[P005] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P03-391] Integrated metabolic, transcriptional, and culture engineering enables efficient diacetyl production from glucose in Bacillus subtilis

○Nunthaphan Vikromvarasiri^1,2, Fuya Kato², Akihiko Kondo^1,2,3, Tomokazu Shirai² (1. Engineering Biology Research Center, Kobe University (Japan), 2. RIKEN Center for Sustainable Resource Science (Japan), 3. Graduate School of Science, Technology and Innovation, Kobe University (Japan))

Keywords:

Bacillus subtilis,Diacetyl,Genome-scale metabolic model,Metabolic engineering,Culture engineering

Diacetyl (2,3-butanedione) is a valuable buttery flavor compound widely used in dairy products and the pharmaceutical industry. Increasing consumer demand for natural and sustainable ingredients has driven interest in the bio-based production of diacetyl. Bacillus subtilis, a Generally Recognized as Safe (GRAS), genetically accessible, and industrially robust microorganism, possesses native pyruvate metabolism that can be redirected toward diacetyl biosynthesis, making it an attractive platform for metabolic and process engineering. In this study, a multilevel engineering strategy integrating computational modeling, genome engineering, transcriptional tuning, and culture optimization was developed to enhance diacetyl production from glucose. First, an in silico “step-by-step gene deletion” strategy was performed using flux balance analysis (FBA) and a genome-scale metabolic model of B. subtilis to identify modifications that increase flux toward α-acetolactate, the direct precursor of diacetyl. The simulations predicted that eliminating competing byproduct-forming pathways would significantly enhance carbon flux toward the target compound. Based on the predictions, CRISPR–Cas9 genome editing was employed to delete competing pathway genes (ackA, pta, alsD, lctE, mmgA, ydaP, and bdhA), generating a Δ7 strain. The engineered strain achieved a diacetyl titer of 1.56 g/L, whereas the wild-type strain produced no detectable diacetyl under the same culture conditions. To enhance precursor formation, the most suitable promoter was selected from four variants to increase alsS expression, leading to a diacetyl titer of 2.02 g/L. Finally, culture conditions were systematically optimized by adjusting glucose concentration and temperature to improve metabolic balance and diacetyl production. Under optimized conditions, diacetyl production reached 3.94 g/L, representing a substantial improvement over the pre-optimized strain. Additional optimization is currently underway. Overall, integrating in silico metabolic design with genome editing, transcriptional tuning, and culture optimization effectively translated theoretical pathway design into practical bioprocess performance. This proposed framework provides a robust and scalable strategy for diacetyl production and can be extended to other pyruvate-derived value-added compounds in B. subtilis.