Chalcone isomerase-like impedes the lactone shunt and enhances flux partitioning in a bifurcated pathway towards isoflavonoid biosynthesis

ABSTRACT The reconstitution of biosynthetic pathways in heterologous hosts is often challenged by the switch to a foreign cellular environment, lacking compatible structural or regulatory features. Auxiliary or non-catalytic proteins can play a critical role in modulating metabolic flux and pathway efficiency. 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 by-product formation, such as the aberrant p-coumaroyltriacetic acid lactone (CTAL). Here, we extended the characterization of CHILs to the legume-characteristic isoflavonoid pathway. We assessed four CHIL orthologs from diverse plant lineages: Glycine max (GmCHIL), Oryza sativa (OsCHIL), Selaginella moellendorffii (SmCHIL), and Marchantia polymorpha (MpCHIL). Structural modelling suggested that naringenin (flavanone) entry into the CHIL binding cleft may be sterically hindered compared to catalytic CHIs. Moreover, legume CHIL isoforms possess an additional bulky residue, Tyr48, that is expected to impose further constraints on ligand binding. In vitro, CHS produced up to 60% lactone CTAL instead of its desired output; however, CHIL suppressed this aberrant activity to 10%, concomitantly increasing target compound titers. Combinatorial enzyme and yeast biotransformation assays revealed a critical role for CHIL in conducting flux through chalcone, flavanone, and isoflavone biosynthesis. The inclusion of CHIL in our engineered yeast strains enhanced overall titers and, unexpectedly, promoted carbon flux toward the so-called deoxy-branch (isoliquiritigenin, liquiritigenin, and daidzein) by up to 67%, with a 33% increase in final daidzein titers. By extending CHIL characterization to the isoflavonoid pathway, we have revealed an expanded role for this auxiliary protein and underscored its utility in engineered metabolic contexts. Our findings reiterate the often-overlooked impact of non-catalytic proteins in shaping specialized metabolism. HIGHLIGHTS Combinatorial enzyme assays reveal a species-dependent preference for CHILs from more closely related plant phyla by soybean CHS, which improved chalcone and downstream flavanone output by suppressing aberrant lactone formation. Yeast co-expressing CHIL with the components of the isoflavonoid metabolon exhibited a 67% increase in flux through a legume-characteristic branch of the pathway, resulting in a 33% increase in titers of the major isoflavone, daidzein. CHIL proteins can be included to engineer the output of phenylpropanoid-derived intermediates preferentially toward isoflavonoid biosynthesis. Auxiliary components, such as CHIL, can be designed to refine metabolic composition in bifurcated pathways, as well as enhancing general flux through pathways. Competing Interest Statement The authors have declared no competing interest.