Part:BBa_K2380000
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.
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_K808000) and the other an Anderson Promotor with defined cleavage sites (BBa_K2380025). 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-GloTM 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-GloTM 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 3: 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
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 373
- 1000COMPATIBLE WITH RFC[1000]
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