A synergistic strategy of metabolic engineering and flocculation recycling for enhanced acetoin production in Zymomonas mobilis

Acetoin, a valuable platform chemical, faces sustainability challenges in its traditional energy-intensive synthesis. Microbial fermentation using microorganisms such as Zymomonas mobilis offers a promising alternative. To overcome metabolic limitations and process inefficiencies of economic acetoin production, we integrated strategies of metabolic engineering, transcriptomic-guided analysis, flocculation-based cell recycling, and non-food feedstock utilization. A dominant metabolism compromised intermediate (DMCI) chassis of Z. mobilis was constructed by deleting ethanol production and acetoin degradation pathways. Transcriptomics was then employed to identify and knockout latent competing pathway genes of ZMO0318 and ZMO1576 to enhance acetoin production. The engineered strain also tolerated to inhibitors in lignocellulosic hydrolysates, and fed-batch fermentation achieved an acetoin titer of 73 g/L. Furthermore, self-flocculating phenotype was engineered via ZMO1082 modification to enable efficient cell recycling over multiple batches for production cost reduction. This study thus establishes a synergistic strategy to enhance acetoin production, highlighting the role of combining metabolic engineering, omics analyses, and processing engineering for economic biochemical production. • Recombinant strain of Z. mobilis constructed to produce 73 g/L acetoin. • Transcriptomic analyses guided knockout of competing pathways. • Cofactor engineering in intermediate chassis redirected carbon flux to acetoin. • Self-flocculation applied for cell recycling to reduce downstream processing cost. • High acetoin titer achieved using lignocellulosic hydrolysates.