Team:IISER TVM/project design

IGEM-IISERTVM

Project


Project Design

Fungal cell walls are rigid and made up of complex polysaccharides called chitin and glucans. Chitinases are hydrolytic enzymes that degrade chitin and are secreted by plants, mammals and bacteria as a natural defence against various pathogenic fungi [1]. They are also produced by arthropods and fungi to remould their chitin walls. Chitinases are classified into three families: GH18, GH19 and GH20 glycosidic hydrolase families. Many family 18 chitinases contain a catalytic domain(CD) which has a triosephosphate isomerase barrel(TIM barrel) structure and an additional chitinase insertion domain [2]. Often separate chitin-binding domains(CBD) are present in the carboxy-terminal of the proteins. Studies have shown enhanced activities for chimeric chitinases with additional domains than the native structure [3]. Here, we proposed to develop hybrid bacterial and plant chitinases by fusing domains from different source organisms.


We designed two chimeric bacteria chitinases from three bacterial sources - Pseudoalteromonas sp DL-6, Amycolatopsis orientalis, and Serratia marcescens.


  1. In the first chimeric combination, We replaced the catalytic domain of Serratia marcescens with Pseudoalteromonas catalytic domain with the basic structure of Serratia chitinase.

    Basic outline: Amino Acids at the beginning of Serratia ChiB natural sequence — CBD of S. marcescens Chi B — CD of Pseudoalteromonas Chi A — Chi B natural linker between CD and CBD of S. marcescens — CBD of S. marcescens .


    Fig1. Basic domain structure of Bacterial Chitinase Combo 1


  2. In the second chimeric combination, we chose chitinase from Amycolaptosis orientalis which lacks a chitin-binding domain. So we added the chitin-binding domain of S. marcescens Chi B in the C-terminal of the Amycolatopsis orientalis chitinase.

    Basic outline:Chitinase seq. of Amycolatopsis orientalis — CBD of S. marcescens Chi B


    Fig2. Basic Domain Structure of Bacterial Chitinase Combo 2



We chose Serratia marcescens chitinase B since it has an effective activity spectrum against a range of fungal species[4][5]. But the optimum temperature and pH of action is not around body and room temperature which led to the selection of Pseudoalteromonas catalytic domain since its chitinase is active around body temperature[6]. The catalytic domain is majorly responsible for the temperature and pH dependence for the activity.


The selection of Amycolaptosis orientalis was done at a later phase of the project when the country saw a major outbreak of mucormycosis cases caused by Rhizopus species [7]. There were studies regarding the activity of Amycolatopsis orientalis chitinases in the lysis of cell walls of fungal species from the Rhizopus genus.[8] Thus we decided to incorporate Amycolatopsis orientalis as one of the sources for our therapeutic chimeric chitinase.


Apart from bacterial chitinases, we also chose plant chitinases as per the suggestions from structural biology experts, Prof. M. R. N. Murthy and Prof. H. S. Savithri from IISc Bangalore. The idea supporting this fact was that plants would have evolved an efficient system of chitinases to fight various phytopathogenic fungi. Thus, we also designed two chimeric plant chitinase combinations using chitinases from two sources - Triticum aestivum (wheat) and Hordeum vulgare (Barley). The cloning and the succeeding downstream assay experiments could not be performed due to a lack of time. Both of these plant chitinases show efficient chitinolytic activity and retain activity at body temperature and pH range. [9][10]


  1. In the first combination, we joined the sequences of both barley and wheat chitinases using common (GGGS)3 linkers. Wheat chitinase doesn’t have a chitin-binding domain, so we added barley CBD in its C-terminal in the combination.

    Basic Outline : AA seq before Barley CBD — Barley CBD — Barley CD — AA after CD barley — (GGGS)3 — AA seq before wheat CD — Wheat CD — natural linker from barley — Barley CBD


    Fig3. Basic Domain Structure of Plant Chitinase Combo 2


  2. In the second combination, we created a chimeric chitinase with a wheat catalytic domain flanked by two barley chitin-binding domains.

    Basic Outline : AA before barley chitinase — Barley CBD — natural linker from barley — Wheat CD — natural linker from Barley — Barley CBD.


    Fig4. Basic Domain Structure of Plant Chitinase Combo 2


The sequences that we created are codon-optimized in order to clone and express them in E. coli .


Keys :CBD: Chitin binding domain CD: Catalytic domain AA: Amino acids


  1. Javed, Saleem et al. "Chitinases: An Update". Journal Of Pharmacy And Bioallied Sciences, vol 5, no. 1, 2013, p. 21. Medknow, doi:10.4103/0975-7406.106559.
  2. Stavros, Philemon et al. "The Stability Of The TIM-Barrel Domain Of A Psychrophilic Chitinase". Biochemistry And Biophysics Reports, vol 3, 2015, pp. 108-116. Elsevier BV, doi:10.1016/j.bbrep.2015.07.016.
  3. Matroodi, Soheila et al. "Designing A New Chitinase With More Chitin Binding And Antifungal Activity". World Journal Of Microbiology And Biotechnology, vol 29, no. 8, 2013, pp. 1517-1523. Springer Science And Business Media LLC, doi:10.1007/s11274-013-1318-0.
  4. Harpster, Mark H., and Pamela Dunsmuir. "Nucleotide Sequence Of The Chitinase B Gene OfSerratia marcescensqmb1466". Nucleic Acids Research, vol 17, no. 13, 1989, pp. 5395-5395. Oxford University Press (OUP), doi:10.1093/nar/17.13.5395.
  5. Li, Jincheng et al. "Expression And Characterization Of A Chitinase From Serratia marcescens". Protein Expression And Purification, vol 171, 2020, p. 105613. Elsevier BV, doi:10.1016/j.pep.2020.105613.
  6. Wang, Xiaohui et al. "Characterisation Of A Chitinase From Pseudoalteromonas Sp. DL-6, A Marine Psychrophilic Bacterium". International Journal Of Biological Macromolecules, vol 70, 2014, pp. 455-462. Elsevier BV, doi:10.1016/j.ijbiomac.2014.07.033.
  7. "Mucormycosis (Zygomycosis): Background, Etiology And Pathophysiology, Epidemiology". Emedicine.Medscape.Com, 2021, https://emedicine.medscape.com/article/222551-overview.
  8. "Purifications And Some Properties Of Two Chitinases From Streptomyces orientalis Which Lyse Rhizopus Cell Wall". 2021, p. ., https://www.tandfonline.com/doi/abs/10.1080/00021369.1976.10862407.
  9. Kirubakaran, S. Isaac, and N. Sakthivel. "Cloning And Overexpression Of Antifungal Barley Chitinase Gene In Escherichia Coli". Protein Expression And Purification, vol 52, no. 1, 2007, pp. 159-166. Elsevier BV, doi:10.1016/j.pep.2006.08.012.
  10. Singh, Arpita et al. "Heterologous Expression Of New Antifungal Chitinase From Wheat". Protein Expression And Purification, vol 56, no. 1, 2007, pp. 100-109. Elsevier BV, doi:10.1016/j.pep.2007.06.013.