Metabolic engineering approaches in medicinal plants

By: Naghmeh Poorinmohammad

May 2015

Introduction

• Plants are the richest with secondary metabolites among different organisms (5.000 – 25.000 per plant).

• Plants provide the source material for over half the drugs currently prescribed.

• Often, bioactive compounds in plants are produced in very small quantities.

10,000 kg of Pacific yew bark yields less than 1 kg of the potent anti-cancer compound paclitaxel!

Chandra et al. Biotechnology for Medicinal Plants. Springer, 2014: P275 1

Introduction

• Shikimic acid pathway, non-mevalonate (MEP) pathway and mevalonate (MVA) pathway lead to diverse classes of compounds, which include the terpenoids, monoterpene indole alkaloids, isoquinoline alkaloids, flavonoids and anthocyanins.

Wilson et al. Current opinion in biotechnology 26 (2014): 174-182. 2

Introduction

Challenges

Small quantities

Undesirable properties

Low number of candidates

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Introduction

A good Solution!

Metabolic Engineering

(ME)Enhancement

Suppression

Sequestration or diversification

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ME approaches theory

Farré et al. Annual review of plant biology 65 (2014): 187-223. 5

ME approaches theory

STOP!

We must first gain knowledge about the plant’s metabolism.

Remember!

The use of term “engineering” implies that there is some precise understanding of the system that is being modified.

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Understanding plant metabolism: A systems biological approach

• To be able to manipulate plant metabolism, one must first create a metabolic model.

• Constructing a metabolic model using a systems biological approach requires the four steps:

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation Analytical Investigation

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Understanding plant metabolism: A systems biological approach

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

Step 1 Find enzymes/reactions: - Literature search and databases - Gene annotation - Experimental procedures

Step 2 Identification of inter-compartmental transport reactions:

- The most challenging step as information in this area remains, for the most part, uncharacterized.

Step 3 Determine the reversibility of reactions- There are some obvious reactions- Some others can be concluded thermodynamically- Some algorithms do exist

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Major Databases Containing Metabolic Information on Plants

Baghalian et al. The Plant Cell Online 26.10 (2014): 3847-3866. 9

metacyc.org

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http://omictools.com/13c-fluxomics-c1414-p1.html

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Compartmentation makes plant genome-scale reconstruction challenging: even for Arabidopsis,

the most intensively studied plant species, thisknowledge is far from complete.

de Oliveira et al. Current opinion in biotechnology24.2 (2013): 271-277. 12

Understanding plant metabolism: A systems biological approach

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

• While step one provides a static view of the metabolic network, mathematical methods are required in order to process and integrate heterogeneous omics data and to build a comprehensive metabolic model.

• Computational platforms have been developed to make the mathematical analysis and visualization convenient.

• Mathematical modeling methods:- Network Models- Stoichiometric Models- Genome-Scale Metabolic Models- Metabolic flux analysis (MFA) (moghayesash ba

gene essentiality data)- Kinetic Models

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Definitions of Terms Used in Metabolic Modeling

Baghalian et al. The Plant Cell Online 26.10 (2014): 3847-3866. 14

Stoichiometric Models

Baghalian et al. The Plant Cell Online 26.10 (2014): 3847-3866. 15

Understanding plant metabolism: A systems biological approach

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

• A valid model faithfully reflects the biologically realistic behavior of the metabolic network.

• Lack of concordance between observed and predicted behavior requires re-elaboration of the model to remove inconsistencies.

• A model becomes acceptable as soon as the outcome of future experiments can be predicted.

• It is also an overriding priority to continuously update the validated model by reference to new findings.

• 70 to 90% similarity between experiment and prediction is a good result.

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Understanding plant metabolism: A systems biological approach

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

• A valid model can be regarded as a virtual laboratory, so predictions can be made much faster and more cheaply than by conducting the necessary wet lab experiments.

• The intention is to manipulate a given pathway to produce a specific outcome, applying the model can potentially generate a variety of alternative strategies, which can lead to a directed experimental validation.

