Shailendras@evolva.com; +91 9566281304
“eOleOnions”: Oleaginous onions evolved with n-3 PUFA to maintain health & wellness
1. Executive Summary / Motivation
Diets deficient in omega 3 fatty acids seem to be a “silent killer” in several metabolic syndromes
and in obese population. A number of reviews and publications have pointed to a positive role
for Omega 3/ (n-3) PUFA in preventing diabetes, metabolic disturbances and cardiovascular
(CV) related diseases. Omega 3 fatty acids have been shown to alleviate Adipose Tissue
Inflammation, Insulin Resistance and reduction of plasma triacyl glycerol (TG) level that leads to
an “anti-obesity” effect.
Global statistics in 2010 indicate that close to 4 million lives were lost (mostly in the age group
of 20 to 79 years) which constitute 6.8 % of world population, mostly due to diabetes. In
addition, cardiovascular related diseases has killed more than 17 million lives globally and
obesity related factors seem to be silently contributing to many of these deaths.
Neutraceutical firms often focus on launching products rich in essential fatty acids (since most
mammalian cells are incapable of producing fatty acids such as omega 3 PUFA) and oceanic
fish oil which is normally found to be contaminated with heavy metals and dioxins. In addition,
food habits, population diversity & demographics and several other factors necessitate the
discovery of “highly accessible” sources for such PUFAs which are acceptable universally for
safe consumption. Motivation for developing this proposal lies inherently in the established fact
that fat consumption is having a negative impact on human health and economy globally and
hence the need to create a viable, vibrant and sustainable source for “essential” oil naturally,
which is affordable to all sections of society across the world.
2. Novelty/Idea/Concept in greater detail
To transform this idea into reality, an evaluation of universal acceptability factor was considered
and this indicates to many plant sources for such PUFAs that are also affordable to all sections
of society (rich & poor) & all eating habits (vegans & vegetarians). One such source is the
humble “onion” which is universally consumed in most food preparations and varieties of salads
on daily basis and can be grown globally in optimal conditions. Also, about 100 gram of red,
white, green and yellow onion contains 10g; 0.08g, 0.08g and 0.63 g total fat content in it
respectively. Hence one possibility is to identify red onion for potential “bio-accumulation” to
assimilate sufficient quantities of omega 3 fatty acid which could able to require/provide RDA
value (c. 450mg/day) for consumption.
This project will aim to “evolve” naturally, a variant of “oleaginous onion” rich in omega3 fatty
acid though our proprietary technology platform which is customized for such evolution by
converting omega 6 fatty acids in onion to omega 3. This would be achieved by subjecting
natural red onion variants rich in omega 6 to varying degrees of natural selection pressures
driving towards omega 3 production and our sensitive screening system picks up the rare
variant (amongst several hundred variants) post evolution.
The methodology adopted is to metabolically engineer onion to accumulate nutritious oil by
using a precursor such as α-Linolenic acid (18:3), a native plant fatty acid for extending omega3
pathways using various desaturase and elongase enzymes. Other supportive genes to enhance
Acyl-CoA availability, conversion of DGA into TAG and genes for lipid protein biosynthesis are
essential for biosynthesis and accumulation of Oil in plants1. Oleosins proteins are essential for
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Shailendras@evolva.com; +91 9566281304
formation of lipid particle, prevent their fusion and remain small and stable, providing high
surface to volume ratio. This is crucial phenomena of oil accumulation which is unavoidable
while engineering any oleaginous plant 2 .
Documented bottleneck for biosynthesis of omega-3 fatty acid in plants includes 1) Lack of
native enzymes for conversion of C18 fatty acid in to C 20 fatty acid which involved three nonnative enzymes; 2) lack of essential elongase substrate in the acyl-CoA pool; 3) Fine tuning of
the fatty acid flux between acyl CoA, Phospholipid & TAG to maximize oil accumulation and 4)
localization of endogenous lipid synthesizing enzymes into subdomain specific endoplasmic
reticulum1.
