Tyrosol is a pharmacologically active phenolic compound widely used in the pharmaceutical and chemical industries. Microbial fermentation has potential value as an environmentally friendly approach to tyrosol production, but suffers from low tyrosol yields and the need for expensive media additives. In this study, Escherichia coli MG1655 was modified by integrating an E. coli codon-optimized version of the Saccharomyces cerevisiae phenylpyruvate decarboxylase gene, named ARO10*, into the lacI locus. The resulting strain (YMGA*) produced 0.14 mM tyrosol from 2% glucose without the need for expensive media supplements. Subsequent deletion of E. coli genes designed to eliminate competing metabolic pathways (feaB, pheA, tyrB) or undesirable gene regulation (tyrR) produced a strain (YMGA*R) that produced 3.11 mM tyrosol. Tyrosol production was then increased to 10.92 mM by increasing the ARO10* copy number to five copies (strain YMG5A*R). Finally, tyrosol production was increased to 28 mM (ca. 3.9 g/L) by optimizing fermentation conditions in a 5 L fermenter. Engineering a productive E. coli strain with high tyrosol titer from glucose using a medium that does not require added amino acids, inducer, or antibiotic provides a solid basis to produce tyrosol through microbial fermentation.
Keywords: CRISPR-Cas9; Escherichia coli; integration; metabolic engineering; tyrosol.