CELL CULTURE - Moodle
68
METABOLIC ENGINEERING OF LIPID PRODUCTION IN MICROALGAE CELL CULTURE - MICROALGAE PLATFORM
Transcript of CELL CULTURE - Moodle
METABOLIC ENGINEERING OF LIPID PRODUCTION IN MICROALGAE
CELL CULTURE- MICROALGAE PLATFORM
CONTENT TABLE:
Microalgae introduction
Research history
Cell culture
Lipid production in microalgae
Culture process
My project introduction
Conclusion
Future work
Lab work plan
2
Microalgae introduction
3
Microalgae are unicellular species of differing sizes, shapes, colors and structures.
How do you know about microalgae?
4Bacillariophyceae
Haptophyte Peridinium Chlorarachniophytes
ChlorococcumCryptomonas Euglenoids
Polysiphonia
Oscillatoria
For more sources: http://www.rbgsyd.nsw.gov.au/science/Plant_Diversity_Research/australian_freshwater_algae/algpic/nonmotile_microalgae
Characters
They have the common property of photosynthesis that capture carbon dioxide and assimilate it as a part of their central metabolism.
20%-50%
Recycling cell factory
5
http://www2.hci.edu.sg/y11hci0149/website/algae.htmlhttp://theharborandthehudson.wordpress.com/2010/12/05/new-algae-biofuel-pilot-project-at-the-rockaway-wastewater-treatment-plant/
Microalgae products: biodiesel-from lipids to biodiesel
6
Comparison of lipid production
Soybeans Microalgae
30000L/hectare/year 300L/hectare/year
100-folds
7
http://northcountrypublicradio.net/news/npr/167558840/peak-farmland-some-researchers-say-it-s-here
http://greenglobalmalaysia.blogspot.ca/
"Foster" destroyer
The injection of biofuels is a "algae fuel", a mixture of 50 percent of traditional petroleum products and 50 percent biofuel from algae oil
Cited from Navy times: http://www.navytimes.com/article/20111110/NEWS/111100338/Experimental-Navy-ship-set-alt-fuel-demo
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Microalgae products: nutritional lipids-long chain multi-unsaturated fatty acids: EPA DHA
Previously: fish oil was famous for its omega-3 fatty acid content, however fish don't actually produce omega-3s, instead accumulating their omega-3 reserves by consuming microalgae.
DHA and EPA: improving human cardiovascular health; disease prevention; cancer prevention and treatment; physical and cognitive development of infants;anti-aging;Treatment of mental health disorders; lessening the impact of rheumatoid arthritis, and prevention of liver disease.
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Microalgae products: irons, proteins and other carbohydrates products
eyes
10
Research history
11
overexploitation
Oil crisis
Algae biodiesel
12
United States: “Aquatic Species Program (ASP)”, “Mini-Manhattan Project”
Japan: “Microalgae diesel research program”
Companies: UK “Carbon Trust”, U.S. “SolixBiofule” “Green Fuels technology”, Canada “International energy” etc.
In 2007, Canada International
Energy, Inc. announced that it has
launched its “algae to oil” research
and development initiatives.
Global research wave
13
14ASP final report, 1998
Oleaginous algae strains：
Microalgae lipid content (% dry wt)
Botryoccus braunii 25-75
Chlorella sp. 28-32
Chlorella protothecoides
(autotrophic/heterotroph
ic) 15-55
Dunaliella. Tertiolecta 36-42
Nannochloropsis sp. 31-68
Nannochlosis sp. 15-32
Crypthecodinium cohnii 20
cylindrotheca sp. 16-37
Dunaliella primolecta 23
Neochloris
oleoabundans 35-54
Nitzschia sp. 45-47
phaeodactylum
tricornutum 20-30
Schizochytrium sp. 50-77
Tetraselmis sueica 15-23
Lipid content of some microalgae species
Chlamydomonas reinhardtii has arisen as the hallmark, model organism. Although the lipid content is not high, it’s the first algae species that finished genome sequencing and annotation (Merchant, Prochnik et al. 2007). C. reinhardtii has been widely used to study photosynthesis, cell motility and phototaxis, cell wall biogenesis, and other fundamental cellular processes (Harris 2001). These studies laid a foundation for researches in other species.
