A brief presentation on the topic "Microbial Production of Bioactive Natural Products and Biologics" as emerging in Indian Pharmaceutical Industries

1. Presented By
Nilesh S. More
Department of Medicinal Chemistry
MC/2019/14
राष्ट्रीय औषधीय शिक्षा एवं अनुसंधान संस्थान (नाईपर), हैदराबाद
National Institute of Pharmaceutical Education and Research (NIPER), HYDERABAD
Microbial Production of Bioactive Natural Products
and Biologics
03-12-2019 1

2. Contents
INTRODUCTION
BIOLOGICAL ACTIVITIES OF NATURAL PRODUCTS AND BIOLOGICS
MICROBIAL CELL FACTORIES
EFFORTS IN PRODUCT IMPROVEMENTS AND GENERATION OF NEW
ANALOGS
FUTURE PROSPECTS
REFERENCES
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3. INTRODUCTION
NATURAL PRODUCTS
Secondary metabolites
- Plants
-Animals
-Microorganisms
-Marine
Organisms
Structurally and chemically
diverse molecules
BIOLOGICS Active Pharmaceutical
Ingredients
Derived from living
organisms
-Animals
-Plants
-Microorganisms
-Human blood
products
-Tissue transplants
Too complex to be
produced
Organic Synthesis
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 3

4. BIOLOGICAL ACTIVITIES OF NATURAL PRODUCTS AND BIOLOGICS
BIOLOGICAL
ACTIVITIES
Antibiotic
Antifungal
Agents
Anticancer
and
Antitumor
Immuno
suppressant
Biofilm-
Inhibitory
Agents
Others
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 4

5. Antibiotics
For example
Erythromycin A Tetracyclines Streptomycin
Polyketides
Assembled by
polyketide synthases
(PKS),
Largest classes of
chemically diverse
natural products
Most important
secondary metabolites
Applications in
medicine, agriculture,
and industry
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 5

6. Erythromycin
Tetracycline
Streptomycin
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 6

7. Antifungal Agents
0
20
40
60
80
100
120
140
1966-1975 1976-1985 1986-1995 1996-2005 2006-2015
NumberOfProduct(s)Published
Year Of Publication
Figure 1 : Number of reviewed anti-Candida albicans products published between 1966 and 2015.
First effective polyene antifungal agentNystatin
• A topical antifungal agent in treating oral, gastro-intestinal, and genital candidosis
Utilized against fungal infections caused by Aspergillus speciesAmphotericin B
• Effective in patients who have undergone organ transplantation
Zida A, Anti-Candida albicans natural products, sources of new antifungal
drugs: A review. Journal de mycologie medicale. 2017 Mar 1;27(1):1-9.
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8. Nystatin
Amphotericin B
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
Ieodoglucomide
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9. Anticancer Agents
Isolated from Streptomyces parvulus in 1940Actinomycin
• First antibiotic shown to have anticancer activity
• In particular, actinomycin D, also known as dactinomycin, is approved by FDA , widely used in
clinical practice as an anticancer drug
Glycopeptide produced by Streptoalloteichus hindustanusBleomycin
• Used for squamous cell carcinomas, melanomas, sarcomas, testicular, and ovarian cancer,
hodgkin’s and non-hodgkin’s lymphomas, and testis tumors as an anticancer agent
Important family of polyketides produced by Streptomyces speciesDaunorubicin
• Used in the treatment of acute lymphoblastic or myeloblastic lymphoma
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
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10. Bleomycin Daunorubicin
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
Actinomycin
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11. Immunosuppressive Agents
Also known as SirolimusRapamycin
• Microbial natural products with immunosuppressive properties
• Exhibits synergism with other immunosuppressants
• Developed to coat coronary stents and prevent organ transplant rejection
Also known as TacrolimusFK506
• First discovered in soil samples containing Streptomyces tsukubaensis
• Minimize organ rejection
• Its immunosuppressive activity led to its use in heart, liver, and kidney transplants with
overwhelming success
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 11

12. Anti-inflammatory Agents
Natural products also have anti-inflammatory activitiesFK506
• Shown efficacy in the treatment of refractory rheumatoid arthritis, a chronic inflammatory disease
Inhibits the inflammatory responseRapamycin
• Diminishing the activation and proliferation of inflammatory cells and the expression of
inflammatory cytokines
• Reducing secondary injury in the spinal cord and providing a neuroprotective effect
Isolated from Streptomyces sp. SCSIO 10355Strepsesquitriol
• Have anti-inflammatory activity through the inhibition of tumor necrosis factor-a production in
lipopolysaccharide-activated macrophages
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 12

