ABSTRACT

Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. It is produced from daidzein by fastidious and oxygen-susceptible intestinal bacteria, which hinders their use at an industrial scale. Therefore, expressing the equol production machinery into easily-cultivable hosts would expedite the heterologous production of this compound. In this work, four genes (racemase, tdr, ddr and dzr) coding for key enzymes involved in equol production in Adlercreutzia equolifaciens DSM19450^T were synthesized and cloned in a pUC-derived vector (pUC57-equol) that was introduced in Escherichia coli. Recombinant clones of E. coli produced equol in cultures supplemented with daidzein (equol precursor) and dihydrodaidzein (intermediate compound). To check whether equol genes were expressed in Gram-positive bacteria, the pUC57-equol construct was cloned into the low-copy-number vector pIL252, and the new construct (pIL252-pUC57-equol) introduced into model strains of Lacticaseibacillus casei and Lactococcus lactis. L. casei clones carrying pIL252-pUC57-equol produced a small amount of equol from dihydrodaidzein but not from daidzein, while L. lactis recombinant clones produced no equol from either of the substrates. This is the first time that A. equolifaciens equol genes have been cloned and expressed in heterologous hosts. E. coli clones harboring pUC57-equol could be used for biotechnological production of equol.

中文翻译：

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Adlercreutzia equilifaciens 雌马酚生物合成基因的异源表达

摘要

雌马酚是异黄酮衍生的代谢物，具有最大的雌激素和抗氧化活性。它是由挑剔且对氧气敏感的肠道细菌从大豆苷元中产生的，这阻碍了它们在工业规模上的应用。因此，将雌马酚生产机器表达到易于培养的宿主中将加速该化合物的异源生产。在这项工作中，合成了四种基因（消旋酶、tdr、ddr和dzr），这些基因编码参与Adlercreutzia equilifaciens DSM19450 ^T中雌马酚生产的关键酶，并将其克隆到 pUC 衍生载体（pUC57-雌马酚）中，该载体引入大肠杆菌。大肠杆菌的重组克隆在补充有黄豆苷元（雌马酚前体）和二氢黄豆苷元（中间化合物）的培养物中产生雌马酚。为了检查是否在革兰氏阳性菌中表达雌马酚基因，将 pUC57-雌马酚构建体克隆到低拷贝数载体 pIL252 中，并将新构建体（pIL252-pUC57-雌马酚）引入干酪乳杆菌和乳球菌模型菌株中乳酸菌。携带 pIL252-pUC57-雌马酚的干酪乳杆菌克隆从二氢黄豆苷元产生少量雌马酚，但不从黄豆苷元产生，而乳酸乳球菌重组克隆不从任一底物产生雌马酚。这是第一次在异源宿主中克隆和表达A. equilifaciens雌马酚基因。含有 pUC57-雌马酚的大肠杆菌克隆可用于雌马酚的生物技术生产。