Statistic
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Cell culture
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Culture nutrition:
1. Macro elements : C, H, O, N, Si---Cell synthesis, energy source, cell wall structure
2. Minor elements: P, K, S, Mg----ATP , phosphorylation of proteins, nucleic acids ; cofactor of several enzymes , signal transfer; composed of several amino acids , nucleic acids and protein.
3. Vitamins and hormones, etc.
•Growth factors: organic nutrients incorporated into the cellular structure.
4. Trace elements: Fe, Zn, Cu, Mn, Co, Mo, B----enzyme cofactors
What kind of special culture nutrition and conditions do a “green cell” need?
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Culture mediumComponents Concentration Utility
KH2PO4 0.7g/L Maintain PH, provide PO4 for nucleotidesynthesis.
K2HPO4 0.3g/L
MgSO4.7H2O 0.3g/L
FeSO4.7H2O 0.3mg/L
Glycine 0.1g/L Nitrogen source
Vitamine B1 0.01mg/L Coenzyme and cofactors, regulate metabolism or energy transformation
A5 trace mineral solution
1ml/L
Yeast extract 1.25g/L Amino acids
Glucose 10g/L
A5 trace mineral solution components
Add to 100ml of distilled water
H3BO3 286mg
MnCl2.4H2O 181mg
ZnSO4.5H2O 22mg
CuSO4.5H2O 7.9mg
(NH4)6MoO24.4H2O 3.9mg
Chlorophyta:
Clorella protothecoides belongs to genus Clorella, it is a chlorophyta. Cell size: Culture medium: Modified basal Medium (MBM)
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Carbon utilization
Chlorella protothecoides can use organic carbon source such as glucose, glycerol, starch etc. to live a heterotrophic life, the cell culture density and lipid content
are all greatly increased.
Meanwhile, it can also use inorganic and organic carbon source simultaneously to live mixtrophy life.
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http://www.vi.cl/foro/topic/1071-apuntes-de-biologia-y-quimica/page-42
Nitrogen utilizationstarch
Amylose
Amylopectin
ADPglcG-1-P
G-6-P R-5-P DNA
F6P
CO2
Amino acid
PEP
Acytl-CoALipid
TCA
α-KG
Gln
Glu
NH4+
NO3-
IPP
σ-aminolevulinic
GPP
Chlorophyll
Phytoene Lycopene
β-Carotene
Amino acid
Glc
GA3PRuBP
CO2
R5P
Cell metabolism reprogramming in N limitation
N is an essential nutrient factor foralgae growth. It is the essentialelement formatting cell aminoacids, purines, pyrimidines, aminosugar, and the amine compound.And especially in algae cell, it isalso a essential component ofchlorophyll.
In limited N condition, protein, nucleic acid synthesis slow down so cell division and growth are restricted, most assimilated carbon are led to produce storage and stress-resistant materials such as lipid, starch, beta-carotene.
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Baeillariophyta :
Navicula pelliculosa belongs to genus Navicula, it is a diatom (Baeillariophyta). It’s a freshwater alga
Size: 7.5-10um*4-5.5um
Culture Medium: CHU-10 medium
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http://www.algae.md/Anketa.aspx?id=i355
Silicon utilization in diatoms
Silicon is major component of diatom cell walls. Similar to the lipid trigger effect produced by N deficiency, Si depletion also results in a decrease in cell growth and often is accompanied by an accumulation of lipid within the cells.
Silicon deficiency led to an increase in the lipid content of all strains (although in some cases the increase was small and probably not statistically significant). The mean lipid content of the eight strains increased from 12.2% in nutrient-sufficient cells to 23.4% in Si-deficient cells.
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Carbon utilization
Although it can grow under heterotrophic
conditions, the growth rate is quite low.
There isn’t any mix trophic culture conditions
for this species yet.