13. RapamycinFK506 (tacrolimus)
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 13

14. Biofilm Inhibitory Agents
Parasitic
microorganisms
Cahuitamycins
A-C
Lactobacillus
biosurfactants
• Adhere to solid surfaces
• Forms complex polysaccharide matrix
• Resistance against antibiotics
• Derived from the marine bacterium Streptomyces
gandocaensis
• High anti-adhesive biofilm formation properties against C.
Albicans
• Also prevented biofilm formation of E. Coli, and S. Aureus
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 14

15. Park SR, Discovery of cahuitamycins as biofilm inhibitors derived from a
convergent biosynthetic pathway. Nature communications. 2016 Feb 16;7:10710.
Cahuitamycins A Cahuitamycins B
Cahuitamycins C
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16. Others
Avermectins
 Antiparasitic activity
Significantly lowers the incidence of lymphatic filariasis
Lipstatin
Is a pancreatic lipase inhibitor produced by Streptomyces toxytricini
Combat obesity and diabetes by interfering with the gastrointestinal absorption of fat
Milbemycin
Isolated fermentation product of S. Hygroscopicus
An insecticide and acaricide with GABAergic activity
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 16

17. Avermectin
Lipstatin
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 17

18. Biological Activity of Microbial Biologics
E. coli
Humulin®
Protropin®
Neupeg®
First recombinant biopharmaceutical
as a treatment for diabetes
Used to treat children with growth
hormone deficiency
For the treatment of Tumour
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 18

19. https://www.rcsb.org/
Figure 2 :Crystal structures of (A) Recombinant human insulin (humulin®) (PDB 4F0N ), (B) Granulocyte colony growth factor Pegfilgrastim (neupeg®) (PDB
1HRG), (C) Human interleukin-3 (PDB 5UV8)
(A) Recombinant Human Insulin (B) Granulocyte Colony Growth Factor Pegfilgrastim
(C) Human Interleukin-3
03-12-2019 19

20. Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
Name Origin Biological activity
Antibiotic
Erythromycin A Saccharopolyspora erythraea Antibacterial
Tetracycline Streptomyces rimosus Antibacterial
Streptomycin Streptomyces griseus Antibacterial
Antifungal Agents
Amphotericin B Streptomyces nodosus Antifungal
Anticancer and Antitumor
Bleomycin Streptomyces verticillus Squamous cell carcinomas, Hodgkin’s
lymphomas and testis Tumors
Didaunorubicin Streptomyces peucetius myeloblastic lymphoma
TABLE 1 : Biological activities of microbial-derived natural products and biologics.
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21. 03-12-2019 Park SR, A Review of the Microbial Production of Bioactive Natural
Products and Biologics. Frontiers in Microbiology. 2019;10:1404.
21
Immunosuppressant/Anti-inflammatory Agents
Rapamycin Streptomyces rapamycinicus Immunosuppressive, antifungal,
antitumor, neuroprotective, neuro-
regenerative, and lifespan extension
activities,
Biofilm-Inhibitory Agents
Cahuitamycins Streptomyces gandocaensis Inhibitors of Acinetobacter
baumannii biofilms
Others
Avermectin Streptomyces avermitilis Onchocerciasis and lymphatic
filariasis

22. MICROBIAL CELL FACTORIES
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
Microbial hosts Advantages Disadvantages Compounds
Gram-negative
Escherichia coli  Fast growth
 Simple culture procedures
 High versatility
 Risk of translational
errors
 Expensive
 Challenging
purification Process
 Recombinant
human insulin
 Erythromycin A
 Insulin glargine
 Pegfilgrastim
 Human serum
albumin
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TABLE 2 : Microbial cell factories

23. Gram-positive
Lactococcus lactis  Simplified downstream
purification processes
 Absence of endotoxins
 Generally recognized as
safe (GRAS)
 Per liter secretion
generally less
 IL-2
 Tetanus toxin
fragments
Streptomyces sp.  Rapid growth
 Abundant supply of
secondary metabolite
precursors
 Ability to produce
natural products.
 Efficient protein
secretion
 Forms pellets or
clumps
 Low protein yield
 Streptomycin
 Nystatin
 Rapamycin
 FK506
 Cahuitamycin
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 23

