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Model-Based Design of Superior Cell Factory: An Illustrative Example of Penicillium chrysogenum

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Systems Metabolic Engineering

Abstract

A dynamic model for metabolic reaction network of Penicillium chrysogenum, coupling the central metabolism to growth, product formation and storage pathways is presented. In constructing the model, we started from an existing stoichiometric model, and systematically reduced this initial model to a one compartment model and further eliminated unidentifiabilities due to time scales. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. We used linlog kinetics in representing the kinetic rate equations of each individual reaction. The final parameterization is performed for the final reduced model using previously published short term glucose perturbation data. The constructed model is a self-contained model in the sense that it can also predict the cofactor dynamics. Using the model, we calculated the Metabolic Control Analysis (MCA) parameters and found that the interplay among the growth, product formation and production of storage materials is strongly governed by the energy budget in the cell, which is in agreement with the previous findings. The model predictions and experimental observations agree reasonably well for most of the metabolites.

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Author information

Authors and Affiliations

  1. Delft Bioinformatics Lab, Delft University of Technology, Mekelweg 4, 2628 CD, Delft, The Netherlands

    I. Emrah Nikerel

  2. Department of Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands

    I. Emrah Nikerel, Peter J. T. Verheijen, Walter M. van Gulik & Joseph J. Heijnen

Authors
  1. I. Emrah Nikerel
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  2. Peter J. T. Verheijen
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  3. Walter M. van Gulik
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  4. Joseph J. Heijnen
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Corresponding author

Correspondence to I. Emrah Nikerel .

Editor information

Editors and Affiliations

  1. Inst. Bioverfahrenstechnik, TU Braunschweig, Gaußstraße 17, Braunschweig, 38106, Germany

    Christoph Wittmann

  2. Science and Technology-KAIST, Dept. Biosystems a. Bioinformatics, Korea Advanced Institute, Guseong-dong 373-1, Daejeon, 305-701, Korea, Republic of (South Korea)

    Sang Yup Lee

Appendix

Appendix

8.1.1 Nomenclature Used for Metabolites in the Model

Below is the nomenclature list for metabolites used in the model, following the names in van Gulik et al. [29]

13PG

1,3-Bisphosphoglycerate

glc

Glucose

2PG

2-Phosphoglycerate

gln

Glutamine

3PG

3-Phosphoglycerate

glu

Glutamate

6APA

6-Aminopenicillinic acid

gly

Glycine

6Pgluct

6-Phosphogluconate

GMP

Guanosine monophosphate

8HPA

8-Hydroxypenillic acid

H

Proton

aAd

a-Aminoadipate

H2O

Water

AApool

Pool of free amino acids

H2S

Hydrogen sulfide

AAprotsyn

Amino acids for protein synthesis

HCoA

Coenzyme A

Ac

Acetate

his

Histidine

AcCoA

Acetyl coenzyme A

homcys

Homocysteine

AcHomser

o-Acetylhomoserine

homser

Homoserine

ACV

d-(a-Aminoadipyl)-cysteinylvaline

iCitr

Isocitrate

ADP

Adenosine-5-diphosphate

ile

Isoleucine

aKG

a-Ketoglutarate

IMP

Inosine monophosphate

aKI

a-Ketoisovalerate

ino

Inositol

ala

Alanine

iPN

Isopenicilline

AMP

Adenosine-5-monophosphate

lano

Lanosterol

arg

Arginine

leu

Leucine

asn

Asparagine

linCoA

Linoleoyl coenzyme A

asp

Aspartate

lys

Lysine

ATP

Adenosine-5-triphosphate

m1P

Mannitol-1-phosphate

bIM

b-Isopropylmalate

mal

Malate

biomass

Average biomass composition of glucose-limited cultures

man

Mannitol

carbP

Carbamoylphosphate

met

Methionine

CDPDAcgcl

Cytidine diphosphate-diacylglyerol

METHF

Methylene tetrahydrofolate

chit

Chitine

meva

Mevalonate

chor

Chorismate

MYTHF

Methyltetrahydrofolate

citr

Citrate

NAD

Nicotinamide adenine dinucleotide (oxidized)

CMP

Cytidine monophosphate

NADH

Nicotinamide adenine dinucleotide (reduced)

CO2

Carbon dioxide

NADP

Nicotinamide adenine dinucleotide phosphate

ctl

CTP

cys

E4P

Citruline

Cytidine triphosphate

Cysteine

Erythrose-4-phosphate

NADPH

Nicotinamide adenine dinucleotide phosphate

  