Two groups of transgenic plants called as “group I type” and “group II type” will be developed to
create evolved oleaginous onion following mating and selection method to obtain desired
eOleOnions plants “.
To develop “group I type” transgenic onion, Omega 3 biosynthesis genes identified from various
sources3, 4, 5, 6, 7 kept under suitable promoters and terminators will be synthesized as a big DNA
fragment after plant based codon optimization of CDS along with non-toxic selection marker
such as phosphomannose isomerase and cloned to intermediate vector pBR22.This
recombinant vector will be transformed from E. coli into Agrobacterium using helper plasmid
pRK 2013 by conjugation, results into Agrobacterium containing conjugated Ti-plasmid
harboring complete omega3 biosynthesis pathways. As it has been proven that ssT-DNA can
carry up to 30kb of exogenous DNA8 and method for developing transgenic onion is
demonstrated successfully9, makes us more confidant on our approach to develop transgenic
onion plant.
Fig1: Overview of heterologous genes involvement in 5 step Omega3 biosynthesis pathway.
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Same Agrobacterium-mediated transformation approach will be adapted to develop “group II
type” plant harboring essential genes responsible for conversion of DAG in to TAG 10,11 as well
as for formation for oil body to enhance lipid accumulation12 (Fig 2).
Fig2: Overview of supportive genes required for (A) stability of oil body formation and (B) TAG
biosynthesis.
Transformed Onion callus will be grown on tissue culture media containing specific selection
marker and validated the presence of integration of targeted exogenous genes using PCR
based technique. Later Callus harboring integrated genes will be differentiated using auxin and
cytokinin (phytoharmones) treatment to develop in to transgenic plant.
Onion has got natural cytoplasmic inheritance of male sterile genes with three different
cytoplasm S,C, and T. For this project, 2 pairs of CMS (cytoplasmic-genic male sterility) lines
MS 48 and MS 65, Male sterile lines (Line A) and fertile maintainer lines (Line B) of onions
developed by Indian Institute of Horticulture Research, (ICAR), Bangalore will be selected to
develop omega 3 rich new hybrid. Using the male sterile lines will help us to make hybrid
combinations with their pollinators to develop new hybrids. One of these male sterile maintainer
lines will be used for group I or II type gene cassette insertion. Other group “1type or II type” will
be introduced in male fertile variety. This advantage of natural male sterility of onion is utilized
in this breeding program. There by avoid problems related to introduction of an external male
sterile gene and its threat to environment. Male sterility will help in breeding and maintaining
hybrid onions with high PUFA content lines pure.
To achieve stable and viable “eOleOnions” transgenic plant, “Group I type” & “Group II type” will
be mated and various mixed type transgenic plants will be re mated till next generation. “Mixed
type” transgenic plant harboring omega 3 PUFA biosynthesis pathway along with TAG synthesis
and oil body forming protein will be validated at molecular level as well as analytical level after
lipid profiling to achieve “eOleOnions” transgenic plant with desired omega 3 PUFA . (Fig3)
Onion is a biennial plant and therefore requires at least 2 years completing its life cycle. Mating
of ‘Group1type’ and group II type’ can be achieved after flowering. Seeds set from this mating
can be termed as F1. To produce F1 will take first two years and there is no shortcut for this. F1
plants with desirable characteristics can be identified with markers for male sterility like AcPME
expression12 or 5'cob-marker and orfA501 markers13 . F1 seeds can be grown and fast track
production of onion bulb by controlling temperature and daylight. Selected F1 generation onions
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Shailendras@evolva.com; +91 9566281304
with uniform shape and sizes will be analyzed biochemically. Lipid profiling for the PUFA
content and allyl sulfides and sulfoxides profiling for pungency of onions will be checked.
Fig3: Mating approach to develop stable omega 3 producing “eOleOnions” transgenic plant.
Development & evolution of such oleaginous onions could be an ideal approach to address
increasing demand of lipid neutraceuticals that are highly affordable to various sects of society
This eventually would boost the health & welfare of humans across the globe.
