Production of hydroxytyrosol through whole-cell bioconversion from L-DOPA using engineered Escherichia coli.

Hydroxytyrosol (HT), a polyphenolic molecule of high value, is used in the nutraceutical, cosmetic, food, and livestock nutrition industries. As a natural product, HT is chemically manufactured or extracted from olives; nevertheless, the increasing demand mandates the exploration and development of alternative sources, such as heterologous production by recombinant bacteria. In order to achieve this purpose, we have molecularly modified Escherichia coli to carry two plasmids. For conversion of L-DOPA (Levodopa) into HT efficiently, it is necessary to enhance the expression of DODC (DOPA decarboxylase), ADH (alcohol dehydrogenases), MAO (Monoamine oxidase) and GDH (glucose dehydrogenases). The step that significantly affects the rate of ht biosynthesis is likely to be associated with the reaction facilitated by DODC enzymatic activity, as suggested by the result of in vitro catalytic experiment and HPLC. Then Pseudomonas putida, Sus scrofa, Homo sapiens and Levilactobacillus brevis DODC were taken into comparsion. The DODC from H. sapiens is superior to that of P. putida, S. scrofa or L. brevis for HT production. Seven promoters were introduced to increase the expression levels of catalase (CAT) to remove the byproduct HO and optimized coexpression strains were obtained after screening. After the 10-hour operation, the optimized whole-cell biocatalyst produced HT at a maximum titer of 4.84 g/L with over 77.5% molar substrate conversion rate.