Identification of a novel glucuronyltransferase from Streptomyces chromofuscus ATCC 49982 for natural product glucuronidation
Identification of a novel glucuronyltransferase from Streptomyces chromofuscus ATCC 49982 for natural product glucuronidation
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DOI：
10.1007/s00253-022-11789-2
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发表时间：
2022-01
影响因子：
5
通讯作者：
Jie Ren;C. D. Barton;Kathryn Eternity Sorenson;J. Zhan
中科院分区：
文献类型：
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作者：
Jie Ren;C. D. Barton;Kathryn Eternity Sorenson;J. Zhan
糖基化是增加天然产物极性的有效方法。 UDP-葡萄糖醛酸转移酶 (UGT) 在 II 期药物代谢中经常被观察到并被广泛研究。然而，微生物中的UGTs尚未得到充分研究，这阻碍了此类酶在天然产物的微生物葡萄糖醛酸化中的利用。对5个放线菌菌株的筛选表明，Streptomyces chromofuscus ATCC 49982可以将多种植物多酚转化为极性更强的产物，根据光谱数据将其表征为多种葡萄糖醛酸苷。对该菌株的基因组分析揭示了一个假定的葡萄糖醛酸化基因簇，其中包含一个 UGT 基因 (gcaC) 和两个 UDP-葡萄糖醛酸生物合成基因 (gcaBandgcaD)。克隆gcaC基因并在大肠杆菌BL21(DE3)中异源表达。将纯化的酶与白藜芦醇和 UDP-葡萄糖醛酸一起孵育，产生白藜芦醇-4'-O-β-D-葡萄糖醛酸和白藜芦醇-3-O-β-D-葡萄糖醛酸，使 GcaC 被表征为灵活的 UGT。 GcaC 的最佳体外反应 pH 值和温度分别为 7.5 和 30 °C。 Ca2+、Mg2+、Mn2+可刺激其活性，而Zn2+、Cu2+、Fe2+则表现出抑制作用。此外，GcaC具有广泛的底物特异性，可以与白藜芦醇以外的多种底物进行葡萄糖醛酸化，包括槲皮素、阿魏酸、香草酸、姜黄素、香草醛、白杨素、玉米赤霉烯酮和芹菜素。大肠杆菌中白藜芦醇-4'-O-β-D-葡萄糖苷酸和白藜芦醇-3-O-β-D-葡萄糖苷酸的效价。 3 小时内，114.125 mg/L 白藜芦醇的大肠杆菌 GcaC 分别为 78.381±0.366 mg/L 和 14.991±0.248 mg/L。因此，本工作为生产白藜芦醇的葡萄糖醛酸苷和其他有益健康的天然产物提供了一种有效的方法。 要点•从色褐链霉菌 ATCC 49982 中发现并鉴定了一种新型多功能微生物 UDP-葡萄糖醛酸转移酶。•该 UDP-葡萄糖醛酸转移酶在大肠杆菌中表达，可在体外和体内将白藜芦醇转化为两种葡萄糖醛酸苷。• UDP-葡萄糖醛酸转移酶具有高度灵活的底物特异性，是制备生物活性分子的单或二葡萄糖醛酸苷的有效工具。
Glycosylation is an effective way to increase the polarity of natural products. UDP-glucuronyltransferases (UGTs) are commonly observed and extensively studied in phase II drug metabolism. However, UGTs in microorganisms are not well studied, which hampered the utilization of this type of enzyme in microbial glucuronidation of natural products. Screening of five actinomycete strains showed thatStreptomyces chromofuscusATCC 49982 can convert diverse plant polyphenols into more polar products, which were characterized as various glucuronides based on their spectral data. Analysis of the genome of this strain revealed a putative glucuronidation gene cluster that contains a UGT gene (gcaC) and two UDP-glucuronic acid biosynthetic genes (gcaBandgcaD). ThegcaCgene was cloned and heterologously expressed inEscherichia coliBL21(DE3). Incubation of the purified enzyme with resveratrol and UDP-glucuronic acid led to the production of resveratrol-4′-O-β-D-glucuronide and resveratrol-3-O-β-D-glucuronide, allowing GcaC to be characterized as a flexible UGT. The optimalin vitroreaction pH and temperature for GcaC are 7.5 and 30 °C, respectively. Its activity can be stimulated by Ca2+, Mg2+, and Mn2+, whereas Zn2+, Cu2+, and Fe2+showed inhibitory effects. Furthermore, GcaC has a broad substrate specificity, which can glucuronidate various substrates besides resveratrol, including quercetin, ferulic acid, vanillic acid, curcumin, vanillin, chrysin, zearalenone, and apigenin. The titers of resveratrol-4′-O-β-D-glucuronide and resveratrol-3-O-β-D-glucuronide inE. coli-GcaC were 78.381 ± 0.366 mg/L and 14.991 ± 0.248 mg/L from 114.125 mg/L resveratrol within 3 h. Therefore, this work provides an effective way to produce glucuronides of resveratrol and other health-benefitting natural products.Key points•A novel versatile microbial UDP-glucuronyltransferase was discovered and characterized from Streptomyces chromofuscus ATCC 49982.•The UDP-glucuronyltransferase was expressed in Escherichia coli and can convert resveratrol into two glucuronides both in vitro and in vivo.•The UDP-glucuronyltransferase has a highly flexible substrate specificity and is an effective tool to prepare mono- or diglucuronides of bioactive molecules.