3. Current Stage of development
Given below is where we are at this point of time:
1. Identified genes required for Omega 3 biosynthesis pathway and other related genes
required for Oil body formation and biosynthesis of TAG.
2. Contract researcher identified for codon optimized gene synthesis.
3. Methodology for integration demonstrated in other eukaryotes in our lab.
4. Plant tissue culture and method of its differentiation has been established in our lab.
4. Existing infrastructure and team
In addition to Dr P.M. Murali (M.D. & C.E.O.) & Mr. Shriram Raghavan (Director, Research
Integration), the company has skilled scientific teams with various research domains.
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Evolva have five well experienced scientists in Molecular biology department, one in agriculture
area, one in bioinformatics and 3 in analytical chemistry department. All scientists have broad
exposure and experience to academic and industrial research filed. Few profiles in brief are
mentioned below:
1. Shailendra Sonkar, M.Sc. Marine biotechnology, (PhD)
Senior Scientist, Molecular biology (5 years of industrial research, Scuba diver, former
researcher NIO, Goa)
Accomplishments in Evolva
 Metabolic engineering of TAG pathways to produce fatty yeasts for biofuel production
 Discovery and creation of novel pathways for production of spice molecules in
Saccharomyces cerevisiae
 Sourcing and construction of transcriptome libraries from various marine organisms,
insects, plants and microbes.
2. Gomathi, M.Sc. Botany, PhD
Phytopathaologist, Project Manager (2 decades of experience in plant fungal interactions, model
organism worked Viz. Magnaporthe grisea, Botrytis cinerea Colletotrichum gloeosporioides,)
Breeding for disease resistance with marker assisted selection in rice for Xa21gene.
Accomplishments in Evolva
 Involved in first in kind generation of novel plant antifungal compound inspired from
Evolva’s technology
3. A.S.Sathish kumar, M.Sc. Biochemistry, (PhD)
Team Leader, Molecular Biology (10 years of industrial research; former scientist, Biocon)
Accomplishments in Evolva
 Discovery of genes and pathways for natural sweetener Stevia and metabolic engineer
into Saccharomyces cerevisiae and E.coli
 Discovery and creation of novel pathways for production of spice molecules in
Saccharomyces cerevisiae
Evolva Biotech is equipped with fully functional molecular biology, cell biology, bioinformatics,
and chemistry lab along with supportive admin team. Along with Dr. P. M. Murali`s expertise in
plant tissue culture, we have expert tissue culture team from DCRD, a close partner of Evolva
who will assist in plant tissue culture to achieve the committed objectives of proposal.
Equipment details are provided below:
Cheff mapper PFGE (Biorad),-300C Deep freezer (Thermo), 40C freezer (2 in number),
Horizontal gel electrophoresis unit (5 in number), Digital dry bath (Labnet, 2 in number), Vertex
machine VX100 (Labnet, 2in number), Min-spin centrifuge (2 in number), Master cycler gradient
PCR machine (Eppendorff, 2in number), Centrifuge 5414 (Eppendorff, 2 in number), Cooling
centrifuge 5415R (Eppendorff, 2 in number), UV Trans illuminator (BIORAD), Hot air oven
(Ascension innovation), Water bath (Thermocon), Hot air oven (Samsung), Hot plate (2 in
number), Autoclave machine (2 in number), Ice maker (Blue star), Cold room (Blue star), Speed
Vac evaporator (Thermo), Safe ImagerTM (Invitrogen), Homogenizer (PRO), Cooling centrifuge
5810R (Eppendorff), -800C Deep freezer (Thermo), Electroporator 2510(Eppendorff), MilliQ
water purification system (MILLIPORE), Incubator shaker (Thermocon), Innova 4230
Refregerator incubator shaker system, Laminar flow (Ascension innovation, CRO, 2 in number),
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Shailendras@evolva.com; +91 9566281304
UV spectrophotometer 1700 (Shimazu), Digital weighing machine (Denver instrument), 37oC
incubator chamber (Thermo), Vector NTI (software), Fridge (SAMSUNG), Microscope
(OLYMPUS), Laminar flow hood (ESCO,2 in number), CO2 Incubator (THERMO ,2 in number),
Water Bath (JULABO), Centrifuge (EPPENDORF), Centrifuge (KUBOTA), Plate reader (Biotek),
Rocker (Labnet), Aspirator(Eppendorff and Greiner-bio, 2 in number), Vortex (SCIENTIFIC
INDUSTRIES), Liquid Nitrogen container (THERMO ,2 in number), Hemocytometer, Klearzone
Systems, AKTA prime plus, Q-TOF, CL-1000 UV cross linker (UVP), HB-1000 Hybridizer (UVP),
Trans-blot SD ( BioRad), Incubator Shaker ( Innova 42) and Q-TOF.
