Chalcone isomerase-like impedes the lactone shunt and enhances flux partitioning in a bifurcated pathway towards isoflavonoid biosynthesis
Bibliographic record
Abstract
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, Tyr 48 , 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".