Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Abstract

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 ␤-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T 3) produced 411 mg͞liter scopolamine, which was over nine times more than that in the wild type (43 mg͞liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184 mg͞liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.Agrobacterium ͉ hyoscyamine 6␤-hydroxylase ͉ putrescine N-methyltransferase ͉ scopolamine ͉ transformation

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua

“…Not all transgenic plants had expression levels that were significantly different from those of the controls, and overexpressing a single gene might not be sufficient for raising the content of artemisinin. Similar results have been found in other studies of plant secondary metabolites (Zhang et al, 2004). We conclude that genetic engineering may represent a useful approach for further plant metabolism studies.…”

Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae)

“…However, overexpressing a single gene is not sufficient to increase artemisinin accumulation. This phenomenon is similar to that in other metabolic studies of plant secondary metabolites (Zhang et al, 2004). Based on these results, we conclude that genetic engineering is a useful method in plant metabolism research, but that there is more than one bottleneck in the artemisinin biosynthetic pathway.…”

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…Transcriptomics, coupled with silencing, metabolite, and biochemical analysis, identified a specialized role for ArAT4 in diverting L-Phe, via phenyllactate, into hyoscyamine and scopolamine in A. belladonna, establishing the identity of the first enzyme in this branch of the pathway. There have been many attempts to engineer increased production of hyoscyamine and scopolamine, particularly through overexpression of PMT and H6H (Moyano et al, 2003;Rothe et al, 2003;Zhang et al, 2004), which participate in early and late steps of the pathway, respectively (Figure 1). The identification of Ab-ArAT4 as the first step in the pathway that provides the phenyllactate moiety for the formation of littorine (Figure 1) provides a tool to potentially manipulate tropane alkaloid levels in the Solanaceae.…”