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Chitin synthase NodC from Rhizobium leguminosarum

The Chitin Synthase (CHS) NodC from Rhizobium leguminosarum is an N-acetylglucosaminyl transferase which catalyzes the formation of chitin pentamers by using UDP-N-acetylglucosamine as donor and N-acetylglucosamine as acceptor.

1. Usage and Biology

Besides cellulose, chitin is the most common natural polysaccharide in nature. Chitin is composed of β(1,4) linked 2-acetamido-2-deoxy-β-D-glucose (N-acetylglucosamine). The polymer is a component of fungi cell walls and the exoskeletons of insects and crustaceans, like crabs or shrimps [Dutta et al., 2004; Kumar, 2000]. The extraction of chitin from crustaceans produces a lot of waste and uses a lot of chemicals. But bacteria, like E. coli can produce chitin via a chitin synthase (CHS) in an environmentally friendly manner. The production of chitin appears to be important as it is a useful substance which finds applications in medicinal, industrial and biotechnological research. Chitin, and its derivate chitosan, is non-toxic, biocompatible and biodegradable. Their bioactivities are for example the promotion of wound healing or hemostatic activity, immune enhancement, eliciting biological responses, and antimicrobial activity [Kurita, 2006]. The nodC gene is originating from the gram-negative bacterium Rhizobium leguminosarum and is a homologue to the chitin synthase from yeast. Rhizobium species live in symbiosis with legumes, where the bacteria form nitrogen-fixing nodules in the legume roots. This interaction leads to an activation of the bacterial nodulation (nod) genes and the secretion of Nod factors. nodC belongs to these nod genes which create and modify the Nod factors. The NodC protein has strongly hydrophobic domains which indicate that it is an integral or transmembrane protein.

To learn more about NodC and its part in our project, visit our Wiki: http://2017.igem.org/Team:TU_Darmstadt/project/chitin_synthase.

2. Mechanism

NodC is involved in the synthesis of chitin oligosaccharides, but only with a polymerization degree up to five [Kamst et al., 1995]. NodC uses UDP-N-acetylglucosamine (UDP-GlcNAc) as sugar donor, which is a precursor for the biosynthesis of peptidoglycan and therefore present in growing bacterial cells. The mechanism of elongation proceeds by a successive inverting nucleophilic substitution reaction at C1 of the UDP-GlcNAc – molecule (Figure 1). UDP departs when the O4 atom of the growing sugar chain attacks as a nucleophile [Dorfmueller et al., 2014]. With a low concentration of UDP-GlcNAc NodC produces a mixture of trimers, tetramers and pentamers and with high concentrations of UDP-GlcNAc it produces pentamers solely. It almost exclusively directs the formation of pentasaccharides [Samain et al., 1997].

Figure 1: Mechanism of NodC. The enzyme uses UDP-acetylglucosamine as donor and N-acetylglucosamine as acceptor ad creates chitin pentamers.

Methods

3. Expression

We ordered the nodC gene via IDT sequencing. We inserted the gene into the pSB1C3 vector via the BioBrick system and verified this via sequencing. One pSB1C3 vector has an AraC promoter system (BBa_K2380002) and the other an Anderson promoter with defined cleavage sites (BBa_K2380001). Both vectors have the RBS BBa_K2380024. Afterwards we transformed the vector in E.coli BL21 for expression studies and started the expression, once by induction with arabinose for the AraC promoter system and the other without induction by the constitutive Anderson promoter. To examine the successful expression, an SDS-PAGE was performed.

4. Purification

The next step was the purification of the protein and the verification of the enzyme function. To purify the enzyme, a site-directed mutagenesis was done at the C-Terminus to add a His-taq and the protein was purified via an EKTA in combination with a 1 mL HisTrap column by GE Healthcare.

5. Activity Assays

UDP-Glo^TM Glycosyltransferase Assay

NodC is a N-acetylglucosamine transferase that uses UDP-GlcNAc as donor molecule. To test the functionality of the nodC enzyme, the UDP-Glo^TM Glycosyltransferase Assay from Promega was used. The NodC transfers N-acetylglucosamine from the UDP-GlcNAc to single N-acetylglucosamine bricks. The assay is a homogenous, single-reagent-addition method to detect UDP. In a first reaction the glycosyltransferase adds the UDP-GlcNac to the acceptor molecule and UDP is set free. In a second step the UDP is converted to ATP via a UDP Detection Reagent. This ATP generates light in a luciferase reaction which can be measured using a luminometer.