• NFA is one of the important part of systems biology and has the potential to make predictions on the basis of a functional understanding.

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flux balance analysis of large networks

de Oliveira et al. Current opinion in biotechnology24.2 (2013): 271-277. 18

ME approaches theory

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CASE STUDY 1

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CASE STUDY 1

Hasan et al.  Plant cell reports 33.6 (2014): 895-904. 21

ME approaches theory

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• GGPP, the precursor for both diterpenoids and tetraterpenoids, was accumulated by suppressing the pathway to carotenoids.

• Two genes for the pathway, phytoene synthase (PSY) and phytoene desaturase (PDS) in the first and second step for the pathway of carotenoid synthesis respectively, were silenced

• How?

Partial products of PSY and PDS were prepared by PCR and used to produce the pTRV2:PSY and pTRV2:PDS plasmid.

Transformation of A.tumefaciens strain GV2260 with the plasmids was acomplished by the freez-thaw method.

To confirm successful silencing of the PSY and PDS gene, total RNA was isolated and visualized on agarose gel.

CASE STUDY 1

Hasan et al.  Plant cell reports 33.6 (2014): 895-904. 23

• Identification and quantification of the amounts of taxadiene were conducted by GC–MS.

• Metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9- fold, respectively. In

CASE STUDY 1

Hasan et al.  Plant cell reports 33.6 (2014): 895-904. 24

1. Knocking out gene function by targeted RNA degradation.

2. Interfering with protein function using specific inhibitors or antibodies.

Other methods for gene silencing??

Wurtzel et al. Encyclopedia of Chemical Processing (2006): 2191-2200. 25

CASE STUDY 2

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CASE STUDY 2

• The medicinal plant Catharanthus roseus is of enormous pharmaceutical interest because it contains more than 120 terpenoid indole alkaloids (TIAs):

Ajmalicine antihypertensivevinblastine and vincristine antineoplastic

They are produced only in very low amounts in C. roseus plants (2) and, despite significant efforts, cell cultures are not yet a valid alternative for production.

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 27

CASE STUDY 2

Metabolite Profiling Transcipt profiling

Identify the conditions where differential accumulation of the desired metabolites can be observed.

TIA-targeted metabolite profiling

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

LC/MS Peak filtering 178 peaks

Using an internal library of masses and retention times, TIAs were identified

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 28

CASE STUDY 2

Metabolite Profiling Transcipt profiling

Identify the conditions where differential accumulation of the desired metabolites can be observed.

TIA-targeted metabolite profiling

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

LC/MS Peak filtering 178 peaks

Using an internal library of masses and retention times, TIAs were identified

cDNA-AFLP technique

Sequencing BLAST with a library (European Molecular

Biology Laboratory)

Less than 10% gave a perfect match. Thus, the vast majority

of the tags are undescribed.

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 28

CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

• The accumulation profiles of the 178 metabolite peaks retained were combined with the expression profiles of the 417 transcripts for integrated analysis.

• principal component analysis (PCA) was performed first to explore the variability structure of the data.

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 29

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619.

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CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

• Correlation network analysis was used to establish gene-to-gene and gene-to-metabolite co-regulation patterns

• The Pearson correlation coefficient between each pair of variables (either gene or metabolite) across the profiles, including all time points and conditions, was calculated.

• TOM SAWYER VISUALIZATION 6.0 visualization

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 32

CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619.

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CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619.

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CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 35

CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 36

CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

Rischer et al.  Proceedings of the National Academy of Sciences 103.14 (2006): 5614-5619. 37

CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

• The power of this approach was also successfully demonstrated by other studies of taxol biosynthesis and by the author’s own study of tobacco nicotine biosynthesis.

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CASE STUDY 2

Establishment of the Metabolic

Network

Convert reconstruction to

Mathematical Model and

Visualization

Validation

Analytical Investigation

A publishable full research data provided

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CASE STUDY 2

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CASE STUDY 2

Zhou et al. Biotechnology and applied biochemistry 52.4 (2009): 313-323. 41

CASE STUDY 2

• Genes encoding rate-limiting enzymes and some key transcription factors can be used to improve TIA production by overexpressing them in transgenic C. roseus cultures.

