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Keywords:

  • Biphasic fermentation;
  • Gene knock-down;
  • Microbial phenol production;
  • Phenol in E. coli;
  • Synthetic sRNA

Abstract

Phenol is an industrially versatile commodity chemical and is currently produced from fossil resources. Phenol's biological production from renewable resources has been limited due to its toxicity to microorganisms. Here, we simultaneously engineered 18 Escherichia coli strains for the production of phenol using synthetic regulatory small RNA (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 (TPL) 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 (i.e. the immediate precursor for phenol), TPL activity, and tolerance to phenol. Among the engineered E. coli strains, the BL21 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 hours, 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.