Department of Bioengineering, Huainan Normal University, Huainan, 232038, Anhui Province, China.
Key Laboratory of Bioresource and Environmental Biotechnology of Anhui Higher Education Institutes, Huainan Normal University, Huainan, 232038, Anhui Province, China.
Plant Mol Biol. 2021 May;106(1-2):145-156. doi: 10.1007/s11103-021-01134-y. Epub 2021 Mar 10.
TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by AT mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A residue in TwPDR1 by sequence alignment and confirmed that AT mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.
雷公藤多萜醇 1 型转运蛋白（TwPDR1）是从雷公藤（Tripterygium wilfordii Hook.f.）中分离得到的 PDR 转运蛋白，能够将雷公藤内酯酮及其类似物外排，其稳定性可通过 AT 突变增强。雷公藤内酯酮是雷公藤中的一种贝壳杉烷二萜，具有多种药理活性。然而，由于其在天然植物中含量较低，且缺乏替代生产方法，因此雷公藤内酯酮供应短缺且价格昂贵。转运蛋白工程通过增加体外培养系统中次生代谢产物的细胞外分泌，是代谢工程中的一种有效策略，但很少有报道。本研究通过生物信息学分析，鉴定出多药耐药型 ATP 结合盒转运蛋白 TwPDR1 是二萜类化合物的最佳外排泵候选蛋白。TwPDR1 位于质膜上，在不定根中高度表达，并受茉莉酸甲酯诱导。通过瞬时表达在烟草 BY-2 细胞和在天然宿主中通过 RNA 干扰下调，证实了 TwPDR1 对雷公藤内酯酮的外排功能。然而，TwPDR1 的过表达对雷公藤内酯酮的分泌影响有限。如前所述，单个氨基酸突变可能通过增加蛋白质稳定性来增加 TwPDR1 的丰度。我们通过序列比对确定了 TwPDR1 中的 A 残基，并证实 AT 突变可以增加 TwPDR1 的表达，从而使突变体中雷公藤内酯酮的释放比例（78.8%）高于对照（60.1%）。TwPDR1 的鉴定和功能表征不仅为雷公藤内酯酮的代谢工程提供了候选基因材料，也为未来的其他转运蛋白工程研究提供了指导。
