Chalcone isomerase-like (CHIL) impedes the lactone shunt and modulates flux partitioning in isoflavonoid biosynthesis

The reconstitution of biosynthetic pathways in heterologous hosts is often challenged by the transition to a foreign cellular environment, lacking compatible structural or regulatory features. Auxiliary or non-catalytic proteins can play a critical role in guiding metabolic flux. Chalcone isomerase-like (CHIL) is a non-catalytic protein known to serve as a partner to chalcone synthase (CHS) in flavonoid biosynthesis, rectifying its promiscuous activity and preventing the formation of by-products, such as p-coumaroyltriacetic acid lactone (CTAL). Here, we extended the functional analysis of CHILs to the legume-characteristic isoflavonoid pathway. We assessed CHIL orthologs, using sequence analysis and structural modelling to predict their respective binding capacities, followed by functional characterization. The addition of CHIL to enzyme assays containing CHS, alone or with downstream enzymes, chalcone reductase (CHR) and chalcone isomerase (CHI), reduced CTAL levels (up to 42 %) while simultaneously increasing the output of desired intermediates. Combinatorial yeast biotransformation assays revealed that CHIL plays a crucial role in directing metabolic flux through chalcone and flavanone pathways, and, with co-expression of isoflavone synthase (IFS), in isoflavone biosynthesis. The inclusion of CHIL in engineered yeast strains enhanced overall titers and, unexpectedly, promoted partitioning toward the deoxy-branch (isoliquiritigenin, liquiritigenin, and daidzein) up to 50 %, with a 33 % increase in final daidzein titers. Therefore, we have revealed an expanded role for CHIL as an auxiliary protein in isoflavonoid biosynthesis and underscored the utility of non-catalytic proteins in metabolic engineering.