Construction of an Escherichia coli cell factory for de novo synthesis of tyrosol through semi-rational design based on phenylpyruvate decarboxylase ARO10 engineering.

Tyrosol (2-(4-hydroxyphenyl) ethanol) is extensively used in the pharmaceutical industry as an important natural product from plants. In previous research, we constructed a recombinant Escherichia coli strain capable of de novo synthesis of tyrosol by integrating the phenylpyruvate decarboxylase ARO10 derived from Saccharomyces cerevisiae. Nevertheless, the insufficient catalytic efficiency of ARO10 required the insertion of multiple gene copies into the genome to attain enhanced tyrosol production. In this study, we constructed a mutation library of ARO10 based on a computer-aided semi-rational design strategy and developed a high-throughput screening method for selecting high-yield tyrosol mutants by introducing the heterologous hydroxylase complex HpaBC. Through multiple rounds of screening and site-saturation mutagenesis, we ultimately identified the two optimal ARO10 mutants, ARO10D331V and ARO10D331C , which respectively achieved a tyrosol titer of 2.02 g/L and 2.04 g/L in shake flasks, both representing more than 50 % improvement compared to the wild-type. Our study demonstrates the great potential of computer-based semi-rational enzyme design strategy in metabolic engineering. The high-throughput screening method for target compound derivative possesses a certain level of generality. Ultimately, we obtained promising mutants capable of achieving industrial-scale production of tyrosol, which also lays a solid foundation for the efficient synthesis of tyrosol derivatives.