MSc Agriculture Biotechnology, Kerala Agricultural University

2. Blue biotechnology
Deepali Rahi
CPBMB
KAU

3. 3Branches of biotechnology
• Blue biotechnology
• Marine organism
• White
biotechnology
• Industry
• Red biotechnology
Medicine
• Green
biotechnology
• Agriculture

4. Outline
 Introduction
 Role of blue biotechnology
 Applications
 Transgenic fishes
 Summary
 Future line of work
4

5. Blue biotechnology
 Technique that uses marine living organism-make/modify
products
 Develop organism – specific use
5

6. 6
Tools &
diagnostics
for
reproduction
& growth
Genetics,
physiology,
biochemistry,
ecology
Bioactive
compounds-
function &
mode of
action
Role of blue biotechnology

7. Role of biotechnology in enhancing production
 Aquaculture
 Mariculture
 Primary & secondary source
 20-25% - weight
 Demerits
7
Fish
Algae
CrustaceansMollusks

8.  Oysters, mussels, shrimp, lobsters – sea food
 Culturing in tanks to floating platforms
 Reproductive cycles- hydrogen peroxide and Gamma Amino
butyric acid (GABA)
8…role of biotechnology in enhancing production
(Ledu and Mccombie, 2003)
4-aminobutanoic acid

9. 8
 Crassostrea gigas - diploid
 Eggs - cytochalasin B
 Disrupt actin - three sets of chromosome inside the eggs – triploid
 Summer mortality
 Oyster-sterile
9
(Ledu and Mccombie, 2003)8
Oyster food

10. Applications of blue biotechnology
 Food ingredient
 Algal products
 Biofuel
 Single cell protein
 Biosensors
10
GM fish
Algal food
Biosensor chips

11. Sources for marine derived food ingredients
Macro & micro
algae
Extremophiles Marine
sponges
Fish & sea food by-products
11
(Rasmussen and Morrissey, 2007)

12. Marine-derived food ingredients 12
Photosynthetic
pigments
Protein
Lipids Polysaccharides
Enzymes
(Rasmussen and Morrissey, 2007)

13. 13
Algal
products

14. Types of algae
Rhodophyta
(Red algae)
Chrysophyta
(green algae)
Phaeophyta
(brown algae)
14

15. 15
 Nutraceuticals (food)
 Pharmaceuticals
 Biological labs
 Bio diesel
 Stabalizing agents-Chondrus crispus
 Bioremediation – Cr, Ni, Cu, Zn, Pb
(Shanker et al., 2000)
 Pollution indicator
 Production of Hydrogen-
C. reinhardtii (Hans Gaffron, 1939)
Uses of algae
Azolla-cattle feed
Tofu-Japanese cuisine
Gel electrophoresis

16. Algal-biofuel
 Fossil fuel – finite source
 Alternatives - corn, sugarcane based ethanol
 Drawbacks
 Algae –
 Grow quickly
 High lipid
 Adaptability
 Compatibility
16
(Khan et al., 2009)
50%
20%
7%
11%
7%
5%
Sales
Transport
Plantation
Power generation
Industry

17. Demerits of Biodiesel
 Low biomass concentration
 Insufficient oil content
 Open system - photobioreactor
 More complicated and costly
17

18.  Pyrosequencing
 Gene encoding biofuel
 BLASTx
 KEGG
(Kyoto Encyclopedia
of Genes & genomes)
18
(Yazd et al., 2011)
34%
18%8%
3%
1%
36%
Volvox carteri 26 Chlamydomonas reihartii 25
Kordia algicida 13 Dunaliella salina 6
Dunaliella viridis 2 Micromonas sp. 1

19. 19
…Transcriptome sequencing and annotation of the
microalgae Dunaliella tertiolecta: Pathway description
and gene discovery for production of next-generation
biofuel
Enzymes in TAG biosynthesis Pathway for TAG synthesis
(Yazd et al., 2011)
Triacylglycerol (TAG) biosynthetic enzymes
1. Glycerol kinase GK
2. Glycerol 3-phosphate 0-
acyltransferase
GPAT
3. 1-Acyl-sn-glycerol-3-phosphate
O-acyltranferase
AGPAT
4. Phosphatidate phosphatase PP
5. Diacylglycerol O-
acyltransferase
DGAT
6. Phospholipidacylglycerol
acyltransferase
PDAT

20. Single cell protein
 Bacteria, fungi, algae
 Unicellular microorganism
 Microbes ferment waste
 Diluted – centrifuge, precipitate, filtered
 Weight loss, cardiovascular disease, blood
pressure, eyes, skin, hair
20
SCP - Chlorella

21. …single cell protein
 Central Food Technological Research Institute, Mysore (CFTRI)
 National Botanical Research Institute, Lucknow (NBRI)
21
Composition of groups by % dry weight Spirulina tablet

22. Industrial bio-products
 Taq-Thermus aquaticus
 Vent-Pyrococcus furiosus
 DNA ligase - Thermococcus fumicolans
 GFP – Aequorea victoria
 Shrimp alkaline phosphatases – Pandalus borealis
22

