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INSTITUTE OF PLANT BIOCHEMISTRY AND PHOTOSYNTHESIS
University of Seville and CSIC
CicCartuja-Spain
ENHANCEMENT OF FATTY ACIDS AND CAROTENOIDS PRODUCTION BY
CLASSICAL (RANDOM) MUTAGENESIS AND GENETIC ENGINEERING
IN MICROALGAE
Dr. Herminia Rodríguez
Professor of Biochemistry and Molecular Biology
Madrid, 13 -15 December 2016
COMMERCIAL APPLICATIONS OF CAROTENOIDS
NATURAL DYES
- Food dyes
- Feed additives (aquaculture and poultry farming, pet food
ingredient)- Cosmetic Industries
THERAPEUTIC AGENTS
- Health-food, nutraceuticals (antioxidant properties)
- Vitamin A precursors
- Skin diseases (as UV-induced skin damage)
- Cataracts and age-related retinal degeneration
- Age-related degenerative diseases
- Cancer preventing properties
- Atherosclerosis
- Enhancement of inmune response
- Anti-inflamatory properties
- Cardiovascular diseases
PRICE AND MARKET VALUE OF THE
DIFFERENT CAROTENOIDS
-CAROTENE (Dunaliella)
The fastest-growing type in the carotenoids market
Market value was US$ 270 million in 2013
Price is approximately US$ 300-3,000 Kg-1
ASTAXANTHIN (Haematococcus)
Market value reached US$ 280 million in 2013
Astaxanthin price is approximately US$ 2,500 Kg-1
LUTEIN (Marigold: Tagetes erecta and Tagetes patula)
Global market accounted for US$ 240 million in 2013
THE WORLDWIDE MARKET VALUE FOR CAROTENOIDS WAS
ABOUT US$ 1.24 BILLION IN 2016 AND PROJECTED TO REACH 1.53 BILLION BY 2021
Price of dispersible powders containing 5-10% active carotenoids (synthetic)
300 a 3,000 $ USA Kg-1
In 2015, Europe (especially Germany and France) accounted for the largest market share in
carotenoids market, followed by U.S. and Asia-Pacific.
THESE ARE THE PRICES FOR SYNTHETIC CAROTENOIDS
THE PRICES FOR NATURAL ONES ARE VERY MUCH HIGHER
NATURAL AND SYNTHETIC
ß-CAROTENE
Synthetic ß-carotene: Stereoisomer all-trans (coloring agent in food E160)
Natural ß-carotene: Mixture of two estereoisomers all-trans and 9-cis
According to Nutritional Studies
- Preferential accumulation of the
mixture of stereoisomers
- 9-cis carotene acts as in vivo
antioxidant more efficiently than all-
trans ß-carotene
NATURAL AND SYNTHETIC ASTAXANTHIN
MicroalgaHaematococcus : 3S,
3’S steroisomer (the same as in
salmon and crustacean). Steryfied
with fatty acids (more stability).
Yeasts (Phaffia): 3R, 3’R
stereoisomer. Free (less stable)
Synthetic astaxanthin : A mixture
of the three isomers: 3S, 3’S; 3R,
3’R; and 3R, 3’S. Free (less stable)
Haematococcus
Phaffia
Synthetic: A mixture of all
isomers
The BestOne
MOST USED MICROALGAE FOR THE PRODUCTION OF CAROTENOIDS
Haematococcus pluvialisDunaliella salina
ß-carotene Astaxanthin
APPLICATIONS OF SHORT CHAIN SATURATED FATTY ACIDS IN
DETERGENTS , COSMETICS AND PHARMACEUTICAL INDUSTRIES
LAURIC ACID
(C12:0)
MYRISTIC ACID
(C14:0)
PALMITIC ACID
(C16:0)
OCCURRENCEPalm kernel, coconut
oil, cinnamon oil
Seeds of the family
Myristicaceae
(Myristica fragrans),
cinnamon oil, coconut
and palm kernel oil
The commonest saturated fatty acid in plants and
animals
Vegetal oils (palm oil,
peanut, soybean, corn, coconut) and marine-animal
oils
USES Similar to Myristic Acid
Care Chemicals:
Soaps, detergents,
shampoos, facial
creams and lotions
Pharmaceutical
industry for its good
antimicrobial and
antiviral properties
Care Chemicals:
Shampoos, soaps, shaving
soaps, shaving and anti-aging
creams, lipstick, mascara,
facial cleanser, facial
moisturizer treatment
Food additive
DRAWBACK: THESE PLANT SOURCES CONTAIN ALSO LONG CHAIN FATTY ACIDS NOT ADECUATE FOR THESE APPLICATIONS
MICROALGAE AS AN ALTERNATIVE SOURCE OF SHORT CHAIN SATURATED FATTY ACIDS
After a big Screening of different microalgae, we have selected a marine diatom, a
strain of Chaetoceros calcitrans sp., as a new source of Myristic (C14:0) and Palmitic
(C16:0) Acids
Chaetoceros calcitrans sp.