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CO2/O2 ratio:
Photorespiration and Calvin cycle Using metabolic flux modeling with measured rates for each steady state we were able to quantify the ratio of the oxygenase reaction to the carboxylase reaction of Rubisco and the fluxes through the photorespiration pathway. This showed that the observed decrease in yield can be explained from an increase in oxygenase activity of Rubisco and the resulting process of photorespiration, up to 20.5% of the carboxylase activity. In conclusion, this study shows that elevated oxygen concentrations and the corresponding increase in the ratio of O2 versus CO2 common in photobioreactors leads to a reduction of the biomass yield on light of the microalgae C. reinhardtii.
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http://en.wikipedia.org/wiki/Photorespiration
Metabolic flux analysis
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Source from the course “Biochemical Engineering” given by professor Mario Jolicoeur
Light：
Light is essential for microalgae growth under autotrophic and mixtrophic conditions. Since light can active the enzyme of Rubisoco (carbon fixation) and relative enzymes to synthesis chlorophyll.
Light cycle: photoperiod
Cell growth:
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Chlorella
Lsochrysis
Platymonas
Dunaliella viridis
http://fhs-bio-wiki.pbworks.com/w/page/12145771/Factors%20effecting%20the%20rate%20of%20photosynthesis
Liu Qing, 2006
Microalgaespecies
Chlorophyll content in unit volume
Photo period
Light cycle: Chlorophyll synthesis under different photoperiod
Chlorophyll content per cell
Chlorophyll Chlorophyll Chlorophyll Chlorophyll Total amountTotal amount
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Liu Qing, 2006
limitation of light for high cell densities is critical for growth and lipid production Wen Zhiyou et al. (Wen Zhiyou 2003), but excessive light can also cause “photoinhibition” (Jack Myers 1940).
Light intensity:
Microalgaespecies
Chlorophyll content in unit volumePhoto intensity (lx)
Chlorophyll content per cell
Chlorophyll Chlorophyll Chlorophyll Chlorophyll Total amountTotal amount
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Lipid production in microalgae
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lipid metabolism
ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; CoA, coenzyme A; DAGAT, diacylglycerol acyltransferase; DHAP, dihydroxyacetone
phosphate; ENR, enoyl-ACP reductase; FAT, fatty acyl-ACP thioesterase; G3PDH, gycerol-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate
acyltransferase; HD, 3-hydroxyacyl- ACP dehydratase; KAR, 3-ketoacyl-ACP reductase; KAS, 3-ketoacyl-ACP synthase; LPAAT, lyso-phosphatidic
acid acyltransferase; LPAT, lyso-phosphatidylcholine acyltransferase; MAT, malonyl-CoA:ACP transacylase; PDH, pyruvate dehydrogenase complex;
TAG, triacylglycerols.
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Randor Radakovits, 2010
Lipid observation: Nile red fluorescent
According to the pre-scan of excitation and emission characteristics of neutral lipid standards, the excitation and emission wavelengths of 530 nm and 575 nm were selected.
31
Freddy Guihéneuf, 2013
Culture process
32
Flask culture in photo incubator:
33http://www.psi.cz/products/growth-chambers-and-incubated-shakers/
Fig. 2 Schematic overview of the photobioreactor. A: annular gap, C: condenser, D:
internal down comer, DO dissolved oxygen sensor, GA gas analyzer, I inner cylinder, L
spherical light sensor, M motor, MFC mass flow controllers for both N2 and CO2, pH pH
control connected to acid pump, S sparger, T temperature control connected to cryostat
and cooling jacket (not shown)
Photo-bioreactor
34Anna M. J. Kliphuis, 2011
Laboratory scale:
The most critical scale-up and operational parameters:Light penetrating Mass transfer, mixing problemO2 inhibition problem- Homogeneity of environmental conditions ,- Meet the CO2 demand of the cells remove the oxygen producedShear force
These parameters are closely interrelated and they determine the productivity and efficiency of the system.
what should we consider for choosing a photo bioreactor?
35
36http://bbi-biotech.com/en/produkte/fermenter-bioreaktoren/algenbioreaktor-xcubio-pbr/http://www.kaznu.kz/en/4867
http://www.labs.chem-eng.utoronto.ca/allen/about/research-equipment/
Air-lift bioreactorAdvantages:
Excellent transfer of oxygen and other gases
Mechanical simplicity of the bioreactor
May have an immobilized biomass (fluidized)-homogeneous
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Air-lift bioreactor: Oxygen transfer efficiency (kLa):
kLa = 0,32 * U𝑠𝑔0.7 (s-1)
U𝑠𝑔: the section surface of photobioreactor.