• Tdc cDNA driven by CaMV 35S (cauliflower-mosaic-virus 35 S promoter) was introduced into C. roseus by means of A. tumefaciens-mediated gene transformation.

.

Zhou et al. Biotechnology and applied biochemistry 52.4 (2009): 313-323. 42

CASE STUDY 2

Zhou et al. Biotechnology and applied biochemistry 52.4 (2009): 313-323. 43

CASE STUDY 2

• Genes encoding rate-limiting enzymes and some key transcription factors can be used to improve TIA production by overexpressing them in transgenic C. roseus cultures.

• Tdc cDNA driven by CaMV 35S (cauliflower-mosaic-virus 35 S promoter) was introduced into C. roseus by means of A. tumefaciens-mediated gene transformation.

• Overexpression of Tdc was not necessary to achieve high levels of alkaloid accumulation, but only enhanced tryptamine levels, and constitutively high TDC activity seemed to be detrimental to C. roseus growth.

• The Str-overexpressing cultures showed a 10-fold higher STR activity than wildtype, resulting in higher TIA accumulation.

Zhou et al. Biotechnology and applied biochemistry 52.4 (2009): 313-323. 44

Other methods for increasing expression??

1. Introduce genes into the plant.

2. Promoters to direct gene expression in the appropriate spatial and temporal landscape.

Wurtzel et al. Encyclopedia of Chemical Processing (2006): 2191-2200. 45

• Although most early examples of metabolic engineering involved single-gene interventions, this approach suffers from limitations such as the inability to increase the activity of multimeric enzymes with heterologous subunits, and the inability to target multiple metabolites simultaneously.

Multigene transfer

Farré et al. Annual review of plant biology 65 (2014): 187-223. 46

Multigene transfer

Farré et al. Annual review of plant biology 65 (2014): 187-223. 47

Increasing metabolic diversity

• The common building blocks of the diterpenoid pathways include various diterpene synthases, cytochrome P450 monooxygenases, glycosyltransferases, acyltransferases, methyltransferases, and oxidoreductases that act specifically on different parts of the skeleton.

• Combining these enzymes in a common background should make it possible to generate novel forms of decoration (or at least novel combinations of decoration) on a single diterpene skeleton.

• Similar building blocks are required for the synthesis of triterpenes.

• For example, the production of avenacin 1 requires many enzymes with different roles, but only five of the corresponding genes have been isolated.

• These genes have been expressed in different combinations in heterologous plants—e.g., the expression of AsMT1, AsUGT74H5, and AsSCPL showed that it was possible to achieve the acetylation of the triterpene backbone.

Farré et al. Annual review of plant biology 65 (2014): 187-223. 48

Challenges

Farré et al. Annual review of plant biology 65 (2014): 187-223. 49

Summary

Metabolic engineering is in principle a simple process of enhancing the capacity of existing pathways, controlling the distribution of flux, and, if necessary, bolting on additional functionalities so that novel compounds can be produced.

In practice, there are at least four major challenges that need to be overcome:

- Gaining enough knowledge of the endogenous pathways to know the best intervention points.

- Identifying and sourcing the genes that can be used to modify the targeted metabolic pathway.

- Expressing those genes in such a way as to produce a functional enzyme in a relevant context (such as the correct subcellular compartment).

- Achieving the primary goals of the metabolic engineering strategy without affecting endogenous metabolism to the extent that the plant cannot grow and develop normally.

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Summary

The valuable pharmacological and nutritional properties of many secondary metabolites combined with their low levels of production in nature mean that active research into novel strategies for metabolic engineering will become increasingly important.

The evolution of metabolic engineering from single- to multistep approaches, along with high-throughput methods for gene discovery and functional analysis and novel platforms for combinatorial testing of heterologous pathways in plants, will increase the predictive accuracy of early development stages.

Thus helping to refine engineering strategies and reduce the need for trial-and-error testing.

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Thank You