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ADVANCES IN MEDICINAL
PLANT BIOTECHNOLOGY
COMBINATORIAL
BIOSYNTHESIS
Contents:
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Medicinal Plants
Status of Medicinal Plants
Importance
Secondary metabolites in plants and functions
Techniques used for medicinal plants
Combinatorial biosynthesis
Combinatorial biosynthesis for Terpenoids
Combinatorial biosynthesis for Alkaloids
Combinatorial biosynthesis for Drug Discovery
Advantages
Challenges and prospective
References
Medicinal Plants
 Plants
that have a recognized medicinal use
 Medicinal
herbs are good alternatives for curing
many diseases

Low cost and less side effects
Status of Medicinal plants

According to WHO 70-80% population in world using
herbal formulations for curing ailments
 In Pakistan 60% population is dependent on herbal
remedies
 30% raw material for modern medicines
 350-400 plants approved
 75 plant based drugs in market
Why Medicinal Plants are
important so?
 Pharmacological
activity of plants is due to
presence of secondary metabolites
 Commonly
 They
called active constituents of plants
perform different functions
 Different
in different plant species
 Concentration
in different plants also vary
Secondary metabolites in plants
and their Functions
PHENOLS
Antiseptic
Antiinflammatory
ALKALOIDS
Antioxidant
Anticancer
FLAVANOIDS
Defense,
Improve blood
Circulation
Secondary metabolites in plants
and their Functions
TERPINOIDS
Antiseptic,
Anti-microbial
MUCILAGES
Soothing effect,
Strengthen
tissues
TANNINS
Tissue
contraction,
Defense
Secondary metabolites in plants
and their Functions
SAPONINS
Steroids,
Expectorant
Antiinflammatory
GLYCOSIDES
Cardio-active
Laxative
Analgesic
VITAMINS &
MINERALS
Vital functions,
growth,
development
Techniques used for Medicinal
Plants

The In vitro plant cell cultures have potential
commercial exploitation of secondary metabolites

Micro-propagation and Agrobacterium transformation
are also common methods for transformation of many
important medicinal plant

The production of secondary metabolites can be
enhanced by using bioreactors
for
Techniques used for Medicinal
Plants

Bioreactors provide more precise control of plant growth
 Bioreactor based propagation of plants can increase the
rate of multiplication and growth of culture as well as it
also reduce :
 Energy
 Labour requirements in commercial micro-propagation
of medicinal plants
 Significant amount of sanguinarine was produced in
cell suspension culture of Papaver somnifera using
bioreactors
Contd……

During the past decade remarkable progress in medicinal
plant genetic-transformation have been witnessed

Rapid progress has resulted in constant flow of new and
improved transformation protocols for many medicinal
plant species

Combinatorial biosynthesis strategies were introduced
for efficient production of secondary metabolites
Combinatorial Biosynthesis

Combinatorial biosynthesis is emerged as a new tool in
generation of novel natural products.

As well as for production of rare and expensive natural
products.

There are several pharmaceuticals that are highly
expensive because :
I.
These compounds are found in rare plants
II. Extreme low concentration
Combinatorial Biosynthesis

Combinatorial Biosynthesis
interesting alternatives

Utilized for important classes of natural products such as :

Alkaloids (Vinblastine, Vincristine)

Terpenoids (Artimisin, paclitaxel)

Flavonoids
are
expected
to
yield
Combinatorial Biosynthesis

The basic concept of Combinatorial Biosynthesis is to
combine metabolic pathways in different organisms at
genetic level

Genes of interest from plants inserted into microorganisms
for production of new and interesting plant secondary
metabolites

New drugs can be added or

Existing drugs can be improved
Combinatorial Biosynthesis for
Terpenoids

Large and important class of natural products

More than 30,000 different structures

Artemisin and zingiberene are of great economic value

Artemisin is anti-malarial drug obtained from Artemisia
annua plant

Yield is 0.5-1.16% on total dry weight

Alternatives could be produced via transgenic plants

Full education of biosynthetic pathway must be known
Combinatorial Biosynthesis for
Terpenoids

Amorphadiene synthetase is the enzyme required for
the synthesis of artemisin

The genes encoding this enzyme has been expressed in
E. coli

Precursors like Artemisinic alcohol and arteminic
aldehyde yield artemisinic acid respectively
Combinatorial Biosynthesis for
Terpenoids

Paclitaxel commonly called taxol

Taxol is well known anticancer drug

The first intermediate Taxadiene can now be produced
in E. coli

The genes encoding enzyme taxadiene synthetase from
Taxus brevifolia species isolated and expressesed in E.
coli
Combinatorial Biosynthesis for
Aklaloids

Vinblastin and Vincristine alkaloids obtained from
Catharanthus roseus plant these are collectively known
as vinca alkaloids

High importance but low yield from plants (3mg per
kg)

They are considered as trace compounds

In vitro application to improve alkaloid yield in plant
has been studied by many scientists
Contd..

It is estimated that for production of 3 kg of Vinca
alkaloids , which is annual need worldwide, around 300
tons of plant material has to be extracted

Production of vinca alkaloids in plant cell culture did not
lead to significant improvement in yield

Today it is accepted that biotechnological approaches in
plant cell culture may not provide an instant solution to
this problem
Contd..

