2. COMBINATORIAL BIOSYNTHESIS ADVANCES IN MEDICINAL PLANT BIOTECHNOLOGY

3. Contents: 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

4. Medicinal Plants  Plants that have a recognized medicinal use  Medicinal herbs are good alternatives for curing many diseases  Low cost and less side effects

5. 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

6. Why Medicinal Plants are important so? 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

7. Secondary metabolites in plants and their Functions PHENOLS ALKALOIDS FLAVANOIDS Antiseptic Anti- inflammatory Antioxidant Anticancer Defense, Improve blood Circulation

8. Secondary metabolites in plants and their Functions TERPINOIDS MUCILAGES TANNINS Antiseptic, Anti-microbial Soothing effect, Strengthen tissues Tissue contraction, Defense

9. Secondary metabolites in plants and their Functions SAPONINS GLYCOSIDES VITAMINS & MINERALS Steroids, Expectorant Anti- inflammatory Cardio-active Laxative Analgesic Vital functions, growth, development

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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.

25. Strategies For Combinatorial Biosynthesis  There are three major strategies for combinatorial biosynthesis 1) Precursor-directed biosynthesis 2) Enzyme-level modification, which includes swapping of the entire domains, modules and subunits, site-specific mutagenesis, and directed evolution 3) Pathway-level recombination.

26. 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.

28. 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

29. 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.

30. 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.

31. 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.

36. 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.

37. Combining two pathways Hybrid Pathway

38. 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.

39. 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.

41. 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

42. 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

43. 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.

44. 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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