NH4

Ammonia

  

O2

Oxygen

  

OAA

Oxaloacetate

ergo

Ergosterol

OPC

6-Oxopiperidine-2-carboxylic acid

ery

Erythritol

PAA

Phenylacetic acid

ESE

Ergosterolester

PAACoA

Phenylacetyl coenzyme A

ExPept

Excreted peptides

PAPS

3-Phosphoadenosine-5-phosphosulfate

f16P

Fructose-1,6-bisphosphate

penG

Penicillin-G

f6P

Fructose-6-phosphate

PEP

Phosphoenolpyruvate

FAD

Flavine adenine dinucleotide (oxidized)

PHchol

Phosphatidylcholine

FADH2

Flavine adenine dinucleotide (reduced)

phe

Phenylalanine

FTHF

Formyltetrahydrofolate

PHeta

Phosphatidylethanolamine

fum

Fumarate

PHino

Phosphatidylinositol

g6P

Glucose-6-phosphate

phospht

Phosphatidate

GAP

3-Phosphoglyceraldehyde

PHser

Phosphatidylserine

gcl3P

3-Phosphoglycerol

Pi

Orthophosphate

pro

Proline

PROT

Protein

PRPP

a-5-Phosphoribosylpyrophosphate

psacch

Polysaccharides

pyr

Pyruvate

Rib5P

Ribose-5-phosphate

Ribu5P

Ribulose-5-phosphate

RNA

Ribose nucleic acid

SAM

S-Adenosylmethionine

sed7P

Sedoheptulose-7-phosphate

ser

Serine

succ

Succinate

succCoA

Succinyl coenzyme A

t6P

Trehalose-6-phosphate

THF

Tetrahydrofolate

thr

Threonine

tre

Trehalose

trp

Tryptophane

tyr

Tyrosine

UDP

Uridine-5-diphosphate

UDPglc

Uridine-5-diphosphoglucose

UMP

Uridine monophosphate

UTP

Uridine triphosphate

val

Valine

Xylu5P

Xylulose-5-phosphate

8.1.2 Data for the Kinetic Model

In the following, three tables are specified the finally used reactions, the input data, the assumed equilibrium constants and data on metabolite concentrations.

Table 8.15 Assumed equilibrium constants
Full size table
Table 8.16 Assumed metabolite concentrations
Full size table

8.1.3 P/O Ratio Calculations

The reactions describing the oxidative phosphorylation in [29] have to be updated/adjusted not only because the mitochondria compartment has been removed, but also, in [28], the experimentally determined P/O parameter was 1.84. The P/O parameter is changed in such a way to have the same theoretical yields for the reduced model as the original model. Using the Herbert-Pirt substrate utilization equation as a quality assessment to these reduction steps, we compared the Herbert-Pirt equation for the three compartment and the one compartment model.

We first considered the stoichiometric network presented in [29], which consists of 155 metabolites and 156 reactions located in three compartments (cytosol, mitochondria and peroxisome). The Herbert-Pirt substrate utilization relation can be written as:

$$ {q_S} = 5.17{q_{\rm{Pen}}} + 0.288\mu + 1.37\,{10^{{ - 3}}} $$

The P/O value is calculated from reaction r6.1 and r6.4. The first adjustment is to multiply the theoretical stoichiometric coefficients of mitochondrial and cytosolic protons, with the ratio of the estimated P/O ratio to the maximum P/O ratio

$$ \begin{array}{lll} 8.360{H_m} + NAD{H_m} + 0.5{O_2}\;{{\longrightarrow}^{{l6.1}}}\;\;\;\,7.360{H_c} + {H_2}{O_c} +NA{D_m} \hfill \\5.416{H_m} + NAD{H_c} + 0.5{O_2}\;{{\longrightarrow}^{{l6.2}}}\;\;\;\,4.416{H_c} + {H_2}{O_c} +NA{D_c} \hfill \\4.416{H_m} + FAD{H_{{2,m}}} + 0.5{O_2}\;{{\longrightarrow}^{{l6.3}}}\;\;\;\,4.416{H_c} + {H_2}{O_c} +FA{D_m} \hfill \\ \end{array} $$

Subsequently the formation of ATP which is driven by the inward flux of cytosolic protons into the mitochondria (r6.4) is lumped with the water transport and the ADP/ATP shuttle (r10.3 and r10.1 respectively) in order to eliminate all mitochondrial reactants with exception of Hm.

