Omics analysis revealed multiple stress responses of lager

Omics analysis revealed multiple stress responses of lager yeast in the process

of autolysis Wang Jinjing

Lab of Brewing Science and Technology Jiangnan University, China

2016.4.22

Autolysis of lager yeast often happens in post-fermentation period, which is a global and irreversible process.

Background

Background

Influence of yeast autolysis on beer quality

Foam stability

Colloid stability

Flavor stability

Background

Autolysis process of lager yeast

What causes the autolysis? 1. Wort quality

2. Inorganic ions

3. Temperature

4. pH value

5. Tank pressure Non

-bio

logi

cal f

acto

rs

Biological factors 1. Yeast strain

2. Yeast generation

3. Inoculum of yeast

4. Yeast recovery

5. Microbial contamination

Autolysis Evaluation

How to evaluate autolysis?

Release fatty acids, proteins

vs. growth

Morphology Study

Oxidation-Reduction Potential vs. growth

Nucleic acids vs. death rate

Strain Protein concentration (mgN/L) 0 h 24 h 48 h 72 h

Q2

total protein / 260.31 426.67 726.56

high molecular protein / 7.31 21.34 52.44

middle molecular protein / 22.88 78.45 137.25

low molecular protein / 230.12 326.88 536.88

G-03

total protein / / 324.68 618.31

high molecular protein / / 7.09 15.44

middle molecular protein / / 31.58 69.75

low molecular protein / / 286.01 533.13

C-03

total protein / / 298.46 548.63




F52

total protein / / 248.46 468.63




Protein analysis of yeast autolysates

24 hr 48 hr

72 hr

Oxidation-Reduction Potential vs. Incubation Time

Fig.4 Changes of oxidation-reduction potential of brewing yeast strain during fermentation

Time/ (hr)

stable

unstable

Nucleic acid vs. Death rate

Time (h) Q2 (%) G-03 (%) C-03 (%) F52 (%)

0 0.00 0.00 0.00 0.00

12 4.15 4.13 3.73 2.88

24 8.91 9.89 6.72 5.49

36 11.81 15.11 11.08 8.86

48 13.68 17.00 13.05 10.41

60 15.98 19.62 15.06 12.48

108 32.34 35.45 30.53 17.91

156 56.41 56.21 51.24 23.52

204 79.26 79.85 75.46 28.50

252 97.89 98.41 96.38 35.31

1.8

1.82

1.84

1.86

1.88

1.9

1.92

1.94

1.96

1.98

2

12 24 36 48 60 108 156 204 252

Nuc

leic

aci

d(A2

60/A

280)

Time(h)

Q2

G-03

C-03

F52

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48 60 108 156 204 252

Deat

h ra

te(%

)

Time(h)

Q2

G-03

C-03

F52

Death rate of different strains during autolysis

Nucleic acid releasing of different strains during autolysis

Fig.5 Released nucleic acid and death rate of yeast during autolysis

Time (h) Q2 G-03 C-03 F52

12 1.872 1.903 1.867 1.814

24 1.887 1.906 1.942 1.905

36 1.901 1.912 1.961 1.938

48 1.932 1.929 1.969 1.953

60 1.949 1.931 1.969 1.963

108 1.960 1.944 1.968 1.966

156 1.964 1.962 1.969 1.965

204 1.967 1.974 1.969 1.968

252 1.971 1.976 1.970 1.970

Development of autolysis index measurement in lager yeast

Verification of autolysis index measurement in different lager yeast

Autolysis index development

Beginning from 17 hr, all the strains’ Nucleic acids/death rate varied in the range of 45-60, declined along with the increase of death rate.

Autolysis genes

2013/5/22

RNA

qRT-PCR

Yeast sample

microarray

Protein

2DE-PAGE SDS-PAGE

12.21%

18.26%

69.54%

19.02%

14.48%

66.50%

a b

Gene regulation pattern during autolysis

Down-regulation: fold≤0.5 Up-regulation: fold≥2 Other: stable expression a. lager yeast Q2； b. lager yeast 5-2

Microarray analysis of two lager yeast

Up-regulated gene

Down-regulated gene

Stable gene

上

稳

5.25%

1.18%1.86%

3.21%

3.05%

2.37%1.52%

2.54%

9.14%

1.52%3.55%

1.69%4.91%

8.8%

8.8%

9.64%

15.4% 15.57%

cellular process physiological process metabolism cell part cell organelle biological regulation organelle part regulation of biological process catalytic activity macromolecular complex response to stimulus binding establishment of localization localization transporter activity membrane-enclosed lumen other items

