Yamada Yasuyuki, Urui Miya, Oki Hidehiro, Inoue Kai, Matsui Haruyuki, Ikeda Yoshito, Nakagawa Akira, Sato Fumihiko, Minami Hiromichi, Shitan Nobukazu
Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University, Motoyamakita-machi, Higashinada-ku, Kobe, 658-8558, Japan.
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa, 921-8836, Japan.
Metab Eng Commun. 2021 Sep 14;13:e00184. doi: 10.1016/j.mec.2021.e00184. eCollection 2021 Dec.
Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing strain into which the multidrug and toxic compound extrusion transporter AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites metabolic engineering.
微生物可通过代谢工程改造来生产特定的植物代谢产物。然而，这些方法受到低产量和代谢产物细胞内积累的限制。我们试图利用转运工程来生产小檗碱，它是生物碱生物合成途径中的一种重要中间体。在本研究中，我们构建了一个生产小檗碱的菌株，并向其中引入了多药和有毒化合物外排转运蛋白AtDTX1。选择AtDTX1是因为它在其中有合适的表达及其小檗碱转运活性。AtDTX1的表达使小檗碱产量提高了11倍，并且所产生的小檗碱被分泌到培养基中。AtDTX1的表达还赋予了高质粒稳定性，并导致与生物过程相关的几个基因上调或下调，包括小檗碱生物合成的代谢途径，从而导致高水平小檗碱的产生和分泌。成功利用转运蛋白进行生物碱生产表明，所提出的转运工程方法可能会改善特定代谢产物的生物合成而非代谢工程。
