Pioneering the Formation of 2-Carboxylic Anthraquinone: CRISPR/Cas9-Mediated Functional Validation of Octaketide Synthase and Polyketide Reductase Genes in Aloe vera

Abstract Aloe vera is an authentic medical plant abundant in aromatic polyketides, including the crucial hexaketides aloenin, aloesin, and barbaloin used in pharmaceuticals yet the enzymatic basis of their biosynthesis remained incompletely understood. While it has been suggested that octaketide synthases (OKS) initiates anthraquinones biosynthesis, heterologous expression of OKS alone consistently produces shunt polyketide products, and the mechanism underlying this derailment was uncertain. To comprehend the mechanism of anthraquinone biosynthesis, we combined biochemical constitution, structural characterization and CRISPR/Cas9-mediated editing of key genes in Aloe vera . It was for the first time demonstrated that the inclusion of a PKR (polyketide reductase) redirected the reactive intermediate toward formation of 2-carboxy anthraquinone (C 16 H 12 0 5 ). The identity of reaction product was confirmed by spectroscopic analysis which additionally rendered it clear from compounds previously misannotated in the literature. Alongside, CRISPR/Cas9-based genome editing of OKS and PKR genes resulted in significant reduction (upto 2.54 fold) in aloin content in edited lines compared to the non-edited control aloe line. Together these findings endorsed the presence of tailoring enzyme ketoreductase for the efficient and appropriate formation of anthraquinones and establish a mechanistic framework for polyketide biosynthesis in aloe plants that sustain as an indigenous herb for mankind. Highlights This study provides direct biochemical and genetic evidence that a tailoring enzyme is required to prevent derailment of polyketide intermediates and enable correct anthraquinone formation in plants.