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COMBINATORIAL BIOSYNTHESIS

ADVANCES IN MEDICINAL

PLANT BIOTECHNOLOGY

Contents:Medicinal Plants Status of Medicinal PlantsImportance Secondary metabolites in plants and functionsTechniques used for medicinal plantsCombinatorial biosynthesisCombinatorial biosynthesis for Terpenoids Combinatorial biosynthesis for Alkaloids Combinatorial biosynthesis for Drug DiscoveryAdvantages 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 called active constituents of plants

They perform different functions

Different in different plant species

Concentration in different plants also vary

Secondary metabolites in plants and their Functions

PHENOLS

ALKALOIDS

FLAVANOIDS

AntisepticAnti-

inflammatory

Antioxidant Anticancer

Defense, Improve blood

Circulation

Secondary metabolites in plants and their Functions

TERPINOIDS

MUCILAGES

TANNINS

Antiseptic,Anti-microbial

Soothing effect, Strengthen

tissues

Tissue contraction,

Defense

Secondary metabolites in plants and their Functions

SAPONINS

GLYCOSIDES

VITAMINS & MINERALS

Steroids, Expectorant

Anti-inflammatory

Cardio-activeLaxative Analgesic

Vital functions, growth,

development

Techniques used for Medicinal Plants

The In vitro plant cell cultures have potential for

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

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 plantsII. Extreme low concentration

Combinatorial Biosynthesis Combinatorial Biosynthesis are expected to yield

interesting alternatives

Utilized for important classes of natural products such as :

Alkaloids (Vinblastine, Vincristine)

Terpenoids (Artimisin, paclitaxel)

Flavonoids

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

1) Precursor-directed biosynthesis2) Enzyme-level modification, which includes

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 producing various natural product analogs.

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 site-specific 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-isorumbrinStrong 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 high-throughput 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

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