Shikimate crosstalk enables record-level homogentisic acid production in Yarrowia lipolytica

Homogentisic acid (HGA; 2,5-dihydroxyphenylacetic acid) is an aromatic compound with diverse applications in pharmaceuticals, food, and functional materials. Industrial HGA production still relies mainly on multistep chemical synthesis routes, which are associated with safety risks and environmental burdens. Here, we report that truncated HMG-CoA reductase (tHMGR), a key enzyme of the mevalonate pathway, unexpectedly serves as a dominant metabolic driving force that enhances shikimate-derived HGA biosynthesis in Yarrowia lipolytica. Through a targeted single-gene screen of the mevalonate pathway, tHMGR emerged as the determinant of HGA overproduction, and copy-number optimization increased the shake-flask titer 2.65-fold to 1766.43 mg/L. Transcriptomic analysis and metabolic network evaluation revealed that the massive NADPH consumption driven by tHMGR perturbs the intracellular redox balance. This cofactor depletion triggers a compensatory upregulation of l-tryptophan biosynthesis and shikimate-associated genes, uncovering a previously unrecognized mevalonate-shikimate metabolic crosstalk. The generality of this tHMGR-driven effect was further validated using p-coumaric acid as a shikimate-derived reporter. Finally, fed-batch fermentation achieved 33.71 g/L HGA in a 2-L bioreactor, representing the highest titer reported to date, and establishing tHMGR as a powerful metabolic lever for enhancing shikimate-derived aromatic production in Y. lipolytica.