Exogenous feeding of immediate precursors reveals synergistic effect on picroside-I biosynthesis in shoot cultures of Picrorhiza kurroa Royle ex Benth.

Varun Kumar,Neha Sharma,Hemant Sood,Rajinder Singh Chauhan

doi:10.1038/srep29750

Varun Kumar, Neha Sharma + Show 2 more

Open Access

https://doi.org/10.1038/srep29750

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Journal: Scientific Reports	Publication Date: Jul 15, 2016
Citations: 24	License type: CC BY 4.0

Affiliation: Jaypee University of Information Technology

Abstract

In the current study, we asked how the supply of immediate biosynthetic precursors i.e. cinnamic acid (CA) and catalpol (CAT) influences the synthesis of picroside-I (P-I) in shoot cultures of P. kurroa. Our results revealed that only CA and CA+CAT stimulated P-I production with 1.6-fold and 4.2-fold, respectively at 2.5 mg/100 mL concentration treatment. Interestingly, feeding CA+CAT not only directed flux towards p-Coumaric acid (p-CA) production but also appeared to trigger the metabolic flux through both shikimate/phenylpropanoid and iridoid pathways by utilizing more of CA and CAT for P-I biosynthesis. However, a deficiency in the supply of either the iridoid or the phenylpropanoid precursor limits flux through the respective pathways as reflected by feedback inhibition effect on PAL and decreased transcripts expressions of rate limiting enzymes (DAHPS, CM, PAL, GS and G10H). It also appears that addition of CA alone directed flux towards both p-CA and P-I production. Based on precursor feeding and metabolic fluxes, a current hypothesis is that precursors from both the iridoid and shikimate/phenylpropanoid pathways are a flux limitation for P-I production in shoot cultures of P. kurroa plants. This work thus sets a stage for future endeavour to elevate production of P-I in cultured plant cells.

Highlights

We report a series of experiments designed to determine the effect of cinnamic acid (CA) and catalpol (CAT) alone and in combinations on the flux limitations leading to picroside I (P-I) biosynthesis in in vitro grown shoots of P. kurroa
To address metabolic basis of P-I biosynthesis in P. kurroa, we applied metabolic flux analysis with feeding of late precursors i.e. CA and CAT alone and in combination to establish the route of P-I production in vivo and to determine the limiting moiety in the end metabolite
The results observed in this study showed that exogenous CAT increased P-I content when CA is not limiting indicating that addition of CAT caused rapid utilization of CA due to synergistic effect

Summary

Introduction

Available on what mechanism controls the flux of P-I biosynthesis in P. kurroa It is still unclear which out of the different modules is limiting for the P-I supply? Crucial to understand the mechanism behind P-I biosynthesis in P. kurroa prior to designing a suitable rational metabolic engineering approach. We address this issue here through feeding of different precursors in the context of P-I biosynthesis in P. kurroa. We report a series of experiments designed to determine the effect of cinnamic acid (CA) and catalpol (CAT) alone and in combinations on the flux limitations leading to P-I biosynthesis in in vitro grown shoots of P. kurroa

Methods

Results

Conclusion