The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

Ying Xiao,Peng Di,Wansheng Chen,Lei Zhang,Shouhong Gao,Saengking Saechao,Junfeng Chen,Vladimir N Uversky

doi:10.1371/journal.pone.0029713

Abstract

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors.

Highlights

Rosmarinic acid is a common hydroxycinnamoyl ester accumulated in the plant species of Boraginaceae and Lamiaceae
The successful cloning of tat, hppr, c4h and 4-hydroxyphenylpyruvate dioxygenase gene from S. miltiorrhiza [12,13,14] in our previous study was another great motivation for the exploration of metabolic engineering as an effective approach to appraise the specific role of enzyme(s) in activation of the rosmarinic acid (RA) pathway by enhancing rate-limiting steps or by blocking competitive pathways, leading to an increase in the yield of target compounds (RA and lithospermic acid B (LAB))
Some transgenic roots turned brown and aged considerably faster than wild-type root cultures

Summary

Introduction

Rosmarinic acid (alpha-O-caffeoyl-3, 4-dihydroxyphenyllactic acid; RA) is a common hydroxycinnamoyl ester accumulated in the plant species of Boraginaceae and Lamiaceae. In our previous study [10], a gene-to-metabolite network was constructed by searching for correlations between the transcripts of RA biosynthetic genes and the accumulations of both RA and its important derivative, lithospermic acid B (LAB) (Figure 1) [11], in MeJA-induced S. miltiorrhiza hairy root cultures.

Results

Conclusion