Myristic Acid
Palmitic Acid
ISOLATION OF
STRAINS
(Microbiology)
SCREENING
(Physiology)
OPTIMIZATION OF
CULTURE AND
PRODUCTION
CONDITIONS
(Physiology and
Biochemistry)
DESIGN AND
DEVELOPMENT
OF BIOREACTORS
(Engineering)
GENETIC
IMPROVEMENT(Classical or
Random
Mutagenesis)
STUDY OF METABOLIC
PATHWAYS AND
REGULATION
(Biochemistry, Physiology
and Molecular Genetics)
GENETIC
IMPROVEMENT
(Genetic Engineering) GMO
OPTIMIZATION OF
CULTURE CONDITIONS
OF MUTANTS
(Physiology)
OPTIMIZATION OF
CULTURE CONDITIONS OF
TRANSFORMANTS
(Physiology)
ALGAE CULTURE IS A VERY
MULTIDISCIPLINARY SCIENCE
GENETIC IMPROVEMENT IS NECESSARY TO
MEET THE ECONOMICAL REQUIREMENTS
TWO DIFFERENT WAYS FOR GENETIC IMPROVEMENT
- CLASSICAL OR RANDOM MUTAGENESIS
- GENETIC AND METABOLIC ENGINEERING
Mutants
Transformants, GMO
CLASSIC OR RANDOM MUTAGENESIS IN MICROALGAE
- Mutants grow more slow and worse than the wild strains?
- Mutants are very unstable?
- Do random mutants fit with regulations and are accepted by industries and people?
- Dangerous for the Environment?
REALITY/ADVANTAGES
- No new or exogenous genes are introduced. We only accelerate Natural Selection. Random mutations occur constantly in a natural way in Algae Cultures and in Natural Environments.
- They are very stable if things are properly done mutants (Stable For Years). VERY GOOD SCREENING METHOD IS NECESSARY.
- Mutants obtained by classic mutagenesis can grow as well or even better than the wild strains, if growth rate is taken as a selection parameter (and not only cellular content in the desired products) and relatively high viability is used when several rounds of mutagenesis are performed.
Productivity = Growth rate x Cellular content in the desired compound
- No transformation method is required
- Not necessary to know the genome of the algae
- Accepted by people and industries, while genetic engineered transformants microalgae not yet, specially in Europe.
- More friendly with the Environment than GMO’s
FEARS/CONCERNS
GENETIC ENGINEERING
Manipulation of the genomic material of microalgae by a big set of technologies to produce novel or
improved strains. A microalgae strain that has been produced by Genetic Engineering is considered
a Genetic Modified Organism (GMO)
More accurate than random mutagenesis
Many different sophisticated techniques
Still in development. Not very developed for microalgae
Very few strains show stable transformation (Transformation methods need to be developed for
commercially interesting strains)
Few algae genomes are fully sequenced
Problems of acceptance by consumers and very strict legislations, especially in Europe
The development of these techniques is very important, since with time GMO microalgae will be
accepted
Although biotechnological processes based on transgenic microalgae are still their infancy,
researchers and companies are considering the potential of microalgae as green cell-factories
to produce value-added metabolites.
