Metabolic engineering of Escherichia coli for efficient production of linalool from biodiesel-derived glycerol by targeting cofactors regeneration and reducing acetate accumulation

Linalool is a valuable monoterpenoid compound commercially used in cosmetics and personal care industry as well as a 'drop-in' biofuel. Direct plant extraction or chemical synthesis of linalool suffers from insufficient abundance in plant and inefficient purification procedures. Herein, a powerful green cell factory has been developed to meet the increasing demands for more sustainable and efficient production of this terpene alcohol. The de novo production of linalool from biodiesel-derived glycerol was achieved by engineered Escherichia coli harboring the exogenous mevalonate pathway followed by coupling with geranyl diphosphate synthase and linalool synthase (LIS). Then, the Mentha aquatica LIS showing the most efficient linalool biosynthesis was identified, and subsequently tuned by ribosomal binding site (RBS) engineering and site-directed mutagenesis to facilitate linalool production. A further engineered strain equipped with an engineered NADP+-dependent pyruvate dehydrogenase complex (PDH) and phosphoketolase bypass, remarkably decreasing the by-products formation together with boosting substrate utilization efficiency. A final titer of 1.07 g/L linalool with a yield of 0.10 g/g glycerol was gained in flask culture and further increase to 4.16 g/L in 2-L fed-batch fermentation, which is the highest reported linalool titer and yield in E. coli to the best of our knowledge. In addition, the engineering strategies established here not only lay a potential production platform for linalool and its derivatives, but also provide references to minimize by-products secretion and improve carbon use efficiency.