Development of robust dominant-metabolism compromised intermediate chassis of ethanologenic Saccharomyces cerevisiae for non-ethanol biochemical production.

Robust microbial cell factories (MCFs) are pivotal for sustainable biomanufacturing. The industrial model chassis Saccharomyces cerevisiae is often constrained by its dominant ethanol biosynthesis to produce other biochemicals. To overcome this limitation, we employed a dominant-metabolism compromised intermediate (DMCI) chassis strategy to reconfigure the central metabolism of S. cerevisiae. We first constructed a non-ethanol-producing DMCI chassis by integrating a heterologous 2,3-butanediol (2,3-BDO) pathway and sequentially deleting all three pyruvate decarboxylase genes (PDC1, PDC5, and PDC6). Genome-scale modeling was utilized to verify and optimize the flux distribution towards 2,3-BDO synthesis as DMCI chassis. This platform was subsequently engineered to produce various C2-C4 chemicals, including d-lactate, acetoin, and malate. Fed-batch fermentation demonstrated the industrial viability of these MCFs, achieving titers of 100, 80, 55, and 30 g/L for 2,3-BDO, d-lactate, malate, and succinate, respectively. Furthermore, to enhance the suitability for organic acid production, we employed adaptive laboratory evolution (ALE) to obtain a weak-acid tolerant mutant B3-ALE. Genomic and functional analyses of B3-ALE identified mutations in genes related to cell wall integrity, membrane trafficking, and oxidative stress response with six weak-acid tolerant biological parts. Collectively, this work establishes a versatile and robust non-ethanol producing yeast chassis with improved environmental resilience, offering a practical yeast platform and strategic guidance for the cost-effective production of value-added biochemicals.