Figure 2: Principle of the UDP-GloTM Glycosyltransferase Assay. After the glycosyltransferase reaction, UDP Detection Reagent is added and UDP is converted to ATP. This converts UDP to ATP and generates light via a luciferase reaction.

Results

The nodC gene was expressed under the control of constitutive Anderson promoter (BBa_K2380025) and by arabinose-inducible AraC promoter (BBa_K808000) in E.coli Top10. Due to the E.coli cells own proteins it was not possible to verify the NodC protein clearly. To verify the expression of the NodC enzyme a SDS-PAGE was done after the purification instead (Figure 3). The fractions after the ÄKTA purification were collected and the purity was examined via a SDS-PAGE. The SDS-PAGE shows a band at 21 kDA (Figure 3, red arrow) which fits to our protein size. The verification was done by using these fractions for a functional assay for the enzyme. The assay shows enzyme activity and therefore the successful expression.

Figure 3: SDS-PAGE after ÄKTA purification. The SDS-PAGE shows fraction 17 and 18 from the ÄKTA purification. A band at the size of 21 kDa can be seen (red arrow). This band was verfied to be our enzyme by the following activity assay. Marker is the Page Ruler Prestained Ladder from Thermo Fischer.

To verify the functionality of the NodC enzyme, the UDP-Glo^TM Glycosyltransferase Assay was performed. The UDP standard curve shows increasing luminescence with increasing UDP concentration. With this curve the conversion to free UDP can be calculated. The evaluation of the assay with sample 17 (vgl Figure 3) shows that the NodC enzyme converts the UDP-GlcNAc to free UPD and a growing oligo-GlcNAc-chain. So the assay shows that the NodC enzyme can create chitin oligomers. All data were measured as duplet and the standard deviation is shown.

Figure 4: UDP standard curve. The standard curve shows the linearity and sensitivity of the UDP-Glo™ Lycosyltransferase Assay. The curve was prepared over the indicated range of UDP concentration in 25 µl buffer. The luminescence was measured after 1 hour of incubation with a Tecan200 Infinite Pro plate reader. Values represent the mean of two replicates and the standard deviation is shown. For better visibility the values for the small concentrations are shown in the additional graph. The following assay (Figure 5) is based on this standard curve. RLU = relative light units.

Figure 5: Activity assay of NodC. The NodC (40 ng) was titrated in 1X glycosyltransferase reaction buffer the presence of 100μM of UDP-N-acetylglcosamine and 10mM N-acetylglucosamine (GlcNAc) as an acceptor substrate. The reaction was performed as described before and the luminescence was measured after 1 hour of incubation with a Tecan200 Infinite Pro plate reader. Each point is an average of two experiments, and the error bars represent the standard deviations. RLU = relative light units.

Improved Part

Northwestern 2010 (http://2010.igem.org/Team:Northwestern/Project)
The iGEM Team from Northwestern ran the project “SCIN – Self-regenerating Chitin Induction” in 2010. For this project they tried to produce the chitin synthase CHS3 from Saccharomyces cerevisiae in E. coli. This CHS3 is found as the major enzyme in its family. For our project, we also need an enzyme which produces chitin. We considered using this CHS3 from Northwestern, but after taking a closer look we decided otherwise. The CHS3 is a transmembrane protein originating from eukaryotes, which is challenging to integrate in prokaryotes, although the Northwestern team used the cDNA. Furthermore, the enzyme is post-translationally glycosylated, which makes it nearly impossible to express a functional enzyme [1].
The sequence of the CHS3 further contains forbidden PstI and XbaI sites (typical standard RFC[10]) inside of the gene (XbaI at 1117 and 1674 and PstI at 2318), so that it is impractical for usage as BioBrick part [2].

[1] Cos, T., Ford, R. A., Trilla, J. A., Duran, A., Cabib, E., and Roncero, C. (1998) Molecular analysis of Chs3p participation in chitin synthase III activity. The FEBS Journal, 256, 419 – 426 DOI: 10.1046/j.1432-1327.1998.2560419.x
[2] iGEM Registry, Part: BBa_K418007, by Northwestern University 2010 https://parts.igem.org/wiki/index.php?title=Part:BBa_K418007

Sequence and Features