characteristics of the kinase mutant tpk2 in bioreactors
DESCRIPTION
PKA has 2 catalytic subunits (Tpk) which are encoded by 3 genes: tpk1,tpk2 and tpk3. On binding cAMP, PKA is able to phosphorylate some proteins. However, in mutants like ours, it can be assumed that some of that proteins may not be phosphorylated. PKA serves as a central regulator of the metabolic and transcriptional status of the yeast cell.TRANSCRIPT
Beatriz Barrera
Borja Garnelo
Juan Carlos López
Elliott James Williams
Mr. Lobster
Characteristics of the kinase mutant
TPK2 in bioreactors
TEAM 3
Strain TPK2
• YCK401 is a background strain which has the tpk2 gene deleted. • We know that PKA has 2 catalytic subunits (Tpk) which are encoded by 3
genes: tpk1,tpk2 and tpk3. On binding cAMP, PKA is able to phosphorylate some proteins. However, in mutants like ours, it can be assumed that some of that proteins may not be phosphorylated. We’ll see it later.
PKA pathway
(Zaman S, et al., Annu. Rev. Genet., 2008)
PKA pathway
As we have said, PKA is a heterotetramer composed of two catalytic subunits and two regulatory subunits. Tpk1, tpk2, tpk3 genes encode the catalytic subunits and seems to be an important check point in protein phosphorylations.
The activation of PKA via Ras is in response to intracellular acidification, which helps activating Cyr1 by phosphorylation. Cyr1 modifies ATP into cAMP, that is going to bind the regulatory subunits, freeing Tpk.
Tpk participates in a negative feed-back. So, when we have high levels of Tpk, it can activate Pde1,2 (transforms cAMP into an activated form) and inhibite Cyr1, so free levels of PKA decreases.
Some of the well-characterized substrates for these kinase subunits include proteins involved in metabolism of storage carbohydrates, enzymes in glycolysis and gluconeogenesis, and transcription factors regulating stress response, ribosomal biogenesis, and carbohydrate metabolism. So, PKA serves as a central regulator of the metabolic and transcriptional status of the yeast cell.
We diluted 3 times,
0.33 x 1.5 = Volume x 4.24
so
Volume = 120 ml
VOLUME INOCULATED
Substrate in fermenter as time goes by
0,00001,0000
2,00003,0000
4,00005,00006,0000
7,00008,0000
9,000010,0000
0,00 2,00 4,00 6,00 8,00
10,0012,0
014,0
016,0
018,0
020,0
022,0
024,0
026,0
028,0
030,0
032,0
034,0
036,0
038,0
040,0
042,0
044,0
0
Time (h)
Am
ou
nt
of
sub
stra
te (
g/l)
glucose
galactose
ethanol
Amount of substrate being used in Mr Lobster by the cells
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0,001,002,003,004,005,006,007,008,009,0010,0
011,0
012,0
013,0
014,0
015,0
016,0
017,0
018,0
019,0
020,0
021,0
022,0
023,0
024,0
025,0
026,0
027,0
028,0
029,0
030,0
031,0
032,0
033,0
034,0
035,0
036,0
037,0
038,0
039,0
040,0
041,0
042,0
043,0
044,0
045,0
0
Time (h)
Am
ou
nt
of
sub
stra
te (
g/l)
glucose
galactose
ethanol
Accumulation of substrates in cells
0
2
4
6
8
10
12
glucose
galactose
ethanol
Filter 1 No
Filter 1
weight Filter 2 No
Filter 2
weight Filter 1 No
Filter 1
weight Filter 2 No
Filter 2
weight Filter 1 Filter 2
0.00 0.2810176 0.4215264
1.00 0.367483 0.5512245
2.00 0.2893735 0.43406025
3.00 0.3126247 0.46893705
