(670a) DNA-Guided Spatial Modulation of Key Pathway Enzymes and Respiration Chain Engineering for Improved N-Acetylglucosamine Production By Bacillus Subtilis

In our previous work, an N-acetylglucosamine (GlcNAc)-overproducing Bacillus subtilis was constructed via systematic engineering of GlcNAc synthetic and catabolic pathways. However, GlcNAc yield remained low owing the intrinsic inefficiency of the GlcNAc synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved GlcNAc production through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a DNA-guided scaffold system was first used to modulate the activities of glucosamine-6-phosphate synthase and GlcNAc-6-phosphate N-acetyltransferase, increasing the GlcNAc titer from 1.83 g/L to 4.55 g/L in a shake flask. Next, sporulation was blocked by respectively deleting spo0A (gene encoding the initiation regulon of sporulation) and sigE (gene encoding RNA polymerase sporulation-specific sigma factor). Deletion of sigE more effectively blocked sporulation without altering cell growth or GlcNAc production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting cydB and cydC, genes encoding cytochrome bd ubiquinol oxidase (subunit II) and ATP-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced 6.15 g/L GlcNAc in a shake flask and 20.58 g/L GlcNAc in a 3-L fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a DNA-guided scaffold system in B. subtilis.