6.29%

1.24%1.46%1.57%1.57%2.25%

2.25%

3.48%

3.6%4.27%

4.38%4.72%4.94%

6.97%

6.97%

9.89%

16.74%17.42%

a

b c

cellular processphysiological process

metabolismcell part

cellorganelle

biological regulationorganelle part

regulation of biological processcatalytic activity

macromolecular complexresponse to stimulus

bindingestablishment of localization

localizationtransporter activity

membrane-enclosed lumenother items

0 10 20 30 40 50 60Gene number

a. GO of 196 genes with the same regulation pattern (black: up-regulation; red: down-regulation)

b. GO of 99 up-regulated genes c. GO of 97 down-regulated genes

Genes with similar regulation pattern

Red: up-regulated gene Green: down-regulated gene

CPD1 FRE7 YHB1

Respiration MBA1

MRPL1 COQ10 PPA2 SLS1

Pentose phosphate pathway GND1

Energy metabolism GDB1,GPH1 TPS3,UGP1

PGM2 Glycolysis/glyconeogenesis

ARO10,GPM2, PYK2, ACS2

ADH2

Fatty acid metabolism

EHD3

Electron transportation

Eighteen genes of total twenty “energy” related genes are down-regulated during autolysis process, indicating the energy metabolism was depressed.

Genes with similar regulation pattern

RPI1 AVO1 RLM1

DFG5 KNH1 WSC2

SVS1 SSA2 YPS6 FIT2

Cell wall component

Cell wall synthesis

DNA damage

NEJ1,PPH3 UFO1,RAD2 RAD16,RSC2

RAD9

Stress

ASG1,SSA3 CCH1,GRX6 TPS3, GND1 SSA2,MGA1 TSA2,YHB1 LAS17,ISC1 CIN5,SVS1

Heat shock WSC2

Starvation

PHM8

pH

NRG1

Cell wall related genes

Stress response related genes

Genes with similar regulation patterns

Genes related to the cell wall reconstruction were up regulated. Genes related to the cell wall components were down regulated. Results indicated protein synthesis were depressed during autolysis.

25 genes were found to be related to cell stress responses. 12 genes related to oxidized stress, osmotic pressure, chemical stress, heat shock were down regulated. 13 genes related to DNA damage, pH, starvation response were up regulated.

Red: up-regulated gene Green: down-regulated gene

Gene Symbol Regulate Ratio Gene Title

SSA3 up 12.25 ATPase involved in protein folding and the response to stress; plays a role in SRP-dependent cotranslational protein-membrane targeting and translocation; member of the heat shock protein 70 (HSP70) family; localized to the cytoplasm

CIN5 down 0.12 Basic leucine zipper (bZIP) transcription factor of the yAP-1 family; physically interacts with the Tup1-Cyc8 complex and recruits Tup1p to its targets; mediates pleiotropic drug resistance and salt tolerance; nuclearly localized under oxidative stress and sequestered in the cytoplasm by Lot6p under reducing conditions

ISC1 down 0.14 "Mitochondrial membrane localized inositol phosphosphingolipid phospholipase C, hydrolyzes complex sphingolipids to produce ceramide; activated by phosphatidylserine, cardiolipin, and phosphatidylglycerol; mediates Na+ and Li+ halotolerance"

LAS17 down 0.15 "Actin assembly factor, activates the Arp2/3 protein complex that nucleates branched actin filaments; localizes with the Arp2/3 complex to actin patches; homolog of the human Wiskott-Aldrich syndrome protein (WASP)"

SSA2 down 0.11

"ATP binding protein involved in protein folding and vacuolar import of proteins; member of heat shock protein 70 (HSP70) family; associated with the chaperonin-containing T-complex; present in the cytoplasm, vacuolar membrane and cell wall; 98% identical with Ssa1p, but subtle differences between the two proteins provide functional specificity with respect to propagation of yeast [URE3] prions and vacuolar-mediated degradations of gluconeogenesis enzymes"

TPS3 down 0.16 "Regulatory subunit of trehalose-6-phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; expression is induced by stress conditions and repressed by the Ras-cAMP pathway"

YHB1 down 0.10 "Nitric oxide oxidoreductase, flavohemoglobin involved in nitric oxide detoxification; plays a role in the oxidative and nitrosative stress responses"

Genes response to stress

a. 264 genes were regulated oppositely in two strains with different autolytic rates. Black: genes up regulated in 5-2 but down regulated in Q2; Red: genes up regulated in Q2 but down regulated in 5-2

b. GO of genes up regulated in 5-2 but down regulated in Q2.

c. GO of genes up regulated in Q2 but down regulated in 5-2.

