| Summary: | Abstract Background 2-Phenylethanol (2-PE) is a higher aromatic alcohol that is widely used in the perfumery, cosmetics, and food industries and is also a potentially valuable next-generation biofuel. In our previous study, a new strain Enterobacter sp. CGMCC 5087 was isolated to produce 2-PE from glucose through the phenylpyruvate pathway. Results In this study, candidate genes for 2-PE biosynthesis were identified from Enterobacter sp. CGMCC 5087 by draft whole-genome sequence, metabolic engineering, and shake flask fermentation. Subsequently, the identified genes encoding the 2-keto acid decarboxylase (Kdc) and alcohol dehydrogenase (Adh) enzymes from Enterobacter sp. CGMCC 5087 were introduced into E. coli BL21(DE3) to construct a high-efficiency microbial cell factory for 2-PE production using the prokaryotic phenylpyruvate pathway. The enzymes Kdc4427 and Adh4428 from Enterobacter sp. CGMCC 5087 showed higher performances than did the corresponding enzymes ARO10 and ADH2 from Saccharomyces cerevisiae, respectively. The E. coli cell factory was further improved by overexpressing two upstream shikimate pathway genes, aroF/aroG/aroH and pheA, to enhance the metabolic flux of the phenylpyruvate pathway, which resulted in 2-PE production of 260 mg/L. The combined overexpression of tktA and ppsA increased the precursor supply of erythrose-4-phosphate and phosphoenolpyruvate, which resulted in 2-PE production of 320 mg/L, with a productivity of 13.3 mg/L/h. Conclusions The present study achieved the highest titer of de novo 2-PE production of in a recombinant E. coli system. This study describes a new, efficient 2-PE producer that lays foundation for the industrial-scale production of 2-PE and its derivatives in the future.
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