Part:BBa_K2139001
Coding Sequence of Endo5a
BBa_K2139001 encompass the coding region for an endo-beta-1,4-glucanase known as Endo5A. Endo5a catalyzes the conversion of cellulose into smaller fragments by making internal cuts in the cellulose chain. It can be used in conjunction with exo-beta-1,4-glucanases for the direct conversion of cellulose into monomeric glucose. The is a monomeric protein with an optimal temperature of 50 degrees Celsius and an optimal pH ranging from 6 to 8. The genbank ascension number for this protein is HQ657203.1 (DNA), AEB00655.1 (amino acid), and a PDB code of 1VRX (homolog). This construct was synthesized by IDT for use in Caulobacter crescentus and is codon optimized for the expression in this species.
To confirm expression of surface layer fusion protein, C. crescentus cultures were grown and western blot analysis were performed on surface proteins from low pH extraction and on the cell lysates. Coomassie Brilliant Blue staining and western immunoblotting was performed. Western blots were probed with primary rabbit anti-RsaA polyclonal antibodies at 1/30,000 dilution. Goat anti-rabbit IgG was used as secondary antibody at 1/50,000 dilution. Fluorophore was detected by Odyssey Infrared Imaging System.
Figure 1. (top) Western Blot of C. crescentus cellulase expressing strains, ran on SDS-PAGE and blotted with anti-RsaA. Left to right: (1) Thermofisher ladder, (2) Gluc1C low pH extracted proteins, (3) Endo5A low pH extracted proteins, (4) E1_399 low pH extracted proteins, (5) E1_422 low pH extracted proteins, (6) ΔGCSS (ΔrsaA) low pH extracted proteins, (7) P4A723 (wildtype) low pH extracted proteins.
150 uL of each two day old culture culture was aliquoted into a clear 96 well plate(Corning) and 150 μL of assay mix (0.1 mg/ml DNPC in 50 mM pH 5.5 potassium acetate buffer) was added to the each well. OD 400 nm and OD 600 nm was measured every 30 minutes for several hours by a VarioScan plate reader (Thermo). Between measurements the culture was incubating at 30°C. Data was normalized by dividing the OD400nm measurement by the OD600nm measurement. The enzyme activity is confirmed by the increased absorbance compared to the wild type control.
Figure 2. Assay for cellulase activity with DNPC substrate.
Triplicate 5mL PYE-CM starter cultures of P4A723 (ΔrsaA C. crescentus complemented with wildtype ΔrsaA in p4A723), E1_399, E1_422, Gluc1C and Endo5A were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining cultures with PYE. 100-fold dilution was used to inoculate the cultures in 200 μL well clear plate containing M2 with 0.2% w/v carboxymethylcellulose (CMC). The data indicates that the cellulase enzymes expressed, and specifically Endo5A, were able to hydrolyse cellulose for C.crescentus growth. There was no growth in the wild type C.crescentus
Figure 3. Cellulase activity and growth assay results for C. crescentus displaying cellulases compared to C. crescentus expressing wildtype RsaA (P4A723).
C.crescentus expressing Endo5A was able to support it's own growth with cellulose as the sole substrate in the system. In addition, these results imply C.crescentus expressing Endo 5A cellulases were able to hydrolyze cellulose into glucose for the growth of E.coli and secondary metabolite product formation in E.coli. The wildtype control had no growth on the same substrate. This shows that the tools of our system, the Endo5A cellulase, was successful in it's purpose.
Figure 4. Consortium growth on cellulose as the only substrate
Literature data for this part can be found http://www.sciencedirect.com/science/article/pii/S1046592812003154
Functional Information: BGU-Israel
While doing the research about Cellulases' activity and specifically Endo5a cellulase functionality, our team (BGU-Israel 2020 Wipeout) discovered a scientific article that shows how to perform enzyme optimization by peptide linkers. In the article, it was stated that Glycine-serine (GS) linkers can be used to construct bifunctional fusions of beta-glucanase and xylanase and show improved catalytic efficiencies of both enzymes in Escherichia coli. Fusions of glucanase (Glu) and xylanase (Xyl) enzymes, constructed with 8 different peptide GS linkers were expressed in Escherichia coli. The catalytic activities of the fusions were compared to the parental enzymes.
Regarding the Endo5a glucanase in topic, the construction with the GS linkers showed 304-426% increase in catalytic efficiency, in comparison to parental enzymes. In the study, the peptide linker "GGGGS GGGGS" (referred as S2 in the article) was best for linking the Glu and Xyl among the tested peptides.
[[File:T--BGU-Israel--Gslinker.jpeg|400px|thumb|center|Figure 1: Kinetic parameters of the parental Glu and Xyl and the moieties in each fusion enzyme separately expressed in E. coli BL21. source: Lu, P., & Feng, M. G. (2008). Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Applied microbiology and biotechnology, 79(4), 579-587.]]
Based on the article's results, we chose the optimized peptide linker to separate the catalytic domain of Endo5a from the cellulose-binding domain (CBD) we used in our project. This way, we intended to optimize the functionality of our cellulase, without changing its sequence.
We recommend future iGEM team to include peptide linkers in their cellulose hydrolyzing complex's designs for improved functionality. Reference: Lu, P., & Feng, M. G. (2008). Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Applied microbiology and biotechnology, 79(4), 579-587. Sequence and Features| None |