0.9%1.08%

6.32%

1.81%2.17%1.81%

2.53%

3.79%

6.86%

4.69%2.17%

5.42%7.58%

7.58%

11.37%

17.15%16.79%

cellular process physiological process metabolism cell part cell organelle biological regulation organelle part catalytic activity macromolecular complex binding establishment of localization localization membrane-enclosed lumen other items response to stimulus negative regulation of biological process

11.11%

1.39%

4.17%

4.17%

2.78%2.78%

4.17%4.17%

2.78%6.94%

8.33%

8.33%

8.33%

15.28% 15.28%

cellular processphysiological process

metabolismcell part

cellorganelle

biological regulationorganelle part

catalytic activitymacromolecular complex

bindingestablishment of localization

localizationmembrane-enclosed lumen

other itemsresponse to stimulus

negative regulation of biological process

0 10 20 30 40 50 60 70 80 90 100Gene number

a

b c

Physiological process, cellular process, metabolism, cell components, and catalytic activity represented 67.33% of the total genes. Ribosomal function related genes accounted for 31.95% of the total genes.

Genes with different regulation patterns

2DE page analysis of cellular protein of Q2

Proteomic analysis of lager yeast

Death rate=10% Death rate=0%

Proteomic analysis of lager yeast

Blue: up-regulated proteins ； Red: new proteins emerging during autolysis Green: the proteins in PAGE b are the modified product after translation of proteins in PAGE a.

a b Stress response

Ssb1，Hsp26 Rhr2，

YJL055w Ubc4，Sod2

Sod1

Carbon hydrates and energy metabolism

Suc2，Dap1，Sec53，Ipp1，Ara1，Lsc2， Eno2，Pdc1，Fba1，Ald4，Qcr2，Tpi1，

Cor1，Erg10，Gpd3，Pgk1，Adh2

Unknown function

Ycp4 Protein

processing

Amino acid metabolism

Sam2，Spe3 Arg1，Dug1 Gdh1，Leu2 Pro2，Ilv5

Sba1，Rpt5 Pre8，Prb1

Tfs1

Nucleotide metabolism

Cdc33，Adk1 Guk1

Stress response

Tsa1

Carbon hydrates and

energy metabolism

Adh1

Protein processing Protein

hydrolysis Pep4，Prc1

Pup3

Rpk1 Tom40


Gsv3 Atp14，Ntf2 Ade17，Cof1 Cpr1，Rim1

Gsp1

Central carbon metabolism

The synthesis of carbon hydrates was depressed during autolysis, so was energy metabolism.

a. Arginine and proline metabolism network；

b. Valine, isoleucine, cysteine and methionine metabolism network

a b


These proteins’ expression would inhibit the synthesis of amino acids and caused the shortage of nutrient.

Proteins response to stress

Protein Description

Ssb1 Stress-Seventy subfamily B

ATPase involved in protein folding, export of ribosomal subunit from nucleus, regulation of translation fidelity, rRNA processing; associated with polysomes and localized in the cytoplasm

Hsp26 Heat Shock Protein Response to heat; Small heat shock protein (sHSP) with chaperone activity; forms hollow, sphere-shaped oligomers that suppress unfolded proteins aggregation;

Ubc4 UBiquitin-Conjugating

Proteasome complex subunit that binds and transfers ubiquitin; involved in protein polyubiquitination and cellular response to heat as well as ubiquitin-dependent protein catabolism via mutlivesicular body sorting pathway

Rhr2 Glycerol-3-Phosphate Phosphatase

Constitutively expressed DL-glycerol-3-phosphate phosphatase; also known as glycerol-1-phosphatase; involved in glycerol biosynthesis, induced in response to both anaerobic and osmotic stress; GPP1 has a paralog, GPP2, that arose from the whole genome duplication

Sod2 SuperOxide Dismutase

Mitochondrial manganese superoxide oxidoreductase involved in the metabolism of reactive oxygen species, cell aging, and the response of non-dividing cells oxidative stress

Sod1 SuperOxide Dismutase

uperoxide dismutase involved in copper and zinc homeostasis, superoxide metabolism, cell aging, cell wall organization, regulation of cellular respiration, and regulation of transcription in response to oxidative stress; localized to the nucleus, cytosol, and mitochondrial intermembrane space

