used KEGG.

Firstly, there is the guanosine monophosphate synthase (GMPS), an enzyme from the class of ligases that form carbon-nitrogen-bonds with glutamine as an amido-N-donor acceptors (see KEGG for more information). It is also known as ‘Guanosine monophosphate synthetase’. GMPS is needed for the amination of XMP (xanthosine monophosphate) to create GMP and possibly iso-GMP in the case of C. tiglium. Besides, GMPS can be found in many organisms apart from Croton tiglium, including Homo sapiens and E. coli. The transcriptome assembly contained two sequences that displayed a strong similarity to known GPMS encoding sequences. These sequences encode peptides of 314 amino acids and molecular mass of approximately 59.46 kDa. GMPS is a promising candidate, since it may not only be able to catalyze the reaction of XMP to GMP but also to iso-GMP.
Another interesting enzyme from the purine metabolism is the Inosine monophosphate-dehydrogenase (IMPDH) that matched three sequences in the transcriptome assembly. 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 (see KEGG). The different forms of IMPDH encoded by sequences in the transcriptome assembly have a molecular mass of 53-58 kDa and amino acid lengths between 500 to 550. In the purine metabolism, IMPDH is the catalyst of the synthesis of XMP out of inosine monophosphate (IMP). Therefore, it could enable the biosynthesis of an isoform of XMP that might then even be a substrate for the production of iso-GMP.
Furthermore, the cytidine deaminase (CDA) seemed to be of immense potential. The CDA, which belongs to the class of hydrolases acting on carbon-nitrogen bonds different from peptide bonds (see KEGG) is usually applied to deaminate cytidine to uridine. However, there is also the possibility of the reverse reaction catalyzed by CDA. A reaction from xanthosine to iso-GMP might be possible. The best matching sequence for CDA in the transcriptome assembly encodes 535 amino acids . The putative gene product has a molecular mass of 33.95 kDa.
Aside from these enzymes, the adenylosuccinate synthetase (ADSS) could be an interesting candidate. The ADSS belongs to the class of ligases, which are forming carbon-nitrogen bonds (see KEGG). Only one matching sequence was identified in the transcriptome assembly. The encoded gene product has a molecular weight of 53.32 kDa and a size of 489 amino acids. In C. tiglium, it is expected to catalyze the reaction of IMP to adenylosuccinate that will then be further processed into AMP.
Finally, we identified the enzyme xanthine dehydrogenase(XDH) as promising candidate. The XDH converts 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 (see KEGG), and could even be matched with six sequences of the trinity assembly. The encoded gene products are expected to have a molecular mass of 64.12 kDa and a size of 587 amino acids.

A modification of the NEB Impact® Kit was used for the purification of heterologous expression in E. coli. The protein purification via the Impact system works with the usage of an intein tag. Impact is short for “Intein Mediated Purification with Affinity Chitin-binding Tag”. In short, the coding sequence of the candidate gene is linked with an intein tag that enables the protein’s purification. As a vector, we used pTXB1 that is responsible for a c-terminal fusion of the intein tag. The expression strain ER2566 with the plasmid pRARE-2 was used. This vector is used to compensate for a bad codon usage as it encodes some rare tRNAs.

Concentrations of purified proteins were estimated based on a modification of the Bradford Assay (Bradford, M., 1976) Roti®-Nanoquant by Carl Roth. Depending on the protein, we reached concentrations from 1.35 up to 5.31 grams per liter (Table 2).

Table 2: Concentrations of the proteins, estimated with Roti® Nanoquant. Replicants were created of the most important enzymes.

Protein name	Concentration in mg/mL
GMPS iso-form 1	4.1775 2.1507 1.4610
GMPS iso-form 2	1.3497
IMPDH form 1	4.4007
IMPDH form 2	4.1763
ADSS	4.2616
XDH	4.1302 1.9349
CDA	5,3092 1,8054 1,3881

Afterwards, we confirmed protein identity via SDS-PAGE and MALDI-TOF-MS.

After we knew that all proteins had been extracted properly, our next step was to test their functionality. Therefore, we used the plate-reader “Tecan infinite® 200” and the program “Tecan i-control, 1.10.4.0”. For all enzyme reactions, we used room temperature to meet the physiological conditions of these plant enzymes. Values were plotted to show the absorption of the main substrate before and after the addition of the enzyme or water, respectively (Figures 2-3 as well as 5-6, also see Final Discussion). Also all of them showed only strong activity within the first hour, we performed over-night enzyme activity assays to reach the final end point. To verify the reaction product, we used the HPLC (high performance liquid chromatography) ”LaChrom Ultra” in combination with the MicroToFQ mass spectrometer. The combination of these separation systems allowed us to separate the substances of the reaction mixtures, analyze their molecular weight and compare them with standards. 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 subtracting the mass of 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) and acetonitril in a ratio of 27 % to 73 %. This was used in isocratic mode with a flow-through of 0.2ml/min. The injection volume was set to 2 µL of the reaction mixture from the corresponding enzyme assay. The separations took place at 40 °C. Since our main goal was to produce iso-GMP or iso-Guanosine using the purified enzymes of Croton tiglium, we focused on the main promising candidate enzymes: both iso-forms of GMPS ( BBa_K220160 and BBa_K220161) and CDA( Part BBa_K220162).

First, we set up an enzyme activity assay for CDA with cytidine to ensure its activity following the protocol by Robert M. Cohen and Richard Wolfenden from 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 containing 50 mM TRIS-HCl buffer (pH 7.5) and 0.167 mM cytidine as a substrate.
We used six replicates with 196 µL of the mixture and measured it for about 20 min (measurement all 30 sec) with the Tecan infinite® 200. We then paused the measurement program to add 4 µL (6 µg) of the previously extracted CDA or 4 µL of water to three samples each. Then, we immediately continued the measurement for about an hour.
As 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.

After confirming general activity of CDA, 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:

Afterwards we set up new activity assays, using 196 µL of the reaction mixture in six of the well plate’s holes. After measuring the absorbance at 282 nm, we added 4 µL of either water or the enzyme (6 µg) to three biological replicates each, continuing the (previous) measurements for about an hour.

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.

GMPS

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