$$\begin{array}{rll}&AD{P_m} + 4{H_c} + P{i_m}\quad \mathop{\longrightarrow}\limits^{{r6.4}}\;\quad AT{P_m} + 3{H_m} + {H_2}{O_m} \hfill \\ &AD{P_c} + AT{P_m}\quad \mathop{\longrightarrow}\limits^{{r10.1}}\quad AD{P_m} + AT{P_c} \hfill \\ &\frac{{{H_2}{O_m}\quad \mathop{\longrightarrow}\limits^{{r10.3}}}\quad {H_2}{O_c}}{{AD{P_c} + 4{H_c} + P{i_m} \mathop{\longrightarrow}\limits^{{l6.4}}\;\quad AT{P_c} + 3{H_m}{H_2}{O_c}}}\end{array}$$

The next step is to eliminate the H in reactions r6.1-3 using l6.4. This yields the following lumped reactions:

$$ \begin{array}{llll} {1.840AD{P_c} + 2.840{H_c} + NAD{H_m} + 0.5{O_{{2,c}}} + 1.840P{i_c}} &\!\! {{ \longrightarrow^{{l6.5}}}} & {1.840AT{P_c} + 2.84{H_2}{O_c} + NA{D_m}} \\{1.104AD{P_c} + 2.104{H_c} + NAD{H_c} + 0.5{O_{{2,c}}} + 1.104P{i_c}} &\!\! {{ \longrightarrow^{{l6.6}}}} & {1.104AT{P_c} + 2.104{H_2}{O_c} + NA{D_c}} \\{1.104AD{P_c} + 1.104{H_c} + FAD{H_{{2,m}}} + 0.5{O_{{2,c}}} + 1.104P{i_c}} &\!\! {{ \longrightarrow^{{l6.7}}}} & {1.104AT{P_c} + 2.104{H_2}{O_c} + FA{D_m}} \\\end{array} $$

Since the model does not contain any inner compartments, reactions l6.5 and l6.6 are parallel to each other, and are therefore lumped by averaging based on their steady state flux distribution (70.5–29.5% respectively). This yields the following lumped reaction:

$$ {1.623ADP + 2.623H + NADH + 0.5{O_2} + 1.623{P_i}\,{\longrightarrow^{{l6.8}}}\;\;\,\;1.623ATP + 2.623{H_2}O + NAD} $$

This shows that the P/O ratio in the simplified model became 1.623 mol ATP/mol O. It should be noted that, the remaining ATP-balance parameters i.e. Kx, KPenG, mATP are kept unchanged in considering black-box balances

After modifying the model, we calculated the substrate Herbert-Pirt equation for the one compartment model.

$$ {q_S} = 6.32{q_{\rm{Pen}}} + 0.291\mu + 2.22{q_{\rm{tre}}} + 1.74\,{10^{{ - 3}}} $$

We conclude that the obtained Herbert Pirt relation for the one compartment model is in agreement with the one for the three compartment model given the standard deviation in the ATP stoichiometry parameters.

8.1.4 Additional Details on the Identifiable Elasticities

List of identifiable elasticities as a function of the original parameters

l1.1

εP1 = ε1

r5.1

εP6 = ε33

 

εP2 = ε1

 

εP7 = ε33

 

εP10 = ε2

 

εpyr = ε31

 

εglc_f = ε3

 

εasp = ε32

r1.3

εP1 = ε4 + 1/8 ε5

l6.7

εP5 = ε35

 

εP2 = ε4 + 1/2 ε5

 

εP10 = ε34

 

εP10 = ε6

l6.9

εP3 = ε37

l1.3

εP1 = 1/4 ε7

 

εP10 = ε36

 

εP2 = ε7

r8.1

εP3 = ε39 + ε40

 

εP3 = ε7

 

εP8 = ε38

 

εP10 = −ε7

 

εP12 = ε40

 

εPEP = ε8

l11.1

εpyr = ε41

r1.9

εP1 =1/8 ε9

 

εile = ε42

 

εP2 =1/2 ε9

 

εval = ε43

 

εP10 = ε11

r11.2

εP8 = ε45

 

εPEP = ε10

 

εgln = ε44

r2.1

εP1 = ε12

l11.2

εP6 = ε50

 

εP2 = ε12

 

εP7 = ε50

 

εP10 = ε13

 

