Production of Phenol from Glucose in Escherichia coli through Metabolic Engineering Approach

Chung, H., Korea Advanced Institute of Science and Technology (KAIST)
Kim, B., Korea Advanced Institute of Science and Technology
Park, H., Korea Advanced Institute of Science and Technology (KAIST)
Lee, S. Y., Korea Advanced Institute of Science and Technology (KAIST)
Na, D., Chung Ang University

Phenol is an industrially versatile commodity chemical and, its annual production exceeded 8 million tons worldwide in 2008. Currently, phenol is produced via the chemical oxidation of cumene derived from benzene. Due to our increasing concerns on global warming and depletion of fossil resources, biological production of phenol has been attracting much attention. However, phenol’s biological production from renewable resources has been limited due to its toxicity to microorganisms and complex biosynthetic network of aromatic compounds. To address these issues, we simultaneously engineered 18 Escherichia coli strains for the production of phenol by taking advantage of synthetic regulatory sRNA technology. sRNA-based knock-down of the two regulators and overexpression of the genes involved in the tyrosine biosynthetic pathway together with tyrosine phenol-lyase in E. coli strains resulted in the production of phenol from glucose. The 18 engineered E. coli strains showed significant differences in the production of tyrosine (the immediate precursor for phenol), tyrosine phenol-lyase activity, and tolerance to phenol. These led to much variation in their phenol-producing capabilities. Among the engineered E. coli strains, the BL21(DE3) strain produced phenol most efficiently: 419 mg/L by flask culture and 1.69 g/L by fed-batch culture. The final titer and productivity were further improved through biphasic fed-batch fermentation using glycerol tributyrate as an extractant of phenol. The concentration of phenol in the glycerol tributyrate phase and fermentation broth reached 9.84 and 0.3 g/L, respectively, in 21 h, which translates into the final phenol titer and productivity of 3.79 g/L and 0.18 g/L/h, respectively. This is the highest titer achieved by microbial fermentation. Although further engineering is required to be competitive with the current petro-based process, the strategies used for the development of the engineered strain and fermentation process will provide a valuable framework for the microbial production of toxic chemicals. [This work was supported by the Intelligent Synthetic Biology Center (2011-0031963) through the Global Frontier Research Program of MEST.]