4.00 7 0.0776 7 0.0806 0.003 0.3 0.3544042 0.5316063
5.00 2 0.0769 6 0.0776 2 0.0788 6 0.0806 0.0019 0.003 0.245 0.6297856 0.9446784
6.00 1 0.078 8 0.0784 1 0.0785 8 0.08 0.0005 0.0016 0.105 0.8187016 1.2280524
7.00 3 0.0772 9 0.0785 3 0.0782 9 0.0832 0.001 0.0047 0.285 0.995992 1.493988
8.00 19 0.0778 24 0.0779 19 0.0855 24 0.0788 0.0077 0.0009 0.43 1.3389472 2.0084208
22.50 12 0.0774 16 0.078 12 0.0992 16 0.0794 0.0218 0.0014 1.16 2.3213104 3.4819656
23.50 11 0.0774 10 0.0778 11 0.0943 10 0.0783 0.0169 0.0005 0.87 2.0074192 3.0111288
24.50 2.2399312 3.3598968
26.50 22 0.078 22 0.1011 0.0231 0.0231 2.31 2.2631824 3.3947736
27.50 25 0.078 26 0.078 25 0.0993 26 0.0996 0.0213 0.0216 2.145 2.3154976 3.4732464
45.50 23 0.0782 21 0.0781 23 0.1033 21 0.1022 0.0251 0.0241 2.46 2.7630832 4.1446248
Average
Weight (*)
Dry weight
(g/l)Biomass (g)
Weight Of Filter Before Weight Of Filter After Calculated Weight Time (h)
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 50,00
Time (h)
Ce
ll c
on
ce
ntr
ati
on
(g
)
Correlation between glucose and DW
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0,0
0
1,0
0
2,0
0
3,0
0
4,0
0
5,0
0
6,0
0
7,0
0
8,0
0
9,0
0
10
,00
11
,00
12
,00
13
,00
14
,00
15
,00
16
,00
17
,00
18
,00
19
,00
20
,00
21
,00
22
,00
23
,00
24
,00
25
,00
26
,00
27
,00
Time (h)
Am
ou
nt
of
glu
cose
(g/
l)
0,1
1
10
Dry
we
igh
t (g
/l)
glucose
Average Dry Weight
CO2 Analysis
Variance of the CO2 respect time
0.01
0.1
1
10
0 500 1000 1500 2000 2500 3000
Time (min)
dC
O2
(%
)
Variance of the CO2 normalised respect time
0,01
0,1
1
10
0 500 1000 1500 2000 2500 3000 3500
Time (min)
dO
2 n
orm
ali
sed
(%
)
Variance of the CO2 respect time
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
0 500 1000 1500 2000 2500 3000
Time (minutes)
dC
O2 (
%)
Variance of CO2 normalised respect time
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 500 1000 1500 2000 2500 3000
Time (minutes)
dO
2 n
orm
alis
ed
(%
)
Specific CO2 growth rate
Max specific growth rate
y = 0,1747e0,1945x
0
5
10
15
20
25
30
35
0,000 5,000 10,000 15,000 20,000 25,000 30,000
Time (h)
CO
2 (
g)
Correlation between O.D and DW
y = 0,545x + 0,2733
0
1
2
3
4
5
0 1 2 3 4 5 6 7 8
OD600
Dry
We
igh
t (g
)
Max Specific growth rate on glucose
y = 0.1057e0.4407x
0.1
1
10
2 3 4 5 6 7 8 9
Time (h)
OD
600
Cell density in the fermenter as time increases
012345678
0,001,0
02,0
03,0
04,0
05,0
06,0
07,0
08,0
09,0
0
10,00
11,00
12,00
13,00
14,00
15,00
16,00
17,00
18,00
19,00
20,00
21,00
22,00
23,00
24,00
25,00
26,00
27,00
28,00
29,00
30,00
31,00
32,00
33,00
34,00
35,00
36,00
37,00
38,00
39,00
40,00
41,00
42,00
43,00
44,00
45,00
T ime (h)
OD
60
0
0
0,5
1
1,5
2
2,5
3
R eal OD600
AverageWeight (*)
Yield Coefficients (Ysx)
Ysx on glucose
y = 0,218x + 0,3879
0
1
2
3
4
5
0,0000 2,0000 4,0000 6,0000 8,0000 10,0000 12,0000 14,0000 16,0000
Glucose (g)
Dry
Weig
h (
g)
Ysx on glucose
y = 0,2378x + 0,0141
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0,5000
Glucose used (Cmols)
Bio
mass (
Cm
ols
)
We calculated the Ysx numerically: Yxs = x / s Yxs = (x2 - x1) / (s2 - s1) Dry Weight (g) · Glucose (g) Yxs = 0.218
Dry Weight (Cmols) · Glucose (Cmol) Yxs = 0.2378
