Construction of a Saccharomyces Cerevisiae Cell Factory for Shikimate Production through Precise Regulatory Strategies.

Shikimate, a key intermediate in the biosynthesis of diverse high-value aromatic compounds, has significant applications in the food and pharmaceutical industries. However, efficient microbial production remains limited by complex pathway regulations and stringent feedback inhibition. Here, an optimized shikimate biosynthetic route was established in Saccharomyces cerevisiae through promoter engineering and rational pathway redesign. Specifically, replacement of the native shikimate kinase with EcaroK, together with promoter optimization, enabled the shikimate accumulation under non-auxotrophic conditions. Moreover, incorporation of feedback-resistant mutants (aro3D154N and aro4K229L) increased titers to 2.16 g/L, while subsequent cofactor engineering identified nde1 as a key regulatory factor, enabling strain TS-17 to achieve 2.33 g/L in shake-flask fermentation. Furthermore, transcriptome analysis indicated that precursor supply, cofactor regulation, and competitive flux toward downstream aromatic amino acids constitute critical bottlenecks to shikimate production. Ultimately, TS-17 achieved a titer of 30.74 g/L in a 5-L bioreactor in fed-batch fermentation, with a yield of 0.18 g/g glucose, representing the highest level reported in S. cerevisiae to date. Collectively, these results validated the growth-production coupling as an effective strategy, overcoming the cost constraints of auxotrophic strains and establishing versatile principles for engineering efficient shikimate-producing yeasts.