De Novo Biosynthesis of Pinosylvin Using Engineered Escherichia coli
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Abstract:
Pinosylvin, a stilbenoid analog of the valuable nutrient resveratrol, exhibits many biological effects, such as cardiovascular disease prevention and anticancer and anti-arthritic activities. Herein, genetic engineering procedures were used to develop a de novo method for pinosylvin synthesis in order to ease the current problem of low pinosylvin supply. A constitutive vector containing the phenylalanine ammonia-lyase, cinnamoyl-coenzyme A ligase, and stilbene synthase genes was transformed into the L-phenylalanine-overproducing host strain Escherichia coli ATCC31884. PCR identification confirmed that a recombinant bacterial strain was obtained. The engineered bacterial cells were grown in shaking culture, and the culture medium was measured by HPLC to confirm that an engineered strain with de novo pinosylvin biosynthetic ability had been successfully created. Testing of culture samples taken at different fermentation times revealed that the rate of pinosylvin production increased more slowly after 12 h of culture, the highest pinosylvin yield of 0.32 mg/L was reached after a 24 h fermentation, and the maximum accumulated amount of cinnamic acid was 52.92 mg/L. These results suggest that the engineered E. coli can employ its own metabolism to synthesize pinosylvin de novo without addition of any other substances. However, the conversion efficiency of cinnamic acid was low, and thus further studies are needed to improve the strain.
