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In-silico Development of Novel Plasmid Construct for Production of Recombinant Chitinase in Saccharomyces cerevisiae.

Contents. Objectives Introduction Research Background Materials and Methods Results and Discussion Conclusion Reference.

Objectives. To develop specialized plasmids with precise regulatory genetic elements to control the expression of target genes. To optimize the expression of heterologous recombinant proteins in yeast. To facilitate the production of new industrial enzymes..

Introduction. Chitin, a robust biopolymer found in the exoskeletons of insects and crustaceans. After cellulose, chitin is the most prevalent biopolymer on earth. It is present in the fungal cell walls as well as the exoskeletons of insects, crustaceans and worms. N-acetyl-D-glucosamine (GlcNAc) units bonded together by β-1,4 glycosidic linkages form the linear polymer known as chitin. Chitinase enzyme catalyze the breakdown of chitin. It has application in biocontrol of pests and diseases, food processing and bioremediation..

Introduction. Chitinases have a wide range of applications, but their production is challenging. S. cerevisiae is a good host for the production of heterologous proteins. Expression of multiple chitinase genes in a single production host is highly expected for industrial production of enzyme..

Research Background. Figure 1: The structure of chitin molecule displays two of the N-acetylglucosamine units that repeat to create lengthy chains in the β-(1→4)-linkage[1]..

Research Background. A. B. Figure 2: 2D (A) and 3D (B) structure of chitinase[2].

Research Background. Classification of chintinase enzyme based on their substrate specificity Endochitinases: Randomly cleave chitin chains at internal glycosidic bonds. Exochitinases: Cleave chitin chains at the ends of the chains. Classification of chintinase enzyme based on their amino acid sequence and catalytic mechanism Family 18 chitinases: Found in all kingdoms of life. Family 19 chitinases: Found in plants and some bacteria..

Research Background. &tysmyrre. Figure 3: Action mechanism of Chitinases. [3].

Research Background. Wild-Type Chitinase A seasoned professional, shaped by natural selection. No fancy tags, no genetic fanfare. Years of adaptation, fine-tuning, and natural selection. Recombinant Chitinase Crafted through the precision of genetic manipulation. A promising competitor. Equipped with the molecular recognition key: Genetic Tag..

Research Background. Advantages of producing recombinant chitinase in yeast Can be used to produce large quantities of chitinase Enzyme production could be increased many fold in compare to wild type chitinase Issues that we focus in this work To increase the production rate of Chitinase in S. cerevisiae. To express multiple genes in single host. To control the regulation of gene expression in yeast..

Research Background. Table1. List of some fungal strains producing natural chitinase enzymes..

Research Background. Fungal strain Temperature (°C) pH Medium composition Trichoderma viride 28 5.0 Mineral salt medium + chitin Talaromyces flavus 28 6.0 Synthetic medium (SMCS) with colloidal chitin and sucrose as carbon sources Aspergillus fumigatus 30 6.0 Potato dextrose broth + chitin Aspergillus niger 26 6.5 Medium with colloidal chitin and shrimp shell Aspergillus terreus 30 6.0 Mineral salt medium with fish scale waste Aspergillus flavus 32 6.4 Wheat bran (CWB) with chitin powder Aspergillus nidulans 32 5.5 Czapek-Dox medium + chitin.

Materials and Methods. List of plasmids constructed in this work Plasmid 1: pESC-TRPGAl1,10Chitinase2 plasmid 2: pESC-TRPPHO5chitinase2.

Materials and Methods. Primer Sequence Length GC Content (%) CHI3_ FOR1 GCGCGAATTCCATCATCACCATCACCACATAAACCATATTGCTGCTATTCACCAAGT 57 44 CHI3_ REV1 GAGCTCCATCATCACCATCACCACCGAAACAAGACGAAACAAAACAACGAAC 52 46 CHI3_ FOR2 CCCGGGCATCATCACCATCACCACATAAACCATATTGCTGCTATTCACCAAGT 53 47 CHI3_REV2 CCGCGGCATCATCACCATCACCACCGAAACAAGACGAAACAAAACAACGAAC 52 50.

