Runguphan Weerawat, Maresh Justin J, O'Connor Sarah E
Department of Chemistry, Massachusetts Institute of Technology, 18-592, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13673-8. doi: 10.1073/pnas.0903393106. Epub 2009 Aug 5.
Natural products have long served as both a source and inspiration for pharmaceuticals. Modifying the structure of a natural product often improves the biological activity of the compound. Metabolic engineering strategies to ferment "unnatural" products have been enormously successful in microbial organisms. However, despite the importance of plant derived natural products, metabolic engineering strategies to yield unnatural products from complex, lengthy plant pathways have not been widely explored. Here, we show that RNA mediated suppression of tryptamine biosynthesis in Catharanthus roseus hairy root culture eliminates all production of monoterpene indole alkaloids, a class of natural products derived from two starting substrates, tryptamine and secologanin. To exploit this chemically silent background, we introduced an unnatural tryptamine analog to the production media and demonstrated that the silenced plant culture could produce a variety of novel products derived from this unnatural starting substrate. The novel alkaloids were not contaminated by the presence of the natural alkaloids normally present in C. roseus. Suppression of tryptamine biosynthesis therefore did not appear to adversely affect expression of downstream biosynthetic enzymes. Targeted suppression of substrate biosynthesis therefore appears to be a viable strategy for programming a plant alkaloid pathway to more effectively produce desirable unnatural products. Moreover, although tryptamine is widely found among plants, this silenced line demonstrates that tryptamine does not play an essential role in growth or development in C. roseus root culture. Silencing the biosynthesis of an early starting substrate enhances our ability to harness the rich diversity of plant based natural products.
天然产物长期以来一直是药物的来源和灵感来源。修饰天然产物的结构通常会提高化合物的生物活性。在微生物中，通过代谢工程策略发酵“非天然”产物已经取得了巨大成功。然而，尽管植物来源的天然产物很重要，但从复杂、冗长的植物途径中生产非天然产物的代谢工程策略尚未得到广泛探索。在这里，我们表明，RNA介导的长春花毛状根培养物中色胺生物合成的抑制消除了单萜吲哚生物碱的所有产生，单萜吲哚生物碱是一类源自两种起始底物色胺和裂环马钱子苷的天然产物。为了利用这种化学沉默的背景，我们将一种非天然色胺类似物引入生产培养基，并证明沉默的植物培养物可以产生多种源自这种非天然起始底物的新型产物。这些新型生物碱没有被长春花中通常存在的天然生物碱污染。因此，色胺生物合成的抑制似乎不会对下游生物合成酶的表达产生不利影响。因此，靶向抑制底物生物合成似乎是一种可行的策略，可用于对植物生物碱途径进行编程，以更有效地生产所需的非天然产物。此外，尽管色胺在植物中广泛存在，但这条沉默的品系表明色胺在长春花根培养物的生长或发育中并不起关键作用。沉默早期起始底物的生物合成增强了我们利用丰富多样的植物源天然产物的能力。