Pilot scale
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John R. Benemann, 2011
Commercial scale
39
My project introduction
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Problem identification
Biomass and lipid synthesis are in conflict: increasing lipid productionalways pay a cost of lower cellular growth. So the total lipid yield isseriously constrained, which becomes the main bottleneck in the research.
Lack of knowledge on algae cellular metabolism: Intracellular metabolismplays fundamental role in culture’s behaviour, but there is still no relativecomprehensive study in Chlorella protothecoides. So we have no detailedunderstanding of how the nutrition distributed in the metabolic processes,how does the related pathways competing with each other.
Lipid content was greatly different along with algae growth stages, cultureconditions and nutritional states, which make the productivity quiteunstable. No dynamic tool to guide the metabolic process control.
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Objectives
Metabolic engineering for lipid improvement: establish a comprehensive dynamic metabolic model, to guide the metabolic process control.
1. Study metabolism characterization of Chlorella protothecoides, reconstruct cell metabolism network.
2. Establish the dynamic model, which can cover most metabolic pathways, key intracellular metabolites to describe cell dynamic behaviors. Introduce the regulation of energies and intermediates on the metabolic pathway.
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3. Develop a cell engineering strategy based on the model prediction and guide our controlling process: model could help at defining optimal culture conditions and genetic engineering strategy.
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Nutrient consumption can be predicted from the model
Production of fatty acids is regulated by acetyl CoA carboxylase (ACCase)(Post-Beittenmiller and Roughan 1992, Bao and Ohlrogge 1999). Cyclotella cryptica ----(ACCase was overexpressed 2-3 folds) -----C. cryptica and Navicula saprophila
Fatty acid synthase has been suggested to be another rate-limiting regulator of lipid production and several studies have been performed where a single enzyme of the FAS complex is overexpressed (Roesler and Shintani 1997).
Overexpress of a certain key enzyme might not be sufficient to enhance the whole lipid synthesis. It tells us the interplay and metabolic balance of every metabolites should be considered as a whole. And preferable but balanced control of metabolic flux is essential to improve lipid synthesis. Metabolic modeling that can simulate flux of fatty acids through TAG biosynthetic pathways and intracellular metabolic networks should play an important part in developing strategies for future genetic manipulation
Methodology
Strain: Chlorella protothecoides
Culture conditions:
- Temperature: 28℃
- Basic medium: MBM; 1% Glucose in mixtrophic and heterotrophic culture
- Other condition: 5% CO2, and 8-W fluorescent light are provided for
autotrophic and mixtrophic culture
Autotrophy: A
Mixtrophy: M
Heterotrophy: H
A M H
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Analysis methods:
1. Glucose, lactate, glutamine, glutamate analysis
Samples were automatically detected by YIS 2700 select, biochemistry analyzer
2. Lipid extraction and analysis: Acetone and sulfosalicylic acid; absorbance
was read at 440nm by UV-VIS determination
3. Other intracellular metabolites: Methanol extraction and analyzed on
HPLC-MS-MS
Glycolysis, photosynthesis, PPP, TCA: Sugar phosphate and organic acid: F6P
R5P G1P G6P Succ Ru5P X5P alpha-keto PEP fumarate pyruvate Gly-1-P
Oxidative phosphorylation, Energy flow: Redox+nucleartide: ATP ADP AMP UTP
UDPG GTP NADPH