MICROALGAL GENOMES
Nuclear, mitochondrial or chloroplast genomes from several microalgae have been
sequenced and several more are being sequenced: C. reinhardtii, Phaeodactylum
tricornutum, Thalassiosira pseudonana, Cyanidioschyzon merolae, Ostreococcus
lucimarinus, Ostreococcus tauri, Micromonas pusilla, Fragilariopsis cylindrus, Pseudo-
nitzschia, Thalassiosira rotula, Botryococcus braunii, Chlorella vulgaris, Dunaliella
salina, Micromonas pusilla, Galdieria sulphuraria, Porphyra purpurea, Volvox carteri, and
Aureococcus anophageferrens and Nannochloropsis gaditana, among others.
GENETIC TRANSFORMATION
Successful genetic transformation has been reported for some green (Chlorophyta), red
(Rhodophyta), and brown (Phaeophyta) algae; diatoms; euglenids; and dinoflagellates.
In some cases, transformation resulted in stable expression of transgenes, but in other
cases only transient expression was observed
ENHANCEMENT OF CAROTENOIDS PRODUCTION AND
CONTENT BY CLASSICAL (RANDOM) MUTAGENESIS
Chlorella sorokiniana Chlorella zofingiensis
Haematococcus pluvialis
CATEGORIES OF CLASSICAL (RANDOM) MUTAGENESIS
IN MICROALGAE
- PHYSICAL
- UV Light (Rather unspecific; no selection method to choose
the desired mutants )
THE ONE WE ARE USING:
- CHEMICAL
- MNNG (N-methyl-N’-nitro-nitrosoguanidine)
- EMS (ethyl methane sulfonate)
Selection by resistance to herbicides or inhibitors of specific
enzymes involved in the biosynthetic pathway.
Flow Cytometry with cell sorting (novel method for selection of
random mutants) It is being assayed by our Group
BIOSYNTHETIC PATHWAY
OF CAROTENOIDS IN
ALGAE AND PLANTS
LUTEIN
ASTAXANTHIN
ISOLATION OF MICROALGAE
MUTANTS WITH INCREASED
CAROTENOIDS PRODUCTION
BY CLASSICAL MUTAGENESIS
NORFLURAZON: Inhibits
Phytoene desaturase
NICOTINE: Inhibits
Lycopene β-cyclase
DIPHENYLAMINE: Inhibits
β-carotene oxygenase
SOME INHIBITORS OF
THE CAROTENOGENIC
PATHWAY
LIQUID CULTURES OF RESISTANT MUTANTS TO
EVALUATE GROWTH RATE (MICROTITER PLATES READER)
AND THE CONTENT IN THE DESIRED COMPOUND ( HPLC, GAS
CHROMATOGRAPHY)
NEW ROUND OF MUTAGENESIS WITH THE SELECTED MUTANTS
INOCULUM
CELL CULTURE IN EXPONENTIAL PHASE
CHEMICAL CLASSICAL MUTAGENESIS (EMS or MNNG)
GROWTH ON SELECTIVE MEDIA AND
OBTENTION OF HERBICIDE-RESISTANT COLONIES
SUBCULTURE OF RESISTANT COLONIES
ON NEW SELECTIVE/ NOT SELECTIVE MEDIA
2nd SUBCULTURE OF RESISTANT COLONIES
ON NEW SELECTIVE/ NOT SELECTIVE MEDIA
PROTOCOL FOR CLASSICAL MUTAGENESIS FOLLOWED BY OUR GROUP
(CAROTENOIDS AND FATTY ACIDS)
SHAKEN ERLENMEYERSIN INDUSTRIAL PROCESSES USUALLY SEVERAL ROUNDS OF MUTAGENESIS ARE PERFORMED
The data represen t t he mean values ± stan dard deviat ions o f 3-6 indepen dent
experim en ts.
