This document discusses genetic engineering to modify fatty acid biosynthesis in plants. It provides background on lipid structure and different types of fatty acids like saturated, unsaturated, and essential fatty acids. The document then discusses strategies for engineering plants to produce very long chain polyunsaturated fatty acids (VLC-PUFAs) like EPA and DHA through the expression of genes encoding fatty acid desaturases and elongases. Optimizing these pathways in transgenic plants could provide sustainable sources of omega-3 fatty acids as alternatives to fish oils.

1. Genetic engineering
for fatty acid
biosynthesis
Roshni.M
IPhD Plant
Biotechnology
PALB6078

2. Lipid = a compound that is insoluble in water, but soluble
in an organic solvent
chemical structure: glycerol + fatty acids

4. Saturated and unsaturated fatty
acids
Saturated: the SFA’s of a lipid have no
double bonds between carbons in chain
Polyunsaturated: more than one double
bond in the chain

5. Corn oil contains 86% polyunsaturated fatty acids
Therefore is a liquid at room temperature.
Olive oil contains monounsaturated fatty acids.
Therefore is a liquid at room temperature, likely to solidify when
refrigerated.
Saturated fatty acids (tropical oils, animal fats) are solids, or
nearly solids at room temperature.

6. Essential Fatty Acids
O Long chain polyunsaturated fatty acids are crucial for
normal cellular and physiological functioning
O These fatty acids require supplementation by diet and
hence referred to as “essential fatty acids” or EFAs
O Two major kinds of EFAs are the omega-3 (n-3) and
omega-6 (n-6) fatty acids.

7. Structures of Essential Fatty
Acids
The n-3 and n-6 fatty acids contain a double
bond that is 3- and 6-carbons away from
the methyl end of the molecule

8. Mónica Venegas-Calerón., 2010
Very long chain polyunsaturated fatty
acids (VLC-PUFAs) are fatty acids of 20
carbons or more in length with three or
more methylene-interrupted double
bonds in the cis position.

11. Good sources of ‘omega-3 fatty acids’
• Oily fish(salmon, mackerel, sardines, tuna)
• Pumpkin seeds, sesame seeds
• soybean oil, flax seeds

13. www.fao.org

15. Good sources of ‘omega-6 fatty acids’
•Most vegetable oil, Sunflower oil, Corn oil, Soybean oil
•Cotton seeds oil
•Pumpkin seeds
•Nuts and cereals
•Poultry, eggs
•Avocado

16. O Excessive amount of omega 6 leads to inflammation,
result in heart disease, cancer, asthma , arthritis and
depression
good sources of omega-3 and omega-6 fats
should be included daily. It is important to take
these two fats in the proper ratio as well.

17. Classes of fatty acids

18. Desaturation and elongation of fatty acids

21. Overview of the main metabolic fates of alpha-linolenic acid in
humans

22. Humans are not capable of de novo
synthesis of LCPUFA because they lack the
two desaturases converting oleic acid (Δ9-
18:1) into linoleic (omega6-Δ9,12-18:2) and
a-linolenic acid (omega3-Δ9,12,15-18:3).

23. Reuters health ltd

24. Aerobic VLC-PUFA biosynthetic pathways.

26. O Over-fishing and concerns about pollution of the
marine environment -alternative, sustainable sources
of very long chain polyunsaturated fatty acids
(VLCPUFAs) such as EPA and DHA.
O A number of different strategies have been considered,
with one of the most promising being transgenic plants
‘‘reverse-engineered” to produce these so-called fish
oils.
O Reconstitution of VLC-PUFA biosynthesis in transgenic
plants
Mónica venegas-calerón et al., 2010

27. Classical plant breeding approaches mutagenesis,
screening and crosses were made between
compatible germ lines
Breeding of low erucic acid rape seed , high oleic
aid sunflower , high stearic acid soybean and low
linolenic acid flax varieties
Loof and Appleqvist ,1972
Stefansson et al., 1961
Urie et al., 1985
Graef et al., 1985

28. METABOLIC ENGINEERING – REVERSE ENGINEERING APPROACH

29. Metabolic engineering in plants involves
the modification of endogenous
pathways to increase flux towards
particular desirable molecules.