24. Fungi/yeast
 Saccharomyces cerevisiae  Fast growth rate
 Technically practical
 Cost-effective
 Advanced
fermentation science
 Lack some required
precursor pathways
 Human serum
albumin
 Recombinant
human insulin
 Paclitaxel
 Aspergillus sp.  Tolerate extreme
cultivation conditions
 Degrade and utilize
diverse biopolymers
 Allowing cultivation
on renewable
resources
 Major Source of
citric acid production
 Highly compact
pellets formed
during submerged
fermentations
 Immunoglobulin G
 Human IL-2
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
03-12-2019 24

25. EFFORTS IN PRODUCT IMPROVEMENTS AND GENERATION OF NEW
ANALOGS
Park SR, A Review of the Microbial Production of Bioactive Natural Products
and Biologics. Frontiers in Microbiology. 2019;10:1404.
Improved
microbial natural
products
Strain
Improvement
Engineering
Precursor
Supply
Pathway
Engineering
Combinatorial
Biosynthesis
Mutasynthesiss
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26. 03-12-2019 https://www.biospectrumindia.com/ 26
TABLE 3 : Bio-Spectrum Top 10 Indian Biopharma companies ( Revenues are in Cr. )
FUTURE PROSPECTS

27. Fungi 23%
Bacteria,
30%Plant,
47%
Other 5%
Figure 3 : The cumulative percentage of FDA-approved natural
product NMEs, separated by environmental source.
Plant
3%
Fungi
46%
Bacteria
51%
Source of FDA-approved antibacterial
natural products
Figure 4 :The cumulative percentage of FDA-approved natural
product antibacterials, separated by environmental source.
Patridge E, An analysis of FDA-approved drugs: natural products and their
derivatives. Drug discovery today. 2016 Feb 1;21(2):204-7.
FDA-approved natural product NMEs, by
environmental source
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28. 0
10
20
30
40
50
Discovery
Pre-Clinical
PhaseI
PhaseII
PhaseIII
Pre-Registration
PRODUCT(%)
STAGES
Figure 5 :Workflow involved in the development of a new drugs and
approximate percentage of recombinant proteins currently in each step
Global Data 2015. http://www.globaldata.com. 2015
Other 19%
Infectious disease
5%
Men
health
5%
Immunology
6%
Women Health
8%
Oncology 15%
Haematological
Disease
18%
Metabolic
dissorder 24%
Figure 6 : Amount of marketed recombinant proteins (expressed in
percentages) applied to each therapeutic area, other therapeutic areas (<5 %
each) include diseases related to cardiology, central nervous system,
ophthalmology and dermatology among others
Sanchez-Garcia L, Recombinant pharmaceuticals from microbial cells: a
2015 update. Microbial cell factories. 2016 Dec 1;15(1):33.
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29. 03-12-2019 29
Summary
Microbial natural products and biologics will continue to broaden their diverse and integral role
in human life
The potential for recombinant drugs is expanding through the utilization of new platforms
and efforts in product improvement
Continued efforts in natural product analog development will provide an avenue for the
discovery of compounds with improved biological activities in comparison to their natural
counterparts

30. REFERENCES
 Park SR, Yoon YJ, Pham JV, Yilma MA, Feliz A, Majid MT, Maffetone N, Walker JR, Kim E, Reynolds JM,
Song MC. A Review of the Microbial Production of Bioactive Natural Products and Biologics. Frontiers in
Microbiology. 2019;10:1404.
 Zida A, Bamba S, Yacouba A, Ouedraogo-Traore R, Guiguemdé RT. Anti-Candida albicans natural products,
sources of new antifungal drugs: A review. Journal de mycologie medicale. 2017 Mar 1;27(1):1-9.
 Park SR, Tripathi A, Wu J, Schultz PJ, Yim I, McQuade TJ, Yu F, Arevang CJ, Mensah AY, Tamayo-Castillo G,
Xi C. Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway. Nature
communications. 2016 Feb 16;7:10710.
 https://www.rcsb.org/
 Patridge E, Gareiss P, Kinch MS, Hoyer D. An analysis of FDA-approved drugs: natural products and their
derivatives. Drug discovery today. 2016 Feb 1;21(2):204-7.
 Global Data 2015. http://www.globaldata.com. 2015
 Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, Villaverde A. Recombinant
pharmaceuticals from microbial cells: a 2015 update. Microbial cell factories. 2016 Dec 1;15(1):33.
 https://www.biospectrumindia.com/
03-12-2019 30

31. is like
(Just don’t lick the spoon)