Synthetic biology of cyanobacteria:

regulation and engineering of chassis

Weiwen Zhang

Laboratory of Synthetic Microbiology

School of Chemical Engineering & Technology

Tianjin University, Tianjin, P.R. China

For “2014 Pacific Rim Summit on Industrial Biotechnology

and Bioenergy”, December 7-9, San Diego, CA

High oil prices

Climate change

National security

Local employment

Green Biofuels and Bioproducts

CO2 + H2O

O2

OH

Renewable

Process

Why Cyanobacteria?

Faster growth

High lipid content (>60-70%)

Amenable for genetic engineering

Grow over a range of temperatures,

pH and nutrient conditions

Synechocystis sp. PCC 6803

Biological questions?

Pathway engineering: novel functions

Chassis engineering: Efficiency? Productivity? Tolerance?

CO2 + H2O

O2

OH

Renewable

Process

Chemicals

“autotrophic cell factory”

CO2

Phenotypes under negative

effects of biofuels

Control

Ethanol

0 200 400 600 800 1000

0

200

400

600

800

10

00

0 200 400 600 800 1000

0

200

400

600

800

1000

0 200 400 600 800 1000

0

200

400

600

800

1000

0 200 400 600 800 1000

0

200

400

600

800

1000

0 200 400 600 800 1000

0

200

400

600

800

1000

0 200 400 600 800 1000

0200

400

600

800

1000

0 200 400 600 800 1000

0

200

400

600

800

1000

0 200 400 600 800 1000

0 200

400

600

800

1000

0 h 24 h 48 h 72 h

0 h 24 h 48 h 72 h

0.25% Butanol 1.5% Ethanol

Systems-level approaches

Although cellular tolerance mechanism to biofuels or organic

solvents well characterized in some native-producing microbes,

very little is know for cyanobacteria

One important finding out of early genome-wide analyses was

that microbes tend to employ multiple resistance mechanisms in

dealing with single biofuel stress

Proteomics

Transcriptomics

Metabolomics

Computational

Cyanobacteria

under biofuels

stress

Targets

Strategies

Research hypothesis -1 ?

Any specific resistance mechanism against

biofuels employed by cyanobacteria?

• Qiao et al., 2012, J Proteome Res. 11:5286-300.

• Qiao et al., 2012, J Bacteriol,194:6358-9.

• Wang et al., 2012, Biotechnol Biofuels. 5:89.

• Liu et al., 2012, Biotechnol Biofuels. 5:68.

• Lei et al., 2012, Biotechnol Biofuels. 5:18.

• Wang et al., 2012, Front Microbiol. 3:344.

• Tian et al., 2013, J Proteomics. 78:326-45.

• Wang et al., 2013, BMC Genomics, 14: 112.

• Zhu et al., 2013, Biotechnol Biofuels. 6:106.

• Qiao et al., 2013, Appl Microbiol Biotechnol. 97: 8253.

• Qiao et al., 2013, Gene, 512: 6-15.

• Huang et al., 2013, Mol Biosystems. 9: 2565-2574.

• Wang et al., 2013, J Proteome Res. 12: 5302–5312.

• Jin et al., 2014, Biotechnol Adv. 32: 541–548

• Wang et al., 2014, Funct Integr Genomics.14:431-440

• Pei et al., 2014, Front Biotechnol Bioeng, 2:48.

• Wang et al., 2014, Microbial Cell Fact. 13:151

3-phosphoglyceric acid Glycine Glycolysis pathway 5

TCA cycle 5

Amino acid biosynthesis 14

Fatty acid metabolism 15

nucleotide metabolism 5

Metabolites identified

Synechocystis under 0.25% butanol stress

gly

coli

c a

cid

L-(

+)

lact

ic a

cid

asp

art

ic a

cid

3-p

ho

sph

og

lyceric

acid

cy

tosi

ne

xa

nth

ine

citr

ic a

cid

met

hy

l pal

mit

ate

Nic

oti

nam

ide

sucr

ose

hy

dro

xyla

min

ela

uri

c a

cid

D-m

alic

acid

py

ruv

ic a

cid

ox

alic

acid

D-g

luco

se-6

-ph

osph

ate

ure

ap

anto

then

ic a

cid

2-m

on

op

alm

itoy

lgly

cer

ol

2-m

on

ost

ear

inm

eth

yl s

teara

tem

yri

stic

acid

pal

mit

ic a

cid

ph

eny

lala

nin

ep

utr

escin

eli

no

leic

acid

cap

ryli

c a

cid

alan

ine

2-h

yd

roxy

pyri

din

eb

enzo

ic a

cid

ph

osp

hor

ic a

cid

pal

mit

ole

ic a

cid

ole

ic a

cid

po

rph

ine

cyti

nd

ine-5

'-m

onop

hosp

hat

egl

uta

mic

acid

2'-

deo

xycy

tid

ine

5'-m

ono

pho

sph

oric

acid

thy

min

egl

uta

mic

aci

d (

deh

ydra

ted)

succ

inic

acid

bet

a-gl

ycer

olp

hos

phat

egl

yce

rol 1

-ph

osph

ate

threo

nin

eu

ra

cil

hy

po

xa

nth

ine

D-(

+)t

reh

alo

sea

den

ine

va

lin

ely

sin

eis

ole

ucin

ele

ucin

eg

lycin

ety

ro

sin

eh

epta

deca

noic

aci

dst

eari

c a

cid

arac

hid

ic a

cid

Th

reo

se

squ

alen

eO

-ph

osp

ho

cola

min

ead

eno

sin

e-5

-mon

oph

osp

hat

e1

-mo

nop

alm

itin

seri

ne

gly

cero

lm

alo

nic

acid

fum

ari

c a

cid

0.4% butanol-

module (red)

