Characteristics of the kinase mutant TPK2 in bioreactors

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

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

Time (h)

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

Time (h)

glucose

galactose

ethanol

Accumulation of substrates in cells

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)

0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 50,00

Time (h)

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

Time (h)

glucose

Average Dry Weight

CO2 Analysis

Variance of the CO2 respect time

0 500 1000 1500 2000 2500 3000

Time (min)

Variance of the CO2 normalised respect time

0 500 1000 1500 2000 2500 3000 3500

Time (min)

Variance of the CO2 respect time

0 500 1000 1500 2000 2500 3000

Time (minutes)

Variance of CO2 normalised respect time

0 500 1000 1500 2000 2500 3000

Time (minutes)

Specific CO2 growth rate

Max specific growth rate

y = 0,1747e0,1945x

0,000 5,000 10,000 15,000 20,000 25,000 30,000

Time (h)

Correlation between O.D and DW

y = 0,545x + 0,2733

0 1 2 3 4 5 6 7 8

Max Specific growth rate on glucose

y = 0.1057e0.4407x

2 3 4 5 6 7 8 9

Time (h)

Cell density in the fermenter as time increases

012345678

0,001,0

T ime (h)

R eal OD600

AverageWeight (*)

Yield Coefficients (Ysx)

Ysx on glucose

y = 0,218x + 0,3879

0,0000 2,0000 4,0000 6,0000 8,0000 10,0000 12,0000 14,0000 16,0000

Glucose (g)

Ysx on glucose

y = 0,2378x + 0,0141

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)

mass (

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 2 4 6 8 10 12 14 16

Galactose used (g)

Ysx on galactose

y = 0,2084x + 0,0314

0 0,1 0,2 0,3 0,4 0,5

Galactose (Cmols)

mass (

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)

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)

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

0,0000 1,0000 2,0000 3,0000 4,0000 5,0000 6,0000 7,0000

Glucose used (g)

CO2 Analysis Variance of the CO2 respect time

0 500 1000 1500 2000 2500 3000

Time (min)

Variance of the CO2 normalised respect time

0 500 1000 1500 2000 2500 3000 3500

Time (min)

TPK2 vs Wild Type

Varianc e of C O2 normalis ed (T E AM 1)

0 5 10 15 20 25 30 35 40 45 50

T ime (h)

CO2 analysis

Variance of CO2 normalised

0 500 1000 1500 2000 2500 3000

Time (minutes)

TPK2 vs Wild Type (Ysx)

Ysx on glucose

y = 0,2378x + 0,0141

0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0,5000

mass (

Ysx on galactose

y = 0,2084x + 0,0314

0 0,1 0,2 0,3 0,4 0,5

Galactose (Cmols)

mass (

Ysx on substrates (TEAM 1)

y = 0,1288x + 0,0682

y = 0.3407x + 0.0008

0 0,05 0,1 0,15 0,2 0,25 0,3

Substrates used (Cmol)

mass (

Glucose(Cmol)

Galactose(Cmol)

TPK2 vs Wild Type

Yse on glucose (Ethanol produced

Y s e on g luc os e (T E AM 1)

y = 0.5599x + 0.0698

0 0,5 1 1,5 2G luc ose used (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)

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)

Y s e on g alac tos e (T E AM 1)y = 0.5292x + 0.529

0,0 2,0 4,0 6,0 8,0 10,0 12,0

G a la c tose used (g /l)

Yse on galactose (Ethanol produced

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 0,4 0,45 0,5

S ubs trates us ed (C mol)

G luc os e(C mol)

G alac tos e(C mol)

Ysc on glucose

y = 0,3795x - 0,2286

0,0000 1,0000 2,0000 3,0000 4,0000 5,0000 6,0000 7,0000

Glucose used (g)

Ysc on glucose (CO2 g)

TPK2 vs SNF1

Variance of CO2 normalised (TEAM 2)

0 10 20 30 40 50 60

Time (h)

Variance of CO2 normalised

0 500 1000 1500 2000 2500 3000

Time (minutes)d

CO2 analysis

TPK2 vs SNF1

Ysx on glucose (TEAM 2)

y = 0.1347x + 0.0094

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05

Glucose (c-mol)

mass (

Ysx on glucose

y = 0,2378x + 0,0141

0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 0,3000 0,3500 0,4000 0,4500 0,5000

mass (

Ysx on glucose (Biomass Cmol)

TPK2 vs SNF1

Yse on glucose (TEAM 2)

y = 0.2116x + 0.2557

0 1 2 3 4 5 6 7 8 9 10

Glucose (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)

Yse on glucose (Ethanol produced

TPK2 vs SNF1

Ysc on glucose (TEAM 2)

y = 0.5157x - 0.0888

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

Glucose (g)

Ysc on glucose (CO2 g)

Ysc on glucose

y = 0,3795x - 0,2286

0,0000 1,0000 2,0000 3,0000 4,0000 5,0000 6,0000 7,0000

Glucose used (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!

Characteristics of the kinase mutant TPK2 in bioreactors

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