Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics
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Bibliographic record
Abstract
BACKGROUND: L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. RESULTS: Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4(FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520 μmol/g DCW or 192 mM in the cytosol, but sustained flux through this pathway was found to depend on the complete elimination of feedback inhibition and degradation pathways. CONCLUSIONS: Our targeted metabolomics approach confirmed a likely regulatory site at DAHP synthase and identified another possible cofactor limitation at prephenate dehydrogenase. Additionally, the genome-scale metabolic model identified design strategies that have the potential to improve availability of erythrose 4-phosphate for DAHP synthase and cofactor availability for prephenate dehydrogenase. We evaluated these strategies and provide recommendations for further improvement of aromatic amino acid biosynthesis in S. cerevisiae.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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) | 0.000 | 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 it