εpyr = ε46

 

εP11 = ε14

 

εile = ε47

r2.2

εP10 = ε16

 

εleu = ε48

 

εP11 = ε17

 

εval = ε49

 

ε6Pgluct = ε15

r11.3

εP8 = ε51

r4.1

εP3 = ε20

 

εP10 = ε53

 

εP6 = ε21

 

εpro = ε52

 

εP7 = ε21

l11.3

εP1 =1/3 ε55

 

εP10 = ε19

 

εP2 = ε55

 

εpyr = ε18

 

εPEP = ε54

r4.4

εP3 = ε22 + ε23

 

εtrp = ε56

 

εP4 = ε22

l11.4

εP1 =1/3 ε58

 

εP5 = ε22

 

εP2 = ε58

 

εP6 = ε22

 

εPEP = ε57

r4.7

εP3 = ε25 + ε26

 

εtyr = ε59

 

εP4 = ε25

l11.5

εP1 =1/3 ε61

 

εP5 = ε25

 

εP2 = ε61

 

εP7 = ε27

 

εPEP = ε60

 

εP8 = ε24

 

εphe = ε62

 

εP11 = ε24

r11.7

εP6 = ε66

r4.8

εP6 = ε29

 

εP7 = ε66

 

εP7 = ε29 + ε30

 

εP8 = ε63

 

εP10 = ε28

 

εP11 = ε63

   

εlys = ε64

   

εaAd = ε65

l11.7

εP9 = ε68

l13.2

εP1 =1/2 ε105

 

εP10 = ε69

 

εP2 = ε105

 

εcys = ε67

 

εP10 = ε108

r11.8

εlys = ε70

 

εasp = ε106

 

εaAd = ε71

 

εAMP = ε107

l11.8

εP10 = ε75

 

εGMP = ε109

 

εasp = ε72

l13.3

εP1 =1/2 ε110

 

εmet = ε73

 

εP2 = ε110

 

εthr = ε74

 

εasp = ε111

 

εhomcys = ε76

 

εAMP = ε112

r11.9

εP1 = 1/4 ε77

l13.4

εP1 = 1/2 ε113

 

εP2 = ε77

 

εP2 = ε113

 

εP3 = ε77 + ε81

 

εasp = ε114

 

εP8 = ε78 + ε79

 

εUTP = ε115

 

εP9 = ε80

r13.6

εCTP = ε116

 

εP10 = −ε77

 

εUTP = ε117

 

εP11 = ε78

r15.1

εP10 = ε118

l11.9

εasp = ε82

l17.1

εP1 = ε119

 

εcys = ε83

 

εP2 = ε119

 

εthr = ε84

 

εt6P = ε120

l11.10

εP1 = 1/2 ε85

 

εAMP = ε121

 

εP2 = ε85

l17.4

εP1 = ε123 + 1/8 ε122

 

εP10 = ε87

 

εP2 = ε123 + 1/2 ε122

 

εhis = ε86

 

εpsacch = ε124

l11.11

εP8 = ε88

 

εAMP = ε125

 

εorn = ε89

r17.7

εt6P = ε126

l11.12

εP10 = ε91

l17.6

εtre = ε127

 

εorn = ε90

l18.2

εP10 = ε128

r11.16

εP3 = ε92

l19.1

εP10 = ε134

 

εP4 = ε92

 

εcys = ε129

 

εP5 = ε92

 

εval = ε130

 

εasp = ε93

 

εaAd = ε131

r11.17

εasn = ε94

 

εPAA = ε132

 

εasp = ε95

 

εpenG = ε133

r11.20

εP3 = ε97

R9.14

εpsacch = ε135

 

εP12 = ε97

R9.8

εpenG = ε136

 

εmet = ε96

R9.11

εPAA_f = ε137

 

εhomcys = ε98

R9.1

εP10 = ε138

r11.21

εpyr = ε99

  
 

εile = ε100

  
 

εval = ε101

  

r11.22

εpyr = ε102

  
 

εala = ε103

  

r11.34

εP10 = ε104

  