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Sugar phosphate and organic acids: Glycolysis, photosynthesis, PPP, TCA
y = 0.772x + 0.2074R² = 0.9674
0
5
10
0 5 10 15
pyruvate
y = 0.9497x + 13.144R² = 0.8553
0
10
20
30
0 5 10 15
gly1P
y = 0.7948x + 4.6489R² = 0.9721
0
5
10
15
0 5 10 15
G6P
y = 0.8038x + 17.464R² = 0.6734
0
10
20
30
0 5 10 15
succ
y = 1.1918x + 3.5046R² = 0.9064
0
5
10
15
20
0 5 10 15
Ru5P X5P
y = 0.7397x + 4.1027R² = 0.8952
0
5
10
15
0 5 10 15
F6Py = 0.7902x + 3.0261
R² = 0.9845
0
2
4
6
8
10
12
0 5 10 15
R5P
y = 0.8648x + 1.1697R² = 0.9766
0
2
4
6
8
10
12
0 5 10 15
G1P
y = 0.6118x + 4.4918R² = 0.9657
0
5
10
15
0 5 10 15
PEP
y = 0.9059x - 0.5468R² = 0.9817
-5
0
5
10
0 5 10 15
fumaric
y = 0.6811x + 0.2283R² = 0.8875
0
2
4
6
8
10
0 5 10
alpha-keto
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Energy state, Redox nucleotide: Oxidative phosphorylation, Energy flow
y = 0.967x + 1.1531R² = 0.9927
0
5
10
0 2 4 6 8
exp
(u
M)
std add (uM)
ADP
y = 0.9219x + 4.1698R² = 0.9631
0
5
10
15
0 2 4 6 8
exp
(u
M)
std add (uM)
AMPy = 1.0386x + 1.1845
R² = 0.989
0
2
4
6
8
0 2 4 6 8
exp
(u
M)
std add (uM)
ATP
y = 0.9909x + 0.9515R² = 0.9899
0
2
4
6
8
0 2 4 6
exp
(u
M)
std add (uM)
UTP
y = 1.2327x + 2.1163R² = 0.9927
0
5
10
15
0 2 4 6 8
exp
(u
M)
std add (uM)
UDPG
y = 1.3155x + 0.9594R² = 0.9945
0
2
4
6
8
10
0 2 4 6
exp
(u
M)
std add (uM)
GTP
y = 1.1807x + 0.8449R² = 0.9948
0
2
4
6
8
0 2 4 6
exp
(u
M)
std add (uM)
NADPH
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Preliminary resultsChapter 1: Culture characterization
48
-5.00E+07
0.00E+00
5.00E+07
1.00E+08
1.50E+08
2.00E+08
2.50E+08
0 50 100 150 200
cell
nu
mb
er (
cell/
ml)
Time (h)
Cell growth
A1 A2 M1 M2 H1 H2
0
20
40
60
80
100
120
140
160
0 50 100 150 200
(mm
ol/
L)
Time (h)
Glucose consumption
H M
0
2
4
6
8
10
12
14
16
18
0 50 100 150 200
% D
W
Time (h)
lipid content
A M H
0.106 (1/h)0.145 (1/h)
0.154 (1/h)
A: 5.04gDW/L
M: 9.54gDW/L
H: 10.32gDW/L
Biomass:Culture behavior
49
48h
120h
144h
AUTO
MIXTRO
HETERO
Chlorophyll-fluorescent-365nm excitation
White light-normal cells
Chlorophyll synthesis
50
0.00E+002.00E-094.00E-096.00E-098.00E-091.00E-081.20E-081.40E-08
0 50 100 150 200 250
G1P
A M H
0.00E+00
5.00E-09
1.00E-08
1.50E-08
2.00E-08
0 50 100 150 200 250
F6P
A M H
0.00E+00
2.00E-09
4.00E-09
6.00E-09
8.00E-09
0 50 100 150 200 250
PEP
A M H
0.00E+00
5.00E-08
1.00E-07
0 50 100 150 200 250
PYR
A M H
Glycolysis and gluconeogenesis
X axle: time-hY axle: [C]-nmol/cell
0.00E+00
2.00E-08
4.00E-08
6.00E-08
8.00E-08
1.00E-07
0 50 100 150 200 250
G6P
A M H
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0.00E+00
1.00E-10
2.00E-10
3.00E-10
4.00E-10
5.00E-10
6.00E-10
0 50 100 150 200 250
R5P
A M H
0.00E+00
5.00E-10
1.00E-09
1.50E-09
2.00E-09
2.50E-09
0 50 100 150 200 250
X5P
A M H
PPP and Calvin cycle
X axle: time-hY axle: [C]-nmol/cell
52
Intermediate metabolites concentration in gluconeogenesis pathway are much higher than that of mixtrophy and heterotrophy, this accumulation further extended to the PPP pathway on R5P and X5P;
53
0.00E+00
2.00E-08
4.00E-08
6.00E-08
8.00E-08
1.00E-07
1.20E-07
0 50 100 150 200 250
MAL
A M H
0.00E+00
5.00E-08
1.00E-07
1.50E-07
2.00E-07
2.50E-07
0 50 100 150 200 250
SUCC
A M H
0.00E+00
5.00E-08
1.00E-07
1.50E-07
2.00E-07
0 50 100 150 200 250
CIT isocit
A M H
0.00E+00
5.00E-09
1.00E-08
1.50E-08
2.00E-08
0 50 100 150 200 250
FUM
A M H
0.00E+00
2.00E-08
4.00E-08
6.00E-08
8.00E-08
1.00E-07
1.20E-07
0 50 100 150 200 250
AKG
A M H
TCA cycle
X axle: time-hY axle: [C]-nmol/cell
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TCA cycle is the starting point for many anabolisms, such as lipid, protein and so on. Therefore, this result explains the metabolic basis that lipid content was significantly higher in heterotrophy and mixtrophy culture than in the autotrophy culture.