23. Shrimp alkaline phosphatases 23
Pandalus borealis
Dephosphorylation by SAP

24. 24
 A Pichia pastoris clone - shrimp alkaline phosphatase gene from
Northern shrimp Pandalus borealis
 Completely and irreversibly inactivated by heating at 65°C for 5
minutes, thereby making removal of rSAP prior to ligation or end-
labeling unnecessary
Pichia pastoris SAP
Recombinant single alkaline phosphatases

25. 25
Biosensors

26. Biosensor
Biological signal Physical signal
26

27. Use of biosensors
 Fish inspection
 Marine environment
monitoring
 Fish pathogen detection
 Bioremediation
27
(Kroger. S, and Law, R. J. 2005)

28. 28
Medical
applications

29. 29
Kahalalide-F
Antitumor
Dolastatin 10
Antimitotic
Didemnin-B
Herpes
simplex virus
Conus magus
Ziconotide
(prialt)
Chronic pain
Trididemnum
solidum
Dolabella
auricularia
Elysia
rufescens
Molinski et al., 2009

30. 30Anti-cancer drugs from marine organisms
Brentuximab-
leukemia
Eribulin
Metastatic
breast cancer
Trabectin-
Soft tissue
carcinoma
Cytarabine
Myelogenous
leukemia
(Chinembiri et al., 2014)
Cryptotethia
crypta
Ecteinascidia
turbinateHalichondria
okadai
Symploca
hydnoides 1998
2007
2010
2011

31. 31

32. 32
Transgenic fish

33. Classes of fish
 Genetic engineering techniques
 To introduce a new trait to the fish which does not
occur naturally in the species
33
• Agnatha
(jawless fish)
• Chondrichythes
(cartilaginous fish)
• Osteichythes
(bony fish)

34. Why GM Fish?
 FAO – 2025 - seven-fold more aquaculture products
 Blue Revolution - technology from the biological sciences and
engineering
 Low cost production independent of proximity to the oceans
 Species - Less invasive to the environment
 Increased growth rates
 Enhanced resistance to disease
 Alteration of breeding cycles
34
(Taylor and Francis, 2009)

35. GM fishes-covers four broad areas
GM
Enhance
trait
Bioreactors
Pollution
indicators
Genomic
studies
36

36. 36
X
Pseudopleuronectes americanus
Antifreeze gene (afa3)
(Hightower et al., 1991)
Lycopersicon
Esculantus

37. …expression of antifreeze proteins in transgenic
plants
 Synthetic Anti freeze
gene
 afa3 gene
 Not effective
 Chimeric antifreeze gene
 Staphylococus protein A X afa5
Spa-afa5
 Recrystallization inhibition
37
CaMV-promoter
Agrobacterium co culture
(Hightower et al., 1991)

38.  AquAdvantage salmon
 AquaBounty Technologies
 US FDA - production, sale and consumption
 First genetically modified animal to be approved for
human consumption
38Transgenic Salmon

39. 39
Atlantic salmon
(Salmo salar)
Ocean pout
(Zoarces americanus)
Antifreeze gene
Chinook salmon
(Oncorhynchus tshawytscha)
Growth hormone gene
X
AquaAdvantage Salmon
39
(Bodnar, 2010)

40. 39
 Allergen
 Carbohydrate, ash, moisture, protein, total fat, vitamin, mineral,
amino acid, and fatty acid - vit. B6 (0.72 to 0.77 mg/gm) – tuna
(0.81mg/gm)
 Omega 3 & 6
 Growth harmone – below 10.40 ng/g of tissue
 Estradiol, testosterone, T3, T4
AquaAdvantage Salmon (Bodnar, 2010)
Risk assessment of AquaAdvantage almon

41. GloFish
 Patented and trademarked brand
 Sold in US only
 Variety
 Not meant for consumption
 $50 – 1/10 of normal zebra fish
41
GloFish

42. 42
 Green fluorescent protein (GFP) - Aequora victoria (jellyfish)
 Caenorhabditus elegans-first
 All kingdoms – GFP
 ANDi (Chan, A.W.S et al)
 GloFish
Fluorescent Drossophila
Fluorescent ANDi
(Stewart, 2006)
...GloFish

43.  Fluorescence pattern
 Nicotiana tabacum cv. Xanthi
 mGFP5er gene
 Promotor - CaMV 35S
 Vascular & young leaves
 Adaxial close to stem
43
(Harper and Stewart, 2000)
Whole plant fluorescence pattern

44. ICAR-CMFRI
Patented products and techniques
44
Shore pearl
culture technology
Green mussels
extract (GMe)
for arthritis
Hatchery
technology for
clown fish

45. Summary
 Blue biotechnology – novel or modified products – by
growing/manipulating organisms
 Raising population
 Food, algal products, single cell protein, cancer applications,
biosensors
 Transgenic fishes
 Role of GFP
45

46. Work-yet to be done!
46
(Mehbub et al., 2014)

47. …work-yet to be done!
 Large supply-challenging
 Advanced technology
 GM-borderline on ethics
47

48. 48
Biotechnology and its products have created some
amazing possibility but also has raised fears. Hence,
we should utilize biotechnology wisely & for a good
cause

49. THANK YOU 49
Thank You