Although the biosynthesis of vincristine and vinblastine
is complex but Strictosidine is the important branching
intermediate for these alkaloids

Seven enzymes and there corresponding genes are
involved for its synthesis four of which have been
expressed in E. coli

Ultimately we can get high yield of these alkaloids to
meet the worldwide demand
Combinatorial Biosynthesis for Drug
Discovery



Natural products have played a significant role in drug
discovery
Because of extraordinary structural diversity
Broad biological activities

Traditionally, chemists have attempted to synthesize
natural product analogs that are important sources of
new drugs.

However, the extraordinary structural complexity of
natural products sometimes makes it challenging for
traditional chemical synthesis
Contd…

Because chemical synthesis involve
 multiple steps
 harsh conditions
 toxic organic solvents
 byproduct wastes.

In contrast, combinatorial biosynthesis provides an
environmentally friendly way to produce natural product
analogs with potential pharmaceutical value.
Strategies For Combinatorial
Biosynthesis

There are three major strategies for combinatorial
biosynthesis
Precursor-directed biosynthesis
2) Enzyme-level modification, which includes
1)
swapping of the entire domains, modules and subunits,
site-specific mutagenesis, and directed evolution
3)
Pathway-level recombination.
Precursor-directed
Biosynthesis

The structural diversity of natural products comes
substantially from diverse building blocks of the natural
product assembly lines.

Precursor-directed combinatorial biosynthesis takes
advantage of the enzymes in the biosynthetic pathways

After detail study of Biosynthetic pathway nonnative
building blocks are incorporated

Consequently
analogs.
producing
various
natural
product
Enzyme Level Modification

Swapping of the entire domains, modules, or subunits has
been the main classical approach for combinatorial
biosynthesis.

This strategy not only enables generation of natural product
analogs, but also allows us to interpret the programmed
biosynthesis of PKS


Ultimately generating novel bioactive polyketides
Further study is required to establish the rules on choosing
domains for combinatorial domain swapping
Site-specific Mutagenesis
 The classical domain swapping approach often leads to
insoluble protein expression, impaired activities and reduced
product yields.
 This is most probably due to disruption of the protein’s
overall structure and thus its function.
 Moreover, the drastic structural changes of intermediates
created by domain swapping may render the intermediates
inaccessible by downstream catalytic domains.
 Modern protein engineering methods, such as site-specific
mutagenesis to substitute specific amino acids,
 Less invasive and offer more effective ways to change the
enzyme function.
Directed Evolution

A powerful enzyme engineering approach, has not
been widely employed on natural product
biosynthetic enzymes.

However, there are significant advantages of
applying directed evolution to combinatorial
biosynthesis.
 Compared
to more conservative changes by sitespecific mutagenesis, directed evolution approaches
can potentially produce more alterations

While restoring the impaired activity due to large
changes in substrate specificity.
Contd…
In contrast to only one enzyme variant obtained with every
successful domain swap, directed evolution methods
significantly increase the throughput of enzyme
variants beneficial for combinatorial biosynthesis.
Last but not least, directed evolution can be accomplished
even when the enzyme catalytic mechanism still
remains elusive.
Pathway-level combinatorial
biosynthesis

The development of molecular and synthetic biology
techniques has enabled the expression of biosynthetic
genes from different species in well characterized host
organisms.

Hybrid pathways have been widely used for production
of novel natural products, especially in the field of drug
discovery.

A novel antibiotic compound, mederrhodin
by
interchanging and combining genes from multiple
species to generate combinatorial pathways.
Combining
two
pathways
Hybrid
Pathway
Advantages

There are three advantages of combinatorial biosynthesis
for drug discovery:

Firstly, combinatorial biosynthesis helps to enrich the
novelty and diversity of the natural product synthesis
which potentially enhances their biological features.

Secondly, Efficient expression of the combinatorial
biosynthetic pathway into genetically different hosts
can increase the concentration of the compound,
eventually resulting in less expensive drugs.
Contd…

Thirdly, combinatorial biosynthesis offers an
environmentally friendly way to produce natural
product analogs, whereas chemical synthesis usually
involves multiple steps, harsh conditions, toxic organic
solvents, and byproduct wastes.
Products of Combinatorial
Biosynthesis
Mithramycin
Binds to DNA and inhibits transcription and protein
synthesis. It has been used for the treatment of several
types of cancers
Micacocidin used to treat Mycoplasma pneumoniae
Infections
3-chloro- and 3-bromo-isorumbrin
Strong anticancer activity as compared to natural
rumbrin
Challenges and Perspective

Combinatorial biosynthesis exploits the shuffling of
anabolic pathways to produce natural product analogs

It has been led to a fundamental change in the field of
classical synthesis.

It will undoubtedly remain very important for drug
discovery programs.

However, production of many of the novel compounds
is still often hampered by low yields, which in turn
hinders their commercialization
Contd ….

The low production could be tackled by enzyme
engineering, finding appropriate expression hosts

Complete knowledge about the biosynthetic pathway of
secondary metabolites must be known because it is a
complex process as many enzymes are involved

Moreover, combinatorial biosynthesis usually generates
large analog libraries, and screening thousands of
compounds consumes time and effort. Hence highthroughput screening methods are urgently needed.
References
Yaseen K. M., S. Aliabbas, V. Kumar, S. Rajkumar. 2009. Recent
advances in medicinal plant biotechnology. Indian Journal of
Biotechnology., 8: 9-22.
Huihua Sun, Zihe Liu, Huimin Zhao, Ee Lui Ang. 2015 Recent
advances in combinatorial biosynthesis for drug discovery. Drug
Design, Development and Therapy, 9: 823–833
Thank You