Abstract
Large amounts of xylose mother liquor (XML) are produced as a byproduct of the xylose industry and are often treated as waste. Salidroside is a high-value glycoside with pharmaceutical and nutraceutical applications, but conventional extraction from plants cannot meet market demand and threatens the sustainability of Rhodiola resources. Here, XML was valorized for de novo salidroside production using engineered Kluyveromyces marxianus. The KmPDC1 disruption promoted pyruvate accumulation, which in turn enhanced the accumulation of a shikimate pathway precursor (phosphoenolpyruvate). Introduction of the salidroside biosynthesis pathway enabled salidroside production in K. marxianus. Additionally, salidroside production was improved through three strategies: expressing feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase in the shikimate pathway, expressing phosphoketolase to boost erythrose-4-phosphate (another precursor of the shikimate pathway), and expressing chorismate mutase/prephenate dehydrogenase to facilitate tyrosine synthesis. The control of pH during fermentation further enhanced salidroside production. The final strain YSAL07 produced 10.17 ± 0.12 g/L salidroside in shake-flask cultivation, with a productivity of 84.75 mg/(L·h), which is higher than previously reported values. In the bioreactor, YSAL07 produced 13.18 ± 0.18 g/L salidroside within 96 h using glucose as the substrate, whereas fermentation with XML achieved a salidroside titer of 15.49 ± 0.69 g/L within 120 h. This study demonstrates the first gram-scale (≥10 g/L) salidroside production in K. marxianus directly from XML, providing a sustainable and economically viable route for the manufacture of high-value natural products.