H . pluv ial is
str ain
Astaxanthin yield (mg l-1)
Astaxanthin
yiel d (mg l-1)
(% r elat ive to
the wild strain)
Dry
weight
(g l-1)
Astaxanthin
content
mg (g dw-1)
Astaxanthin
content
mg (g dw -1)
(% r elat ive to
the wild
strain)
3 days 3 days 6 days 6 days 6 days
Wild strain
CCAP 4,6±1,0
6 days
11,8 ±5,1 100 100 1,22
6 days
10,6 ±4,6 100
Wild strain
SAG 4,9±1,3 10,1 ±2,9 100 100 1,12 9,4± 2,7 100
M S13 9,2±2,8 15,2 ±3,9 188 150 1,12 13,5 ±3,5 144
M C35 10,3±1,5 19,0 ±3,0 224 161 1,31 14,5 ±2,3 137
M C36 8,8±2,7 17,9 ±1,6 191 154 1,25 14,3 ±1,3 135
Mutants exhibiting two-fold volumetric yield of astaxanthin, only 3 days after
induction of the red phase, as compared to the wild strains were obtained. Growth was
similar in WT and mutants (same dry weight). MS13 (nicotine); MC35 and MC36
(norflurazon)
CLASSICAL OR RANDOM MUTAGENESIS
Obtention of Astaxanthin Super-Producing Strains of Haematococcus pluvialis
by Random Mutagenesis (only one round of mutagenesis)
Data obtained
by our Group
(Unpublished)
Banding pattern of RAPD-PCR of Wild type (C) and the mutant MC36 (36)
1+4
C 36
Extra band
MUTANT IDENTIFICATION AND STRAIN STABILITY IN
Haematococcus pluvialis
Mutants Identification Random Mutants can be
distinguished from the WT by
Genomic Finger Printing
Strain stabilityMutants kept resistance to the
herbicide after consecutive
cultures on selective and not
selective medium, as well as
their phenotypes during
several years.
DIFFERENT CELL MORPHOTYPES AND CELL STAGES IN
Haematococcus pluvialis
Green vegetative flagellates
Microzoids
(putative gametes
or sexual cells)
Reddish flagellates Palmelloids
Pre-cysts Cysts or aplanospores
IT IS NECESSARY TO STUDY THE LIFE/CELL CYCLE (Including Sexual Reproduction if present) OF THE
DIFFERENT STRAINS OF MICROALGAE
IN ORDER TO OBTAIN MAXIMAL GROWTH, CONTENT IN THE DESIRED COMPOUNDS AND
PRODUCTIVITY
It has only well studied in Chlamydomonas reinhardtii. The first author (Herminia Rodríguez) has studiedand published about “Gametogenesis and Sexual reproduction in Chlamydomonas reinhardtii, factors and genes involved” at the University of Freiburg (Germany). Our Group has been also studying this topic in H. pluvialis during several years and continues studying it. VERY IMPORTANT INDUSTRIAL IMPLICATIONS.
Chlorella zofingiensis: A LUTEIN AND ASTAXANTHIN PRODUCER
(An alternative to Haematococcus pluvialis for Astaxanthin Production)
Chlorophyceae (green microalga)
Accumulation of either astaxanthin or lutein depending on growth conditions
In batch cultures, lutein accumulates during early stages of culture, whereas astaxanthin accumulates in the late stationary phase
Under moderate stress conditions astaxanthin accumulates, whereas lutein decreases or remains unaffected
Good for metabolic and regulatory studies
Considered as a good alternative algae strain to Haematococcus pluvialis for AstaxanthinProduction
Strain Lutein
(mg L-1)
Lutein
mg g-1 dw
Lutein
(% with respect to
the Wild Strain)
mg L-1 mg g-1 dw
Wild Type 1,64 2,75 100 100
B-39 4,52 4,65 276 170
B-34 3,80 4,50 232 164
B-40 4,16 4,26 254 155
B-29 3,99 4,26 244 155
B-35 3,39 4,12 207 150
B-28 3,43 3,85 210 140
0 2 4 6 8 1010
8
109
1010
DE
NS
IDA
D C
EL
UL
AR
(cé
lula
s L
-1
)
TIEMPO (días)
Silvestre B-28 B-29 B-34 B-35 B-39 B-40
Obtention of Lutein Super-Producing Mutants of Chlorella zofingiensis by
Random Mutagenesis (only one round of mutagenesis)
Data obtained by our
Group (Unpublished)
Wild Type
B-39
Currently our Group is
searching for Super-
Producing Astaxanthin
Mutants
Mutants show higher
growth than Wild Type
Mutants exhibiting almost 3-fold volumetric tield of lutein as
compared to the wild strain were obtained. And cellular
contents close to 2-fold as compared to de wild type.
OPTIMIZATION OF CULTURE CONDITIONS OF THE
WILDE TYPE OF THE LUTEIN PRODUCER
Chlorella sorokiniana
- Maximal specific growth rate and lutein content
were attained at 690 μmol photons m-2 s-1, 28ºC,
2mM NaCl, 40 mM Nitrate and mixotrophic
conditions.