.
5. Phase I Strategy (100k USD for 18 months)
5. I) Phase I Milestones/Deliverables
ii) Timeline (Assume start date of Apr 1st, 2012)
iii) Budget breakup (with timeline)
Phase I milestones converting timeline and budget mentioned below:
6. Phase II strategy (1 Million USD post 18 months)
6. i) Phase II Milestones with Timeline
ii) Brief Budget plans
Phase II milestones converting timeline and budget mentioned below:
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Shailendras@evolva.com; +91 9566281304
7. Business Strategy
Evolva being a b2b company, the immediate benefits could be licensed out to any major
agriculture / nutraceutical companies that are interested in scaling this product to reach mass
market / consumer segments.
There are several options for these companies- such as directly selling the onions into those
consumer segments where approvals are easier to obtain or alternatively making an extract of
the omega 3 fatty acid and package it as “ingredient” to other companies that sell finished
products in nutrition or nutraceutical. The unique selling point of the product is its acceptability
to all sections of society including vegans and koshers and also its flavour.
The product can be positioned attractively due to its affordability.
7. i) IP Strategy
1. Proprietary synthetic biology platform for designing and characterizing new enzymes for
omega-3 pathway in general and for Onions in particular
2. Proprietary eOleonions genotypes producing high value omega-3 metabolites
metabolites at a commercially attractive and affordable price points.
3. Potential discovery of several novel enzymes involved in assembling a functional
omega-3 biosynthesis pathway and related precursors
7. ii) Market Overview and Business Model (as applicable)
Several business reports project that the global market for omega-3-enhanced foods is
expected to touch US $8.2 billion by end of 2012, with a 13.8% annual growth until 2016, with
asia dominating a 34% share mainly due to increased consumer awareness towards healthy life
and specifically increasing trend on cardiovascular diseases.
Marine based omega 3 fatty acids occupies major portion i.e. 85% of the markets, however with
its unpalatable flavor food companies look for alternative source like algae, flax seeds, canola or
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Shailendras@evolva.com; +91 9566281304
walnuts. These non-marine i.e. plants based food items are not still potent enough to meet
either the ever growing demand or they not efficacious (contains only the omega-3 precursors
like alpha linoleic acid) as like marine derived ones.
The extensive growing demand for the omega 3 fatty acids based products is largely attributed
by factors that include increasing scientific evidence on benefits of consuming omega 3 fatty
acids, innovative product preparations and awareness of consumers on new trends and the
technologies.
With advent of technological developments now it is possible to the incorporate these omega 3
fatty acid pathway in different hosts other the marine organisms. In 2009, US Food and Drug
Administration gave GRAS status to Monsanto’s genetically modified (GM) soy bean enriched
with stearidonic acid (SDA). Human body converts SDA obtained through GM soya bean meal
or its derived products to eicosapentaenoic acid (EPA).
