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

(hrm@us.es)

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 hrm@us.es+34 640749579

+34 954 489512