Multilevel metabolic engineering of Bacillus licheniformis for de novo biosynthesis of 2-phenylethanol

Due to its pleasant rose-like scent, 2-phenylethanol (2-PE) has been widely used in the fields of cosmetics and food. Microbial production of 2-PE offers a natural and sustainable production process. However, the current bioprocesses for de novo production of 2-PE suffer from low titer, yield, and productivity. In this work, a multilevel metabolic engineering strategy was employed for the high-level production of 2-PE. Firstly, the native alcohol dehydrogenase YugJ was identified and characterized for 2-PE production via genome mining and gene function analysis. Subsequently, the redirection of carbon flux into 2-PE biosynthesis by combining optimization of Ehrlich pathway, central metabolic pathway, and phenylpyruvate pathway enabled the production of 2-PE to a titer of 1.81 g/L. Specifically, AroK and AroD were identified as the rate-limiting enzymes of 2-PE production through transcription and metabolite analyses, and overexpression of aroK and aroD efficiently boosted 2-PE synthesis. The precursor competing pathways were blocked by eliminating byproduct formation pathways and modulating the glucose transport system. Under the optimal condition, the engineered strain PE23 produced 6.24 g/L of 2-PE with a yield and productivity of 0.14 g/g glucose and 0.13 g/L/h, respectively, using a complex medium in shake flasks. This work achieves the highest titer, yield, and productivity of 2-PE from glucose via the phenylpyruvate pathway. This study provides a promising platform that might be widely useful for improving the production of aromatic-derived chemicals.