BASF and Syngenta are working on creating transgenic plants like canola or soybean enriched
with omega-3 oils and the Du Pont by fermenting microorganisms. Recently Cargill joins BASF
on US$ 190 million to bring genetically modified canola oil rich in EPA and DHA to market by
2020. Packaged Facts projects that the demand for omega-3 market is massive and it is far
from reaching its saturation point. FDA’ GRAS to Monsanto’s GM soy bean is a major
benchmark which will positively lead to more efficient and odourless non-fish omega-3 products
in the market14
Evolva is a B2B company. Once the stable eOleOnion plants created it is likely that outcome of
this innovation is transferred to agro companies for human welfare through business
partnerships
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References (Page 9 and after)
1. Ian A Graham, Tony Larson and Johnathan A Napier, Rational metabolic engineering of
transgenic plants for biosynthesis of omega-3 polyunsaturates. Current Opinion in
Biotechnology 2007; 18:142-147
2. Katerina Giannoulia, Georgios Banilas, Polydefkis Hatzopoulos, Oleosin gene
expression in olive, Journal of Plant Physiology 2007; 164: 104—107.
3. Robert SS, Singh SP, Zhou XR, Petrie JR, Blackbum SI, Mansour PM, Nichols PD, Liu
Q, Green AG: Metabolic engineering of Arabidopsis to produce nutritionally important
DHA in seed oil. Funct Plant Biol 2005; 32:473-479.
4. Damude HG, Zhang H, Farrall L, Ripp KG, Tomb JF, Hollerbach D, Yadav NS:
Identification of bifunctional D12/omega 3 fatty acid desaturases for improving the ratio
of omega 3 to omega 6 fatty acids in microbes and plants. Proc Natl Acad Sci USA
2006; 103:9446-9451.
5. Sayanova O, Haslam R, Guschina I, Lloyd D, Christie WW, Harwood JL, Napier JA: A
bi-functional D12, D15 esaturase from Acanthamoeba castellanii directs the synthesis of
highly unusual n-1 series unsaturated fatty acids. J Biol Chem 2006; 281:36533-36541.
6. Domergue F, Abbadi A, Zahringer U, Moreau H, Heinz E: In vivo characterization of the
first acyl-CoA D6-desaturase from a member of the plant kingdom, the microalga
Ostreococcus tauri. Biochem J 2005; 389:483-490.
7. Kinney, A.J., Cahoon, E.B., Damude, H.G., Hitz, W.D., Kolar, C.W. and Liu, Z.-B. (2004)
Production of very long chain polyunsaturated fatty acids in oilseed plants. E.I. Dupont
De Nemours and Company, WO 2004/071467 A2.
8. Zupan, J. and Zambryski, P., The Agrobacterium DNA Transfer Complex. Critical
Reviews in Plant Sciences1997, 16; 3: 279-295.
9. Chenna Reddy Aswath, Sung Youn Mo, Doo Hwan Kim, S Won Park, Agrobacterium
and biolistic transformation of onion using non-antibiotic selection marker
phosphomannose isomerase. Plant Cell Reports 2006;25,2:92-99.
10. Domergue F, Abbadi A, Heinz E: Relief for fish stocks: oceanic fatty acids in transgenic
oilseeds. Trends Plant Sci 2005, 10:112-116.
11. Stahl U, Carlsson AS, Lenman M, Dahlqvist A, Huang B, Banas W, Banas A, Stymne S:
Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase
from Arabidopsis. Plant Physiol 2004, 135:1324-1335
12. Huo, Y, Miao. J.,, Liu, B., Yangy,Y., Tahara, Y Meng, Q, He, Q, Kitano, H and Wu, X
2011, The expression of pectin methylesterase in onion flower buds is associated with
the dominant male-fertility restoration allele Plant Breeding 131, 211—216
13. Engelke, T.; Terefe, D.; Tatlioglu, T. 2003. A PCR-based marker system monitoring
CMS-(S), CMS-(T) and (N)-cytoplasm in the onion (Allium cepa L.). Theoretical and
Applied Genetics 107: 162–167.
14. Omega-3: Global Product Trends and Opportunities, Packaged Facts, 2011, pp. 190,
2011, Pub ID: LA6385341
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Shailendras@evolva.com; +91 9566281304
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