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首页> 外文期刊>RSC Advances >Transcriptome-guided discovery and functional characterization of two UDP-sugar 4-epimerase families involved in the biosynthesis of anti-tumor polysaccharides in Ornithogalum caudatum
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Transcriptome-guided discovery and functional characterization of two UDP-sugar 4-epimerase families involved in the biosynthesis of anti-tumor polysaccharides in Ornithogalum caudatum

机译：转录的两种UDP-糖4个映酶系列的发现和功能表征参与鸟类抗肿瘤多糖生物合成的抗肿瘤多糖的生物合成

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UDP-glucose 4-epimerase ( UGE ) and UDP-xylose 4-epimerase ( UXE ), two important UDP-sugar 4-epimerases, are well known to be essential for de novo biosynthesis of UDP- D -galactose and UDP- L -arabinose, two universal sugar donors for the formation of four galactose- and arabinose-containing anticancer polysaccharides in Ornithogalum caudatum . However, very little is known about their cDNA sequences. Furthermore, the functional significance of the two epimerases in the biosynthesis of these anticancer polysaccharides in O. caudatum had not been documented. Here, we presented a full characterization of UGE and UXE , which were deemed to be responsible for anticancer polysaccharides biosynthesis in O. caudatum . Specifically, a transcriptome-guided search for the two epimerase genes in O. caudatum was first performed in the present study. A total of 4 unigenes sharing high sequence identity with UDP-sugar 4-epimerases were retrieved from transcriptome assembly. Four full-length cDNAs encoding UDP-sugar 4-epimerases, including two UGE-like and two UXE-like genes, were then isolated by reverse transcription polymerase chain reaction (RT-PCR) from O. caudatum . Bio-informatic analysis indicated the two UDP-sugar 4-epimerase families shared two common conserved domains, namely an N-terminal GxxGxxG motif and a catalytic Ser/Thr-Tyr-Lys triad. A phylogenetic analysis revealed the two members in the same UGE family could be classified into two subgroups, revealing their divergently functional significance. These candidate isoenzymes were screened by functional expression in E. coli individually as standalone enzymes. Two UGE-like cDNAs were identified to be bona fide genes, exhibiting both UGE and UXE activities. To further explore the possible role of these epimerase proteins in polysaccharides biosynthesis, transcript profiles of the four genes were subsequently examined by real-time quantitative PCR in various O. caudatum tissues. OcUGE1 , OcUGE2 and OcUXE1 were therefore assumed to be responsible for the biosynthesis of the four galactose- and arabinose-containing polysaccharides due to their expression profiles in O. caudatum . Taken together, these data provide further comprehensive knowledge for polysaccharides biosynthesis in O. caudatum and broaden the potential application of UGE in metabolic engineering or synthetic biology as a potential gene part.
机译：UDP-葡萄糖4-映异构酶（UGE）和UDP-木糖4-映异构酶（UXE），两个重要的UDP-糖4-截止酶，是必不可少的UDP-D -Galactions和UDP-L的Novo生物合成至关重要 - 阿拉伯糖，两个普通糖供体用于形成四个半乳糖和阿拉伯糖的抗癌多糖中的鸟类毛茛属植物。然而，对其cDNA序列知之甚少。此外，尚未记录过甲板的这些抗癌多糖的生物合成中的两种映异构酶的功能意义。在这里，我们展示了UGE和UXE的全面表征，被认为是对O. Caudatum中的抗癌多糖生物合成负责。具体地，首先在本研究中首先进行对O.剖反酶基因的转录组合搜索。从转录组组件中检索总共4个与UDP-糖4-旁旁旁酶共享高序列同一性的unigenes。然后通过逆转录聚合酶链反应（RT-PCR）分离O. caudatum的四个编码UDP-糖4-截止酶的四个全长CDNA，包括两个uge样和两个类似的基因。生物信息分析表明，两种UDP-糖4-映酶系列共用两个常见的保守结构域，即N末端GXXGXXG基序和催化Ser / Thr-Tyr-Lys Triad。系统发育分析揭示了同一UGE系列中的两个成员可以分为两个亚组，揭示了它们的发作性功能性。将这些候选成分通过在大肠杆菌中单独作为独立酶的功能表达筛选。鉴定了两个uge样CDNA是真正的基因，表现出UGE和UXE活动。为了进一步探讨这些截止酶蛋白在多糖中的可能作用生物合成，随后通过各种O.剖反组织中的实时定量PCR检查四种基因的转录物谱。因此，由于O. Caudatum中的表达谱，因此假设ocuge1，Ocuge2和Ocuxe1负责含四半乳糖和含阿拉伯糖的多糖的生物合成。总之，这些数据为O. Caudatum提供了对多糖生物合成的进一步全面了解，并扩大了UGE在代谢工程或合成生物学中作为潜在基因部分的潜在应用。

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