Engineered EryF hydroxylase improving heterologous polyketide erythronolide B production in Escherichia coli

In the last two decades, the production of complex polyketides such as erythromycin and its precursor 6-deoxyerythronolide B (6-dEB) was demonstrated feasible in Escherichia coli. Although the heterologous production of polyketide skeleton 6-dEB has reached 210 mg l-1 in E. coli, the yield of its post-modification products erythromycins remains to be improved. Cytochrome P450EryF catalyses the C6 hydroxylation of 6-dEB to form erythronolide B (EB), which is the initial rate-limiting modification in a multi-step pathway to convert 6-dEB into erythromycin. Here, we engineered hydroxylase EryF to improve the production of heterologous polyketide EB in E. coli. By comparative analysis of various versions of P450EryFs, we confirmed the optimal SaEryF for the biosynthesis of EB. Further mutation of SaEryF based on the crystal structure of SaEryF and homology modelling of AcEryF and AeEryF afforded the enhancement of EB production. The designed mutant of SaEryF, I379V, achieved the yield of 131 mg l-1 EB, which was fourfold to that produced by wild-type SaEryF. Moreover, the combined mutagenesis of multiple residues led to further boost the EB concentration by another 41%, which laid the foundation for efficient heterologous biosynthesis of erythromycin or other complex polyketides.