YJL055w Uncharacterized Putative protein of unknown function; functions together with HAM1 to mediate resistance to 5-FU; specifically reduces the incorporation of 5-FU into RNA, without affecting uptake or incorporation of uracil into RNA; proposed to be involved in the metabolism of purine and pyrimidine base analogues; deletion mutants are sensitive to HAP and AHA

Genes’ expressions in mRNA level and protein level were

Regression between two kinds of omics analysis

Modifications on yeast autolysis genes

RLM1(UBC4)

RLM1 and UBC4 modifications

PCR UBC4

RLM1

Genomic DNA

pMD19T-uprkl7419 bp

SphI

NheI

ApaIKpnI

SalI

SpeINotI

PO

lacZ

ori

AmpR

lower arm

KanMX

PGK1upper arm

RLM1

NotI ApaI

upper armPGK1

UBC4

lower armKanMX

NotI ApaI

upper armPGK1

RLM1KanMX

lower arm

pMD19T-upukl6092 bp

SphI

NheI

ApaIKpnI

SalI

SpeINotI

P O

lacZ

ori

AmpR

lower arm

KanMX

PGK1

upper arm

UBC4

Knocking out

Recombinant strain Autolytic parameters

H5H-ubc4ΔH/ubc4

H5H-rlm1ΔH/rlm1

Recombinant strain Autolytic parameters

Knocking out of RLM1 and UBC4

RLM1(UBC4) 45bp + pUG6 19bp RLM1(UBC4) 46bp + pUG6 22bp

Primer LF Primer LR

RLM1(UBC4) upper RLM1(UBC4) lower

RLM1(UBC4)

loxP-KanMX-loxP

loxP-KanMX-loxP

pUG64009 bp

KanMX

loxP

loxP

AmpR

TEF

TEF

knock out of RLM1 and UBC4 genes

Positive recombinant

strain

Overexpression of RLM1andUBC4

PCR

PCR

Genomic DNA

UBC4 RLM1 PGK1 Upper arm Lower arm KanMX

pMD19T-simple2692 bp

PO

lacZ

ori

AmpR

pMD19T-uprkl7419 bp

SphI

NheI

ApaIKpnI

SalI

SpeINotI

PO

lacZ

ori

AmpR

lower arm

KanMX

PGK1upper arm

RLM1pMD19T-upukl

6092 bpSphI

NheI

ApaIKpnI

SalI

SpeINotI

P O

lacZ

ori

AmpR

lower arm

KanMX

PGK1

upper arm

UBC4

TA clone

NotI ApaI

upper armPGK1

UBC4

lower armKanMX

NotI ApaI

upper armPGK1

RLM1KanMX

lower arm

Not I，Apa I

pUG64009 bp

KanMX

loxP

loxP

AmpR

RLM1overexpression

UBC4 expression

Resistance analysis of recombinant strains

H5

H-rlm1Δ

H/rlm1

NaCl (1mol/L)

Micafungin (30 ng/mL)

Fluorescer (1mol/L)

H5

H-rlm1Δ

H/rlm1

22°C 28°C 37°C

a b Effect of RLM1 gene modification on yeast autolysis

Table 1 expression levels of RLM1gene in two lager strains

Strains Death rate=0% Death rate=10%

Q2 138.85 781.72

5-2 115.61 581.38

RLM1 gene had negative impact on yeast autolysis, increased expression of RLM1 gene would accelerate the autolysis process.

Resistance analysis of recombinant strains

( )

H5

H-ubc4Δ

H/ubc4

( )

NaCl (1mol/L)

Micafungin (30 ng/mL)

Fluorescer (1mol/L)

H5

H-ubc4Δ

H/ubc4

22°C 28°C 37°C

a b

Table 2 Expression levels of UBC4 gene in two lager strains

Strain Death rate=0% Death rate=10%

Q2 3385.27 1421.81

5-2 3490.65 1756.15

UBC4 gene had positive impact on yeast autolysis, increased expression of UBC4 gene would slow the autolysis process.

Effect of UBC4 gene modification on yeast autolysis

Special thanks to… Prof. Li Qi Dr. Xu Weina Mr. Chen Xi Ms. Li Xin’er

Wang Jinjing, Ph.D School of Biotechnology, Jiangnan University No. 1800 Lihu Ave, Wuxi, Jiangsu, 214122 P. R. China [email protected]

Thank you for your attention!

Acknowledgement

mailto:[email protected]

Documents

Omics analysis revealed multiple stress responses of lager