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Energy state： oxidative phosphorylation pathway
0
10
20
30
40
50
60
70
Autotrophy Mixtrophy Heterotrophy
Energy state: ATP/ADP
48h 120h 192h
Energetic level of the cells within time
56
Amino acids: Nitrogen source metabolism
0
0.5
1
1.5
2
2.5
3
3.5
4
Extracellular AA concentration (mmol/L) 0 hour
57
GLU
G6P
F6P
1mol GA3P(glyceraldehyde-3-P)
3-P-G
PEP
PYR(pyruvate)
Acetyl-COA
COASH
Malonyl-COA
Malonyl-ACP
Acetyl-ACP
Butyrul-ACP
ß -Ketoacyl-ACP ß -Hydroxybutyrl-ACP
Trans-delta-butenoyl-ACP
16:0-ACP
16:1-ACP
(PA)16:0
18:0-ACPStearic acid
18:1-ACP18:1 ω9
Δ9
18:2 ω9 Δ6,9
20:2 ω9 Δ8,11
20:3 ω9 Δ5,8,11
22:3 ω9 Δ7,10,13
22:4 ω9 Δ4,7,10,13
ω9 family
18:2 ω6 LA Δ9,12
18:3 ω6 GLA
Δ6,9,12
20:3 ω6 Δ8,11,14
20:4 ω6 AA
Δ5,8,11,14
22:4 ω6 Δ7,10,13,16
22:5 ω6 Δ4,7,10,13,16
ω6 family
18:3 ω3 GLA
Δ9,12,15
18:4 ω3 Δ6,9,12,15
20:4ω3 Δ8,11,14,17
20:5 ω3
EPA Δ5,8,11,14,17
22:5 ω3 DPA
Δ7,10,13,16,19
22:6 ω3 DHA
Δ4,7,10,13,16,19
ω3 family
DHAP(Dihydroxyacetone-P)
G-3-PGlycero-3-phospate
LPALysophosphatidic
PAPhosphatidic acid
DAGDiacylglycerol
TAGTricylglycer
ide
FFAFree fatty
acids
Acyl-COA
R5P
X5P
R5P
E4PP
CO2
ATP
ADP
ADP+H+
ATP
NADH+CO2
NAD+ COA
HKGCK
G6PI
PFK
PK
PGAM
Δ6desau
Δ6desauΔ6desau
Enlong
Δ5desau
Enlong
Δ4 desau
Enlong
Δ5desau
Enlong
Δ4 desau
Enlong
Δ5desau
Enlong
Δ4 desau
Δ7desau
ACCase
CO2
MAT
Acety-COA
CO2+COA
KASⅢ,I
NADPH+H+ NADP+
KASⅢ,I
H2OHAD
HADNADP+
H+ NADP+
×6cycles
×7cycles
OAT
ACP
Δ9desau
Δ9desau Δ9desau
FAT
ACS
COASH
GPAT LPAAT PP DGTA
PC LPCCPT
PDAG
rPiA
rPiB
rpe
tktA
tktB
S7P
G3P
talB
OAAOxaloacetate
CIT(citrate)
ISO(isocitrate)
α- KGα- Ketoglutarate
SCOASuccinyl-COA
SUCSuccinate
FURFurnatrate
MALMalate
NADPH+H+NADP+ Pi
CO2COASH
NADPH+H+
NADP+ Pi
2 H2O O2
hv ATP+NADPH
Arginine，methionine，Lysine，AsparagineThreoinine
SerineGlycineCysteineLeucine
ChlorophyII
NH4+
NO2-
NO3-
Acyl-ACP
Chloroplast
Endoplasmic reticulum (ER)
Mitochondrion
Component transfer
Enzyme
Starch + Carbohydrates DNA，RNA
NADH ATP NAD+NADPH NADH+NADP
FADH2 ATP ATP+AMP 2ADP
H2O+PPi 2Pi+H+ ATP+H2O ADP+Pi+H+
O2
O2
Sterol
FBP
Glutamate
O2
HKGCK
1
2
3
ATP
ADP
H2O
4
5
6
