Ectopic expression and functional characterization of type III polyketide synthase mutants from Emblica officinalis Gaertn

Abstract

Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173 bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.

Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in Grewia asiatica L

“…It has been observed that CHS shows wide substrate specificity range as it could accept both aliphatic and aromatic CoA esters to produce various reaction products including the unnatural aromatic polyketide [55]. The differential enzyme activity of different CHS isoforms with different substrates has also been reported in Humulus lupulus [56], Gerbera hybrida [57], Emblica officinalis [58] and others. The differential enzyme activity of Ga CHS2 towards different substrates indicates its evolutionary expansion possibly due to gene duplication “Fig 6”.…”

Cross-Tolerance and Autoimmunity as Missing Links in Abiotic and Biotic Stress Responses in Plants: A Perspective toward Secondary Metabolic Engineering

“…Later, the role of flavonoids in the plant–microbe interaction arsenal was proposed by [ 88 ]. Studies have been conducted on Aegle marmelos [ 89 ] and Emblica officinalis [ 90 ] to unveil the role of active site amino acid residues and the phenotypic changes in their mutants. The overexpression of the quinolone synthase gene in Aegle marmelos induced improved growth, chlorophyll content, and photosynthetic efficiency.…”