Yield Coefficients (Ysx)
Ysx on galactose
y = 0,1911x + 0,8633
0
1
2
3
4
5
0 2 4 6 8 10 12 14 16
Galactose used (g)
DW
(g
)
Ysx on galactose
y = 0,2084x + 0,0314
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0 0,1 0,2 0,3 0,4 0,5
Galactose (Cmols)
Bio
mass (
Cm
ols
)
Dry Weight (g) · Galactose (g) Yxs = 0.2175 Dry Weight (Cmols) · Galactose (Cmol) Yxs = 0.2373
Yield Coefficients (Yse)
Yse on glucose
y = 0,5487x + 0,0351
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0,0000 2,0000 4,0000 6,0000 8,0000 10,0000 12,0000 14,0000 16,0000
Glucose used (g)
Eth
an
ol
pro
du
ced
(g)
Yse on galactose
y = 0,4973x + 1,1397
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0 2 4 6 8 10 12 14 16
Galactose Used (g)
Eth
an
ol
Pro
du
ced
(g
)
We calculate the Yse numerically: Yse = e / s Yse = (e2 - e1) / (s2 - s1) Ethanol (g) · Glucose (g) Yse = 0.581 Ethanol (g) · Galactose (g) Yse = 0.58
Yield Coefficients (Ysc)
We calculate Ysc numerically: Ysc = c/s
Ycs = (c2-c1) / (s2-s1)
CO2 (g)
. Glucose Ycs = 0,352
Ysc on glucose
y = 0,3795x - 0,2286
0
0,5
1
1,5
2
2,5
3
0,0000 1,0000 2,0000 3,0000 4,0000 5,0000 6,0000 7,0000
Glucose used (g)
CO
2 (
g)
CO2 Analysis Variance of the CO2 respect time
0,01
0,1
1
10
0 500 1000 1500 2000 2500 3000
Time (min)
dC
O2
(%
)
Variance of the CO2 normalised respect time
0,01
0,1
1
10
0 500 1000 1500 2000 2500 3000 3500
Time (min)
dO
2 n
orm
ali
sed
(%
)
TPK2 vs Wild Type
Varianc e of C O2 normalis ed (T E AM 1)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0 5 10 15 20 25 30 35 40 45 50
T ime (h)
dC
O2
no
rma
lis
ed
(%
)
CO2 analysis
Variance of CO2 normalised
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 500 1000 1500 2000 2500 3000
Time (minutes)
dO
2 n
orm
ali
se
d (
%)
TPK2 vs Wild Type (Ysx)
Ysx on glucose
y = 0,2378x + 0,0141
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0,5000
Glucose used (Cmols)
Bio
mass (
Cm
ols
)
Ysx on galactose
y = 0,2084x + 0,0314
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0 0,1 0,2 0,3 0,4 0,5
Galactose (Cmols)
Bio
mass (
Cm
ols
)
Ysx on substrates (TEAM 1)
y = 0,1288x + 0,0682
y = 0.3407x + 0.0008
0
0,02
0,04
0,06
0,08
0,1
0,12
0 0,05 0,1 0,15 0,2 0,25 0,3
Substrates used (Cmol)
Bio
mass (
Cm
ol)
Glucose(Cmol)
Galactose(Cmol)
TPK2 vs Wild Type
Yse on glucose (Ethanol produced
(g))
Y s e on g luc os e (T E AM 1)
y = 0.5599x + 0.0698
0,0
0,2
0,4
0,6
0,8
1,0
1,2
0 0,5 1 1,5 2G luc ose used (g /l)
Eth
ano
l (g
/l)
Yse on glucose
y = 0,5487x + 0,0351
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0,0000 2,0000 4,0000 6,0000 8,0000 10,0000 12,0000 14,0000 16,0000
Glucose used (g)
Eth
an
ol
pro
du
ced
(g)
TPK2 vs Wild Type
Yse on galactose
y = 0,4973x + 1,1397
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0 2 4 6 8 10 12 14 16
Galactose Used (g)
Eth
an
ol
Pro
du
ced
(g
)
Y s e on g alac tos e (T E AM 1)y = 0.5292x + 0.529
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
0,0 2,0 4,0 6,0 8,0 10,0 12,0
G a la c tose used (g /l)
Eth
ano
l (g
/l)
Yse on galactose (Ethanol produced
(g))
TPK2 vs Wild Type
Ysc on substrates (TEAM 1)
y = 0.478x - 0.0077
y = 0.4589x + 0.1533