NADH + ATP 7
H2O 8
9
10
11
12
13
14 15
16
17
18
19
20
21
22 23
24
25
26
29
30
31
32
33
28
3635
34
R5P
TAG
G6P
Glycine
Chlorophyll
Biomass
Metabolic pathway
58
Conclusion:
1. Developed culture characterizations of the three metabolic
strategies in Chlorella protothecoides.
2. Developed analysis methods, tracked variation of
metabolites on central metabolic pathways, studied flux
distribution in three culture strategy and collected data for
model development.
3. Established Chlorella protothecoides’ metabolic pathway
based on databases.
59
Chapter2: Model development
60
Model development:
We consider only carbon metabolism in the primary work, nitrogen and energy metabolism will be added in our model later.
We assume G6P, glycine, lipid, R5P and chlorophyll make contribution to biomass growth.
we assume all the reactions are only one direction, reverse reactions will be considered later.
No other regulation in the metabolism process, this will also be considered in our latter work.
Model hypothesis:
61
G6P
GA3P
3-P-G
PYR
Acetyl-COA
X5P
CO2
α- KG
R5P
Lipid
G6P
ChlorophyllNH4+ Glutamate
ATP
RuBPrubisco
Glycine
NADH
NAD+ADP
ATP
ADP
ChlorophyllF6P
PEP
RU5P
r5P
ATP
ADP
R5P
ATP
ADP
Lipid
NADP
NADPHATP
ADP
SUCCacid
Fumaricacid
Malac acid
NADH
NAD+NAD+ADP
NADH+ATP
ADP ATP
NADH NAD
O2
Biomass
Glycine
EGLC
r1
r2
r3
r4
r5
r6
r7
r8
r9
r10r11
r12 r13
r14
r14
r15
r16
r17
r18
r19 r20
r21
r22
r23
r24
NADH
NAD+
Metabolic network and stoichiometric: Glycolysis
r1 EGLC+ATP----G6P+ADP
r2 G6P ---F6P
r3 F6P+ATP ----GA3P+ADP
r4 GA3P+ADP+NAD----3PG+ATP+NADH
r5 3PG ----PEP
r6 PEP+ADP -----PYR+ATP
r7 PYR+NAD -----Acetyl-COA+NADH
Lipid synthesis
r8Acetyl-COA+ATP+NADPH ---- lipid+ADP+NADP
Photosynthesis
r9 RuBP---- GA3P
r10 GA3P -----R5P
r11 R5P -----RuBPPPP pathway
r12 G6P -----Ru5P
r13 Ru5P ----- R5P+X5P
r14 R5P+X5P ---- F6P
TCA cycle
r15 Acetyl-COA ----- Malac
r16 Malac +NAD -----α- KG+NADH
r17 α- KG _NAD_ADP-----Succac+NADH+ATP
r18 Succac ------Fumaric
r19 Fumaric ------ Malac
r20 α- KG+NH4 -----Glutamate
Chlorophyll synthesis
r21 Glutamate----Chlorophyll
r22 Glycine----PYR
Oxidative phosphorylation
r23 ATP ---- ADP