8.1.5 Complete List of Estimated Elasticities

#ri.i

Representative transformation

Identifiable elasticities

l1.1

glc → g6P

P1(−0.01), P2(−0.01), P10(0.01), glcf(0.51)

r1.3

f6P → f16P

P1(−0.01), P2(−0.01), P10(−0.15)

r2.2

6PG → Ribu5P

P1(−1.00), 6PG(0.01), P11(2.11)

r4.1

pyr → AcCoA

P1(0.01), P2(0.02), P6(−17.60), P3(−0.01), pyr(0.02)

r4.4

iCitr → aKG

P3(5), P4(0.07), P5(1.16), P6(0.01)

r4.7

aKG → succCoA

P7(11.61), P11(0.01), P3(0.01)

r4.8

succCoA → succ

P8(1.28), P9(0.01), P11(0.01), P3(−0.01)

r5.1

pyr → OAA

P4(−0.01), P5(−0.01), pyr(0.01)

l6.7

H2 + O2 → H2O (FAD)

P5(−0.01), P10(−0.01)

l6.9

H2 + O2 → H2O (NADH)

P3(0.25), P10(−0.25)

r8.1

CO2 → gly

P8(0.01), P12(−1.67), P3(4.59)

l11.1

pyr → val

pyr(0.30), val(−11.99), ile(−3.65)

r11.2

glu → gln

P8(4.87), gln(−0.07)

l11.2

pyr → leu

P6(1.21), P7(0.01), ile(6.30), leu(3.24), val(−1.96), pyr(4.09)

r11.3

glu → pro

P8(8.66), pro(−8.95), P10(0.686)

l11.3

PEP → trp

PEP(2.87), trp(−0.01)

l11.4

PEP → tyr

P1(0.40), P2(0.12), tyr(−0.07), PEP(5.17)

l11.5

PEP → phe

P1(0.59), P2(0.26), phe(−1.89), PEP(3.26)

r11.7

glu → aAd

P6(0.01), P7(0.01), P8(2.05), lys(−1.86), aAd(−0.01)

l11.7

ser → cys

P9(1.11), cys(−0.01), P10(2.64)

r11.8

glu → lys

aAd(0.01), lys(−3.63)

l11.8

asp → homcys

asp(43.03), P10(−35.1), homcys(−0.91), met(18.7), thr(7.08)

r11.9

3PG → ser

P2(0.01), P8(2.33), P3(0.68)

l11.9

asp → thr

P11(−0.96), asp(12.57), cys(0.50), thr(−0.01)

l11.10

gln → his

P1(1.81), P2(0.26), P10(6.79)

l11.11

glu → orn

P8(0.01), orn(−0.01),

l11.12

orn → arg

orn(0.01), P10(0.01), gln(0.61), arg(−0.76)

r11.16

glu → asp

P4(0.83), P5(0.83), asp(−0.01), P3(12.77)

r11.17

asp → asn

asp(2.29), asn(−0.01)

r11.20

met → homcys

met(−0.01), homcys(−1.03)

r11.21

pyr → ile

P1(−0.76), P2(0.16), ile, val(−0.76), pyr(−0.84)

r11.22

pyr → ala

P1(5.32), P2(0.01), pyr(8.67), ala(−2.52)

r11.34

qExPept

P10(1)

l13.2

→ GMP

P1(2.43), P2(3.71), P4(−0.01), P5(−2.62), P10(−0.01), asp(30.69), GMP(−0.38)

l13.3

→ AMP

P1(0.20), P2(11.41), P4(−0.34), P5(−0.01), asp(53.4), AMP(−20.31)

l13.4

→ UTP

P1(2.54), P2(−5.37), asp(0.461), UTP(−0.74)

r13.6

→ CTP

UTP(1.10), CTP(−3.60)

r15.1

mATP

P10(1.00)

l17.1

g6P → t6P

P1(31.85), P2(35.38), t6P(−23.03)

l17.4

g1P → psacch

P1(7.05), P2(0.132), psacch(−0.01), AMP(0.53)

r17.7

t6P → tre

t6P(0.134)

l17.6

qtre

tre(0.91)

l18.2

m

P10(1.00)

l19.1

→ PenG

cys(1.00), val(1.00), aAd(1.00), P10(1.00), PenG(−0.50), PAA(1.00)

R9.14

qpsacch

psacch(1.00)

R9.8

qPenG

PenG(1.00)

R9.11

qPAA

PAAext(1.00)

R9.1

Hext →

P10(1.00)

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Nikerel, I.E., Verheijen, P.J.T., van Gulik, W.M., Heijnen, J.J. (2012). Model-Based Design of Superior Cell Factory: An Illustrative Example of Penicillium chrysogenum . In: Wittmann, C., Lee, S. (eds) Systems Metabolic Engineering. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4534-6_8

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