초록
<P>The production of high-value phenolic acids is of significant importance for sustainability in the pharmaceutical industry. In this study, an artificial thermophilic reaction cascade composed of D-mandelate dehydrogenase (ManDH), phenylalanine 4-hydroxylase (PAH), and hydroxyphenylacetate 3-hydroxylase (HpaH) was constructed to utilize the low-cost phenylpyruvic acid (PPA) and 2-phenylglyoxylic acid (PGA) as dual substrates. The dihydroxyphenolic acids salvianic acid A (SAA) and 3,4-dihydroxymandelic acid (DOMA) were then produced at 84.9% and 90.9% of the theoretical yields from PPA and PGA, respectively. The key to success of the reaction was the novel <I>Tb</I>ManDH from <I>Thermococcus barophilus</I> and <I>Tc</I>PAH from <I>Thermomonospora curvata</I> that exhibited a broad substrate specificity. The newly <I>in situ</I> regeneration of the cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterine (DMPH<SUB>4</SUB>) in combination with nicotinamide adenine dinucleotide (NADH) recycling also contributed to the high production yields. Considering the broad substrate specificity of many other natural enzymes, it is attractive to design such dual- or even multifunctional catalyst systems for efficiently producing value-added chemicals.</P><P>An artificial reaction cascade with a dual catalytic nature was constructed for cell-free production of value-added dihydroxyphenolic acids.</P><BR>[FIG OMISSION]</BR>