r24 ADP +NADH------ ATP+NAD
Biomass synthesis
r25 R5P+lipid+Glycine+G6P+Chlorophyll ---- X
62
Model functions:
2. Cell growth: Multi-Monod-equation
3. Mass balance
1. Biochemical reaction kinetics: Multi-Michaelis-Menten equation
𝑉 = 𝑉max
𝑖=1
𝑁𝑠𝐶𝑆𝑖
𝐾𝑚𝑆𝑖 + 𝐶𝑆𝑖
𝑑[𝐶𝑆𝑖𝑑𝑡=
𝑖=1
𝑀
𝑉𝑖𝑛𝑝𝑢𝑡𝑖 −
𝑗=1
𝑁
𝑉𝑜𝑢𝑡𝑝𝑢𝑡𝑗 − a × 𝑉𝑔𝑟𝑜𝑤𝑡ℎ − 𝐶𝑆𝑖 × 𝑉𝑔𝑟𝑜𝑤𝑡ℎ
𝑉_𝑔𝑟𝑜𝑤𝑡ℎ
= 𝑉_𝑔𝑟𝑜𝑤𝑡ℎ𝑚𝑎𝑥 ×𝑅5𝑃
𝑅5𝑃 + 𝐾𝑚_𝑅5𝑃×
𝐿𝑖𝑝𝑖𝑑
𝐿𝑖𝑝𝑖𝑑 + 𝐾𝑚_𝑙𝑖𝑝𝑖𝑑×
𝐺𝑙𝑦𝑐𝑖𝑛𝑒
𝐺𝑙𝑦𝑐𝑖𝑛𝑒 + 𝐾𝑚_𝑔𝑙𝑦𝑐𝑖𝑛𝑒
×𝐺6𝑃
𝐺6𝑃 + 𝐾𝑚_𝐺6𝑃×
𝐶ℎ𝑙𝑜𝑝ℎ𝑦𝑙𝑙
𝐶ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 + 𝐾𝑚_𝐶ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙
Contribution to Biomass
Cell dilution Extracellular
× 𝐵𝑖𝑜𝑚𝑎𝑠𝑠
63
Simulation results of model and
experimental data
Simulation result：
Biomass
G6P
EGlucose
F6P
PYR AKG
64
G6P
GA3P
3-P-G
PYR
Acetyl-COA
X5P
CO2
α- KG
R5P
Lipid
G6P
ChlorophyllNH4+ Glutamate
ATP
RuBPrubisco
Glycine
NADH
NAD+ADP
ATP
ADP
ChlorophyllF6P
PEP
RU5P
r5P
ATP
ADP
R5P
ATP
ADP
Lipid
NADP
NADPHATP
ADP
SUCCacid
Fumaricacid
Malac acid
NADH
NAD+NAD+ADP
NADH+ATP
ADP ATP
NADH NAD
O2
Biomass
Glycine
EGLC
r1
r2
r3
r4
r5
r6
r7
r8
r9
r10r11
r12 r13
r14
r14
r15
r16
r17
r18
r19 r20
r21
r22
r23
r24
NADH
NAD+
Future work
Nutrition strategy based on the model prediction
-Analysis of the flux distribution and identify key enzymes and nutrient
limitations along the time profile.
-Develop a real-time control nutritional strategy to improve lipid production
based on model prediction.
-Predict hypothesis of knocking off relevant genes, which will also provide a
further strategy to improve the lipid production.
65
Acknowledgement
Thanks for my supervisors Prof. Mario Jolicoeur, Jean-Sébastien Deschênes, Réjean
Tremblay support my research.
Thanks for our technician Jingkui Chen and other partners' help in my work.
Thanks for the supports from the organizations.
66
Lab course plan
- Medium preparation: modified basal medium
- Inoculation: 1.0*106cell/ml
- Microscope observation: cell size, cell density, chlorophyll
- Cell harvest: 3600rpm, 5min, wash cells.
- lipid analysis: Acetone and sulfosalicylic acid; absorbance was read at
440nm by UV-VIS determination
67
Merci!68