- Attaining values of lutein of 35.0 mg L-1 and 5.2 mg
g-1 DW
This lutein values were further enhanced by chemical random mutagenesis
up to 42 mg L-1 and 7.0 mg g-1 DW using MNNG and selecting mutants by:
1) Resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon
2) Their growth rate
3) High lutein content
CLASSICAL (RANDOM) MUTAGENESIS OF Chlorella sorokiniana
Comparison of the
accumulation of lutein of WT
and the mutant MR-16 under
optimized conditions of culture
Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta)
by Random Mutagenesis (only one round of mutagenesis)
- MR-16 exhibits a volumetric lutein production 2-fold than the
wild strain
- DMR-5 and DMR-8 stand out in terms of cellular content in
lutein, reaching values of 7 mg lutein g-1 dw
WTMR-16
Data obtained by our Group
(Cordero et al.
Marine Drugs 9: 1607-1624)
Kinetics of growth of WT and
the mutant MR-16
STRAIN
Maximum
(h-1)
Maximal
Lutein in
the
exponential
phase
(mg l-1)
Maximal
Lutein in
the culture
(mg l-1)
Maximal
cellular
Lutein
content
(mg g-1 DW)
Chlorella
sorokiniana
(WT)0.15 12 21 3.9
Chlorella
sorokiniana
(mutant
MR-16)
0.16 13 42 5.5
GROWTH, MAXIMAL LUTEIN PRODUCTIVITY AND CONTENT OF
C. sorokiniana WT and MUTANT MR-16
UNDER AUTOTROPHIC CONDITIONS
ENHANCEMENT OF SHORT CHAIN SATURATED FATTY
ACIDS PRODUCTION AND CONTENT BY
CLASSICAL MUTAGENESIS
Chaetoceros calcitrans sp.
FA SYNTHESIS
ACC: Acetyl-CoA carboxylase
Biotin carboxilase
Carboxyl transferase
Malonyl-CoA: ACP acyltransferase
FAS: fatty acid synthase (elongation
cycles)
KAS III (β-ketoacyl-ACP
synthase III)
KAS I
KAS II
TAG FORMATION
GPAT (acyl-CoA:glycerol-3-
phosphate acyl-transferase)
LPAT (lysophosphatidate acyl-
transferase)
PAP (phosphatidic acid
phosphatase)
DGAT (acylCoA:diacylglycerol
acyl-transferase)
Malonyl-CoA: ACP acyltransferase (MAT)
KAS IIIKAS IKAS II
TAGFormation
(LPA)
GPAT
(PA)
LPAT
PAP
DGAT
(Malic Enzyme)
(ATP:Citrate Lyase)
(PhosphoenolpyruvateCarboxylase)
(Acetyl-CoA Synthetase)
NADPH NADP+
FA
Synthesis
TAG
Formation
(Kennedy
Pathway)
BIOSYNTHEIC PATHWAY OF FATTY ACIDS
Acetyl-CoA Carboxilase
InhibitorQuizalotrof
VOLUMETRIC YIELD AND CONTENT IMPROVEMENT OF SHORT CHAIN
SATURATED FATTY ACIDS BY CLASSICAL MUTAGENESIS
IN Chaetoceros calcitrans sp. (only one round of mutagenesis)
MIRYSTIC ACID PALMITIC ACID
Data obtained by our Group (Unpublished)
EMS-random mutagenesis was used to obtain Quizalofop-P (inhibitor of the Acetil CoA
Carboxylase) resistant mutant strains.
Mutants exhibiting 4-8 fold volumetric yield and more than 2-8 fold in terms of cellular
content of either mirystic or palmitic acids, as compared to the wild strains, were obtained.