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5
S ubs trates us ed (C mol)
CO
2 p
rod
uc
ed
(Cm
ol)
0
5
10
15
20
25
30
35
40
Tim
e (
h)
G luc os e(C mol)
G alac tos e(C mol)
Ysc on glucose
y = 0,3795x - 0,2286
0
0,5
1
1,5
2
2,5
3
0,0000 1,0000 2,0000 3,0000 4,0000 5,0000 6,0000 7,0000
Glucose used (g)
CO
2 (
g)
Ysc on glucose (CO2 g)
TPK2 vs SNF1
Variance of CO2 normalised (TEAM 2)
0
1
2
3
4
5
6
7
8
9
10
0 10 20 30 40 50 60
Time (h)
CO
2 (
g)
Variance of CO2 normalised
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 500 1000 1500 2000 2500 3000
Time (minutes)d
O2
no
rma
lis
ed
(%
)
CO2 analysis
TPK2 vs SNF1
Ysx on glucose (TEAM 2)
y = 0.1347x + 0.0094
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Glucose (c-mol)
Bio
mass (
c-m
ol)
Ysx on glucose
y = 0,2378x + 0,0141
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0,5000
Glucose used (Cmols)
Bio
mass (
Cm
ols
)
Ysx on glucose (Biomass Cmol)
TPK2 vs SNF1
Yse on glucose (TEAM 2)
y = 0.2116x + 0.2557
0
0.5
1
1.5
2
2.5
0 1 2 3 4 5 6 7 8 9 10
Glucose (g)
Eth
an
ol
(g)
Yse on glucose
y = 0,5487x + 0,0351
0,0000
1,0000
2,0000
3,0000
4,0000
5,0000
6,0000
7,0000
8,0000
9,0000
10,0000
0,0000 2,0000 4,0000 6,0000 8,0000 10,0000 12,0000 14,0000 16,0000
Glucose used (g)
Eth
an
ol
pro
du
ced
(g)
Yse on glucose (Ethanol produced
g)
TPK2 vs SNF1
Ysc on glucose (TEAM 2)
y = 0.5157x - 0.0888
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
Glucose (g)
CO
2 (
g)
Ysc on glucose (CO2 g)
Ysc on glucose
y = 0,3795x - 0,2286
0
0,5
1
1,5
2
2,5
3
0,0000 1,0000 2,0000 3,0000 4,0000 5,0000 6,0000 7,0000
Glucose used (g)
CO
2 (
g)
Comparison table
StrainYsx on
glucose
Ysx on
galactose
Yse on
glucose
Yse on
galactose
Ysc on
glucose
Growth Rate
glucose
Growth Rate
galactose
Growth Rate
Ethanol Biomass
(gg-1) (gg-1) (gg-1) (gg-1) (gg-1) (h-1) (h-1) (h-1) (gl-1)
WT - Team 1 0,296 0,112 0,55 n.a 0,66 0,44 0,045 0,07 2,66
WT - Team 4 0,124 0,376 0,312 0 0,758 0,328 0,057 0 2,8
SNF1 - Team 2 0,12 0 0,21 0 0,51 0,11 0 0 1,61
SNF1 - Team 5 0,12 0 0,24 0 0,23 0,13 0 0 1,2
TPK2 - Team 3 0,218 0,191 0,549 0,49 0,378 0,43 n.a n.a 2,76
TPK2 - Team 6 0,15 0,23 0,23 0,43 0,48 0,26 0,08 0,1 3,83
Carbon balance
INPUT SUM Difference % error
0,9994
0,99939992 0,9770 0,02239906 2,23990569
0,99939992 0,9775 0,02192459 2,19245869
0,99939992 0,9758 0,02362163 2,36216317
0,99939992 0,9732 0,02621192 2,62119219
0,99939992 0,9836 0,01577527 1,57752747
0,99939992 0,9851 0,01426147 1,42614745
0,99939992 0,9989 0,00046984 0,04698416
0,99939992 0,9794 0,02003938 2,00393766
0,99939992 0,8232 0,17633321 17,6333212
0,99939992 0,7895 0,21001142 21,0011419
0,99939992 0,7877 0,2118361 21,1836104
0,99939992 0,7530 0,24658696 24,6586964
0,99939992 0,7229 0,27664874 27,6648736
0,99939992 0,4547 0,54501207 54,5012066
Glucose in Galactose in Biomass Succinato Accetate Glycerol Ethanol Ethanol CO2 CO2
ferm. (Cmol) ferm. (Cmols) (Cmols) (Cmols) (Cmols) (Cmols) produced (g) (Cmols) prod. (g) prod. (Cmol)
0,00 0,4756 0,469451327 0,01832823 0,0027 0,5003 0,0334 0 0,0000
1,00 0,459047551 0,459613822 0,02004453 0,0026 0,5356 0,0357 0,00079691 0,0000