Materials and Methods. Primer Sequence Length GC Content (%) CHI3_ FOR1 GCGCGAATTCCATCATCACCATCACCACATAAACCATATTGCTGCTATTCACCAAGT 57 44 CHI3_ REV1 GAGCTCCATCATCACCATCACCACCGAAACAAGACGAAACAAAACAACGAAC 52 46 CHI3_ FOR2 CCCGGGCATCATCACCATCACCACATAAACCATATTGCTGCTATTCACCAAGT 53 47 CHI3_REV2 CCGCGGCATCATCACCATCACCACCGAAACAAGACGAAACAAAACAACGAAC 52 50 PHO5_FOR GCGCCACCGGTATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTT 48 40 PHO5_REV GCTTCGAACTATTGTCTCAATAGACTGGCGTTGTAATGA 39 41.

Materials and Methods. PCR condition for CHI3 pESC-TRPGAl1,10Chitinase2 PCR condition for pESC-TRPPHO5chitinase2 Cycle 1: 5 m, 95οC; 29 s, 59οC; 20 s, 72 0C; Cycle 1: 5 m, 95οC; 29 s, 59οC; 20 s, 72 0C; Cycle 2 (5 times): 45 s, 950C; 29 s, 590C; 20 s, 720C; Cycle 2: (5 times): 45 s, 95 0C; 29 s, 59 0C; 20 s, 72 0C; Cycle 3 (30 times): 45 s, 95 0C; 29 s, 610C; 30 s, 72 0C; Cycle 3: (30 times): 45 s, 95 0C; 29 s, 610C; 30 s, 72 0C;.

Materials and Methods. Figure 4 : Genetic map of pESC-TRP commercial plasmid where 2µ ori- Yellow, TRP1- Brown, GAL 1,10- White..

Methodology. Primer designed for target DNA fragment/ gene containing suitable restriction sites Restriction digestion of PCR fragment and pESC-TRPGAl1,10Chitinase2 with same pair of restriction enzymes Ligation of PCR fragment and pESC-TRPGAl1,10Chitinase2 using DNA Ligase PCR amplification of DNA fragment / gene.

Methodology. Figure 5 :CHI3 gene amplification and insertion into pESC-TRPGAl1,10Chitinase2.

Methodology. Primer designed for target DNA fragment/ gene containing suitable restriction sites Restriction digestion of PCR fragment and pESC-TRPPHO5chitinase2 with same pair of restriction enzymes Ligation of PCR fragment and pESC-TRPPHO5chitinase2 using DNA Ligase PCR amplification of DNA fragment / gene.

Methodology. Figure 6 : CHI3 gene, Pho5 promoter gene amplification and insertion into pESC-TRPPHO5chitinase2.

Results and Discussion. Figure 7 : Restriction map of CHI3 1 gene where CHI3 gene – Purple..

Results and Discussion. Figure 7 : Restriction map of CHI3 2 gene where CHI3 gene – Purple..

Results and Discussion. Figure 9: Genetic map of pESC-TRPGAl1,10Chitinase2 where 2µ ori- Yellow, TRP1- Brown, GAL 1,10- White and CHI3 gene – Purple..

Results and Discussion. Figure 10 : Restriction map of Pho5 gene where Pho5 gene – Red..

Results and Discussion. Figure 12: Genetic map of pESC-TRPPHO5chitinase2 where 2µ ori- Yellow, TRP1- Brown, GAL 1,10- White, pho5 promoter - Red and CHI3 gene - purple.

Conclusion. Two new plasmid constructs for the production of chitinase enzymes in S. cerevisiae is designed. The chitinase activity of the transformed S. cerevisiae strains with the constructed plasmids would significantly increase chitinase activity. Expression of multiple genes in yeast could be controlled using the constructed plasmids. These results demonstrate that In-silico plasmid construction is a promising approach for the development of new plasmid constructs for production of industrial enzymes..

Reference. https://en.wikipedia.org/wiki/Chitin www. Chemsrc.com., https://www.uniprot.org/uniprotkb/P40954/entry, J D Robertus, A. F. ( 1999). The structure and action of chitinases. EXS, 87:125-35. Amir Sk. Hossain, R. T. (2019). Comparison of two models of surface display of xylose reductase in the. Enzyme and Microbial Technology , 8–14. Rathore, A. S., & Gupta, R. D. (2015). Chitinases from Bacteria to Human: Properties, Applications, and Future Perspectives. Enzyme Research, 2015, 791907. Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria—PMC. (n.d.). Retrieved October 30, 2023. SciELO - Brazil—Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A. (n.d.). Retrieved October 30, 2023. Tippelt, A., & Nett, M. (2021). Saccharomyces cerevisiae as host for the recombinant production of polyketides and nonribosomal peptides. Microbial Cell Factories, 20, 161. Distribution and Biotechnological Applications of Chitinase: A Review. (n.d.). Retrieved October 30, 2023.

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