Redesigned Pathway for <i>De Novo</i> Synthesis of Vanillin and Co-conversion of Multiple Renewable Substrates in <i>Saccharomyces cerevisiae</i>

Vanillin, known as the "queen of flavors", is an extensively used and important aromatic compound with multiple functions, including aldehyde, ether, and hydroxyl. Converting lignocellulosic biomass-derived substrates to natural vanillin is sustainable and economical through artificial cell factories. However, the inefficiency of exogenous enzymes and the toxic effect of vanillin on cells limit its production. In this study, pathway reconstruction and integration site optimization of <i>Saccharomyces cerevisiae</i> enabled the production of 363.0 mg/L of vanillin in just four steps, with chorismate serving as an intermediate, by enhancing the endogenous shikimate pathway. A feeding strategy further yielded a record titer of 533.0 mg/L vanillin using engineered <i>S. cerevisiae</i> in flask shake fermentation. Promoter optimization enabled module adaptation for co-conversion of lignocellulose-derived monomers, including glucose, xylose, vanillic acid, <i>p</i>-coumaric acid, and ferulic acid, toward vanillin. Glycosylation of vanillin enabled the removal of product feedback inhibition, achieving a titer of 1745.5 mg/L glucovanillin through the co-conversion of multisubstrate. As a result, the artificial cell factories achieved the <i>de novo</i> production of vanillin and synthesized 1339.5 and 7476.5 mg/L glucovanillin (equivalent to 3619.4 mg/L vanillin) from glucose in shake flasks and 5-L bioreactors, respectively. The designed artificial cell factories of vanillin provided an alternative strategy for the industrial production of vanillin from sustainable resources.