30. Plants -significant renewable source of fatty acids -many
species accumulate them in the form of triacylglycerol
as major storage components in seeds.
Jay J. Thelen et al., 2002
Metabolic engineering of oilseed fatty acids has become
possible and transgenic plant oils represent some of the
first successes in design of modified plant products

31. Plants represent a large reservoir of fatty acid diversity,
synthesizing at least 200 different types of fatty acids
(Van de Loo et al., 1993)
The abundant fatty acids produced in these major
commercial oilseeds comprise just 4 of the > 200
possibilities, namely linoleate, palmitate, laurate, and
oleate.
The four most important oilseed crops are, soybean, oil
palm, rapeseed, and sunflower.
(Gunstone,
2001)

32. Why Plant Oils Are Attractive Targets for Metabolic Engineering
The fatty acid content and composition of plant
membranes are highly conserved
Fatty acids is tolerant to changes in chemical structure
and is a good target for genetic manipulations
Plant oils are already used for nonfood applications –
familiar with fatty acid chemistry and applications.
The pathways that produce these structures have been
identified
(Voelker and Kinney, 2001)

33. The conversion of native plant fatty acids such
as LA and ALA to VLC-PUFAs requires a
minimum of three sequential non-native
enzymatic reactions (e.g. two desaturations
and acyl- CoA elongation) to generate C20
PUFAs such as ARA and EPA.

35. Hoffmann et al., (2008)

36. O Proof-of concept demonstration that the VLC-PUFA
pathway could function in a transgenic system was first
provided by expression in yeast, with initial data
showing the low accumulation of ARA and EPA and
subsequent experiments
Johnathan A. Napier.,2005

37. O Initial attempts to reconstitute the VLC-PUFA
biosynthetic pathway in a heterologous host
utilised yeast as a simple model system.
O Beaudoin et al. co-expressed the C. elegans C18
Δ6-elongating activity PEA-1 with the borage Δ6-
desaturase and the M. alpina Δ5-desaturase.

38. Microbes as biofactories
O Synthesize VLC-PUFA have been developed and are
economically viable for specific high value applications (such as
infant formula – baby milk – Formulations ) in controlled culture
systems.
O However, such systems are expensive to maintain and have
limited flexibility for significant scale-up
O Requiring the appropriate microbiological facilities (such as
fermenters)
O Sensitive to disruptions of power-supplies and have a significant
environmental footprint.
Carlo Agostoni., 2008

39. Concentrations of PUFA in human breast milk are
relatively stable during the first year of life: ARA is
equivalent to 1% in colostrum and 0.5% in mature
milk, which is equivalent to 14 to 15 mg/dL; DHA is
equivalent to 0.5% in colostrum and 0.25% in
mature milk, equivalent to 7 to 8 mg/dL.

40. Fatty acid biosynthesis in plants.
Plant lipid biochemistry

41. O The modification of lipid metabolism to change the
quantity and quality of fatty acids in plants has
important applications in the food industry, as well as
in the production of detergents, fuels, lubricants,
paints and plastics

42. Crucial issues: optimization the levels of
LCPUFA in transgenic plants
Outlined below are logical approaches which might be
expected to enhance the accumulation of VLC-PUFA in
transgenic plants.
OThe identification of superior desaturases
OIdentification of a VLC-PUFA-specific acyl-exchange
mechanism

43. O Maintenance of a continuous flux of substrates through
the VLC-PUFA biosynthetic pathway without significant
loss to TAG
O Optimizing the fatty acid elongase
O Modulating the acyl-CoA pool
O Co-ordinated expression of transgenes
O Appropriate localisation of transgene-derived activities

44. O 6- desaturase from the higher plant Primula vialii
that has a very strong preference for the ω3
substrate ALA has recently been isolated
O ω6 LC-PUFA can be converted to ω3 LC-PUFA by
the action of novel ω3- desaturases, such as the
nematode FAT-1 D15-desaturase, which converts
C18 and C20 ω6PUFA to their ω3 counterparts

45. Sara Glass et al ., 2017

46. Agne`s E. Ricroch., 2015

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