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 TbManDH from Thermococcus barophilus and TcPAH from Thermomonospora curvata that exhibited a broad substrate specificity. The newly in situ regeneration of the cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterine (DMPH₄) 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.

中文翻译：

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嗜热生物功能多酶级联反应，用于无细胞合成丹参酸A和3,4-二羟基扁桃酸

高价值酚酸的生产对于制药行业的可持续性至关重要。在这项研究中，构建了由D-扁桃酸脱氢酶（ManDH），苯丙氨酸4-羟化酶（PAH）和羟苯乙酸3-羟化酶（HpaH）组成的人工嗜热反应级联，以利用低成本的苯丙酮酸（PPA）和2 -苯基乙醛酸（PGA）作为双重底物。然后分别从PPA和PGA分别以理论收率的84.9％和90.9％产生了二羟基酚酸丹酚酸A（SAA）和3,4-二羟基扁桃酸（DOMA）。到反应成功的关键是新颖铽从ManDH嗜热barophilus和锝PAH从Thermomonospora curvata表现出广泛的底物特异性。辅因子6,7-二甲基-5,6,7,8-四氢蝶呤（DMPH ₄）的新原位再生与烟酰胺腺嘌呤二核苷酸（NADH）回收相结合，也有助于提高产量。考虑到许多其他天然酶的广泛的底物特异性，设计这样的双或什至多功能催化剂体系以有效地生产增值化学品是有吸引力的。