A master thesis from Aalborg University
Expression of presumptive Microbial terpene synthase like genes (MTPSLs) from Lophocolea bidentata (liverwort) in moss Physcomitrella patens
Author(s)
Term
4. term
Education
Publication year
2020
Submitted on
2020-06-03
Pages
67 pages
Abstract
Terpenoids are structurally and functionally diverse natural compounds that are voluminous and more divergent in plants and less abundant in microbes. Terpene synthases are the enzymes that proprietarily produce broad range of terpene metabolites in nature by catalyzing the conversion of prenyl diphosphate precursor molecules. Putative microbial terpene synthase like genes (MTPSLs) were identified predominantly from non-seed plants but not in seed plants. This study is aimed at heterologous expression and biochemical characterization of presumptive microbial terpene synthase like genes (LbMTPSL1,3,4and 5) from untapped liverwort species Lophocolea bidentata in model plant Physcomitrella patens by exploiting its indispensable natural in vivo homologous recombination machinery. A new promising protocol using a blend of enzymes Cellulase R-10 and Macerozyme R-10 was utilized for protoplast preparation from P. patens. Homology based structural models for LbMTPSL1,4 have closest structural similarity to Epi-isozizaene synthase from Streptomyces coelicolor and LbMTPSL3,5 have identical stereoview as that of Selinadiene synthase from Streptomyces pristinaespiralis both belonging to C15 subclass, sesquiterpene synthases. Structure based function analysis predicted the active site binding affinity to Farnesyl diphosphate (FPP) for LbMTPSL1,3,5 and its analogue FsPP (Farnesyl thiopyrophosphate) for LbMTPSL4. The three basic residues along with phenol containing amino acid involved in substrate recognition motif and three aromatic residues with cation-π interactions for stabilizing carbocation intermediates were identified from the active site contour of all enzymes. It is believed that this knowledge on structural and chemical biology of LbMTPSLs built on the basis of computational data analysis could be sourceful for future mechanistic characterization and subsequent manipulations of FPP cyclization trajectory by rational design approach to alter the product profile significantly.
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