BEST MUTANTS
Growth of the selected mutants
was similar or even higher than
WT
ENHANCEMENT OF CAROTENOIDS PRODUCTION AND
CONTENT BY GENETIC AND METABOLIC ENGINEERING
Chlamydomonas reinhartdtii
Dunaliella salinaChlorella zofingiensis
Key GenesPhytoene synthase: psy
Phytoene desaturase: pds
Lycopene β-cyclase: lcyB
Lycopene ε-cyclase: lcyE
Carotene β-hydroxylase: chyB
β-carotene oxygenase: bkt
Zeaxanthin epoxydase: zep
Violaxanthin de-epoxydase: vde
Isolated by our Group
ISOLATION AND CHARACTERIZATION OF
CAROTENOGENIC GENES IN Chlorella zofingiensis
Chlorella
zofingiensis: Model
organism to study
the Carotenoids
Biosynthetic
Pathway and
perform
METABOLIC
ENGINEERING
1.- Insertion of the Czpsy gene in the expression vector pSI105 of Chlamydomonas
2.- Nuclear transformation of Chlamydomonas and selection of transformants resistant
to the antibiotic paramomicin
pSI105-tp
Chlamydomonas
culture
(Exponential phase of
growth)
(TAP+ paromomicin 30mg/ml)
Selective medium
ENHANCEMENT OF CAROTENOIDS BIOSYNTHESIS IN Chlamydomonas reinhardtii BY
NUCLEAR TRANSFORMATION USING A PHYTOENE SYNTHASE GENE (psy)
ISOLATED FROM Chlorella zofingiensis
OVER-EXPRESSION OF CAROTENOGENIC GENES
IN OTHER MICROALGAE: GENETIC ENGINEERING
Cells shaken with
glass beds
ANALYSIS OF TRANSFORMANTS BY HPLC AND qPCR
CAROTENOIDS CONTENT
mRNA RELATIVE
ABUNDANCE OF
ENDOGENOUS Crpsy
AND FOREING Czpsy
Carotenoids: ■ Violaxanthin; ■ Lutein;
■ α-carotene; ■ β-carotene
■ Crpsy; ■ Czpsy
ENHANCEMENT OF CAROTENOIDS BIOSYNTHESIS IN Chlamydomonas reinhardtii BY
NUCLEAR TRANSFORMATION USING A PHYTOENE SYNTHASE GENE (psy) ISOLATED
FROM Chlorella zofingiensis
Data obtained by our Group
Cordero et al. (Appl. Micobiol. Prog. 27:
54-60)
Carotenoids cell content, specially violaxanthin
and lutein, was in some transformants more
than 2-fold as compared to untransformed cells
(WT)
OVER-EXPRESSION OF CAROTENOGENIC GENES
IN OTHER MICROALGAE: GENETIC ENGINEERING
Over-expression of an Exogenous Phytoene Synthase (psy) Gene from Dunaliella salina in
the Unicellular Alga Chlamydomonas reinhardtii Leads to an Increase
in the Content of Carotenoids
Data obtained
by our Group
(Couso et al.
Biotechol. Prog.
27: 54-60)
REGULATION OF THE
CAROTENOGENIC PATHWAY BY
LIGHT AND NITROGEN IN C.
zofingiensis
HIGH-LIGHT STRESS Up-
regulates all genes at
transcriptional level, except lcyB
and lcyE.
NITROGEN STARVATION
Up-regulates all genes at
transcriptional level, except lcyE.
mRNA levels by qPCR
Data obtained by our Group
Cordero et al. Mar. Drugs 10: 2069-2088
REGULATION OF THE
CAROTENOGENIC PATHWAY BY
LIGHT AND NITROGEN IN C.
zofingiens
Lutein, violaxanthin, α-carotene
and β-carotene Accumulate at
low light irradiance and enough
nitrogen availability.
Astaxanthin and Canthaxanthin
Accumulate under high-light
stress and nitrogen starvation.
Carotenoid levels by HPLC
OUR GROUP (From IBVF)
“PRODUCTION OF CHEMICALS OF INDUSTRIAL INTEREST BY
MICROALGAE AND CYANOBACTERIA”
Dr. Herminia Rodríguez
Professor of Plant Biochemistry and Molecular Biology
Dr. M. Ángeles Vargas
Professor of Plant Biochemistry and Molecular Biology
Dr. Irina Obraztsova
Post Doc
Dr. Baldo F. Cordero
Post Doc
Lucía Martín and
José María Jurado
PhD. Students
CicCartuja ~ 400 People
IVBF ~125 People
THANK YOU VERY MUCH FOR YOUR ATTENTION !!
Prof. Herminia Rodríguez [email protected]+34 640749579
+34 954 489512