2,00 0,452636162 0,461264404 0,01578401 0,0018 0,0026 0,0007 0,6397 0,0426 0,00103598 0,0000
3,00 0,440609389 0,461917886 0,01705226 0,0022 0,0026 0,0007 0,7592 0,0506 0,00127505 0,0000
4,00 0,424343979 0,462041649 0,01933114 0,0031 0,0025 0,9289 0,0619 0,00135475 0,0000
5,00 0,399134745 0,463599485 0,03435194 0,0039 0,0022 1,2067 0,0804 0,00135475 0,0000
6,00 0,366107147 0,463174676 0,04465645 0,0047 0,0021 1,5617 0,1041 0,0128568 0,0003
7,00 0,323906116 0,471833846 0,05432684 0,0060 0,0020 0,0016 2,0831 0,1389 0,01684134 0,0004
8,00 0,255707175 0,45674963 0,07303348 0,0072 0,0018 0,0021 2,7337 0,1822 0,02024149 0,0005
22,50 0 0,039912279 0,12661693 0,0342 0,0008 0,0115 9,1454 0,6097 0,01973678 0,0004
23,50 0 0,010723918 0,10949559 0,0357 0,0009 0,0119 9,3072 0,6205 0,01389278 0,0003
24,50 0 0,00927677 0,12217807 0,0349 0,0020 0,0124 9,1026 0,6068 0,00286887 0,0001
26,50 0 0,007590914 0,12344631 0,0350 0,0033 0,0147 8,5337 0,5689 0,00257667 0,0001
27,50 0 0,006428087 0,12629987 0,0344 0,0018 0,0168 8,0572 0,5371 0,00268293 0,0001
45,50 0 0,006743729 0,15071363 0,0300 0,0020 0,0221 3,6456 0,2430 0,00265636 0,0001
Time (h)
Sample for transcription analysis
The sample we took for transcription analysis was the last one of the first
day, assigned as sample number G3.8. It was obtained in the ninth hour of the fermentation process. It should be taken at this time because it’s when all the cells are growing up in an ideal environment.
It is important to know because transcriptome analysis will show what mRNA was present in the cell during that period of time. A cross comparison with the dates shows what proteins were being used by the microorganism when there was glucose in the media.
Other aspects
Crabtree effect. When the level of glucose goes beyond a critical concentration, the ability of the yeast to oxide glucose is diminished and the microorganism begins to express a mixed metabolism which includes a respiration pathway (now limited) and a fermentation pathway too (which is now very active). Nevertheless, there’s no evidence of the Crabtree effect because of the glucose and ethanol levels (they don’t fit as we expected).
We can see glucose repression of growth on galactose. During the time that there is glucose in the media, galactose is not used by the culture because glucose inhibits it. However, when the concentration of glucose arrives to a critical low level, it stops inhibiting galactose´s use and the culture starts to grow up with it.
It may be gluconeogenesis because of the use of ethanol behind the glucose/galactose one.
The strain was able to grow up with both substrates (galactose and ethanol). This can be seen if you look at the decreasing levels of them.
The strain grew up fast because the levels of the different substrates went down easily in comparison to the other strains.
Snf1 mutation
Tpk2 mutation
Snf1 mutation
Tpk2 mutation
Tpk2 mutant
Snf1 mutant
References
Protein phosphorylation and dephosphorylation. Michael J.R. Starks
Online and in situ monitoring of biomass in submerged cultivations. Olsson,L. and J.Nielsen.
How Saccharomyces responds to nutrients. Shania Zaman et al.
So, we got it! Thank you guys!