Pathway-Adapted\nBiosensor for High-Throughput Screening\nof <i>O</i>‑Methyltransferase and its Application\nin Vanillin Synthesis

Vanillin\nis a widely used flavoring compound in the food,\npharmaceutical,\nand cosmetics area. However, the biosynthesis of vanillin from low-cost\nshikimic acid is significantly hindered by the low activity of the\nrate-limiting enzyme, caffeate <i>O</i>-methyltransferase\n(COMT). To screen COMT variants with improved conversion rates, we\ndesigned a biosensing system that is adaptable to the COMT-mediated\nvanillin synthetic pathway. Through the evolution of aldehyde transcriptional\nfactor YqhC, we obtained a dual-responsive variant, MuYqhC, which\npositively responds to the product and negatively responds to the\nsubstrate, with no response to intermediates. Using the MuYqhC-based\nvanillin biosensor, we successfully identified a COMT variant, Mu176,\nthat displayed a 7-fold increase in the conversion rate compared to\nthe wild-type COMT. This variant produced 2.38 mM vanillin from 3\nmM protocatechuic acid, achieving a conversion rate of 79.33%. The\nenhanced activity of Mu176 was attributed to an enlarged binding pocket\nand strengthened substrate interaction. Applying Mu176 to Bacillus subtilis increased the level of vanillin\nproduction from shikimic acid by 2.39-fold. Further optimization of\nthe production chassis, increasing the <i>S</i>-adenosylmethionine\nsupply and the precursor concentration, elevated the vanillin titer\nto 1 mM, marking the highest level of vanillin production from shikimic\nacid in Bacillus. Our work highlights the significance of the MuYqhC-based\nbiosensing system and the Mu176 variant in vanillin production.