2-Phenylethanol (2-PE) is a fragrance widely used in food and cosmetics. Natural 2-PE is preferred in these applications but with high price and very limited availability. Microbial synthesis from glucose or glycerol is an attractive way to produce natural 2-PE, but suffers from low product titer. Escherichia coli NST74-Phe-Sty was engineered to coexpress the L-phenylalanine (L-Phe) biosynthesis pathway and enzyme cascade of L-Phe to 2-PE, producing only 0−0.17 g/L 2-PE from glucose at 22−37 °C due to the incompatibility of the temperature for enzyme expression and activity. Enhanced production of 2-PE (8.4−9.1 g/L) from glucose or glycerol was achieved by coupling of E. coli NST74-Phe expressing the L-Phe biosynthesis pathway for L-Phe production at the optimal temperature of 37 °C with E. coli T7-Sty expressing enzyme cascades of L-Phe to 2-PE at the optimal expression temperature of 22 °C and the optimal biotransformation temperature of 30 °C. The 2-PE titer is 4.7-fold higher than the best reported 2-PE concentration produced from glucose. The coupled strains approach could be generally applicable for enhancing microbial production of useful chemicals from sugars or glycerol.

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Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors.

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