(r=0.64, p=0.002)

0.5% isobutanol-

module (blue)

(r=0.67, p=9e-07)

Modules

Nicotinamide Myristic acid

Hypoxanthine

Arachidic acid

Malonic acid

Linoleic acid

L-Lysine

Phenylalanine

Glycerol-1-phosphate

D-malic acid

Metabolomics

WGCNA network analysis

Module identification

Metabolic network

iTRAQ proteomics RNA-Seq transcriptomics

Cross-species network analysis

Multiple aspects of cellular metabolism

induced Heat Shock Proteins

Transporters

Lipid Synthesis and

Membrane Modifications

Cell Mobility

Regulatory Systems

Oxidative Stress Responses

Central Metabolic Process

DnaJ1 and GrpE

NrtCD-like transporters

ABC-type transporter

Na+/H+ antiporter

Cation-transporting ATPase

Squalene hopene cyclase

Glycerol-3-phosphate dehydrogenase

Peptidylprolyl cis-trans isomerase

UDP-N-acetylglucosamine acyltransferase

O-linked GlcNAc transferase

S-layer-RTX protein

CheY subfamily proteins

Periplasmic WD repeat-containing protein involved in cell motility

Response regulators

Sensory histidine kinase

Rehydrin (Slr1198)

Glutathione peroxidase

Ferredoxin

Geranylgeranyl pyrophosphate synthase

Down-regulation of ribosomal proteins, translation elongation factors, translation

initiation factors, translation trigger factor and tRNA synthetase involved in protein

biosynthesis, polymerases involved in DNA and RNA replication, and isocitrate

dehydrogenase and phosphoenolpyruvate synthase etc

Multiple aspects of photosynthesis induced

in Synechocystis cells

4 proteins from photosystem I and 3 proteins from photosystem II

Six ferredoxin-like proteins

Light-harvesting related phycocyanin alpha phycocyanobilin lyase and a

plastocyanin

Multiple cytochromes, such as cytochrome B6-f complex subunit IV (Slr0343),

cytochrome C-550 (Sll0258) and cytochrome C553 (Sll1796)

Two F0F1 ATP synthase subunits

L-cysteine/cystine lyase (Slr2143) involved in ferredoxin Fe-S cluster formation

4 thylakoid membrane-associated proteins involved in ATP formation during

photosynthesis

Tian et al., 2013, J Proteomics. 78: 326-345

Wang et al., 2013, Biotechnol Biofuels. 5: 89

Conformation and proposed hypothesis for enhanced

photosynthetic activity under biofuel stress

PSII PSIB6f

PC

Fd

FNR

Ferredoxin ATP synthase

Plastocyanin

Plastoquinone

Cytochrome

Oxygen-evolving complex

Cytosome

Thylakoid lumen

Slr0172, Slr0823

Ssr2831, Sll0629

Slr1645

Sll1194

Sll1398

Slr0148, Slr1828, Sll1382

Ssl0020, Slr1205, Ssl3044

Sll1663

Slr0343

Sll0258

Sll1796 Sll1322

Slr1330

ADP ATP

NADP NADPH·OH, 1O2

H2O2

H2O2

·OH, 1O2

Thylakoid

membrane

proteins

Slr0729, Slr1796

Slr1034, Ssl2009

Phycobilisome

Chlorophyll a

concentration in cells

Reactive oxygen

species

Qiao et al., 2012, J Proteome Res, 11: 5286-5300.

Proteomics data Pairwise

correlation

Threshold

correlation matrix

PPI network Co-expression

network

Integrated network

870

412

563 19

33

3840

7 1 169

587 10

5

2

421

653 410

2

3

1 5 223

314

310

8 2 1

0 1

528 1444 336

N-starvation 101

38

42 3

4

1069

2

9 102

0

1

48

114 66

0

0

0 2

20

37

1 1 0

0 0

40 216 41

15

0 Butanol

1147

(205) 752

(79)

582

(75)

4166

(1091) 970

(152) 752

(55)

987

(91)

8221

(1696) 36

(6) 61

(7)

A B

C D

Ethanol Ethanol

N-starvation

Hexane Salt

Ethanol Butanol

Hexane

Salt N-starvation

Hexane Salt

Butanol

Enrichment analysis of biofuel-

specific modules

Common-regulated peptides or

proteins Co-expression and PPI network

Enrichment analysis of biofuel-specific

pathways and modules

Pei et al., 2014, Front Bioeng Biotechnol. 2:48.

Research hypothesis -2 ?

Is tolerance regulated by signal transduction

system directly in cyanobacteria?

PCR product

purification

(96-well)

Transformation

(96-well)

Mutant

screening

Mutant

confirmation

Construction of a mutant library of

signal genes in 6803

96-well based mutant construction protocol:

表：课题已构建完成的突变底盘

突变

株种

类

双组分系统反应调控蛋白 转录调控因子 运输

蛋白

未知功

能蛋白

光合

作用

相关

蛋白

数目 46 40 12 13 4

基因

ID

sll1783, slr1305, slr0687, slr1042, slr1760,

slr2041, sll1292, slr1213, slr1214, slr1837,

sll1624, slr2100, sll1879, slr1983, slr1693,

slr0322, slr0474, sll0474, sll0797, sll0789,

slr1584, slr1588, sll1544, slr6040, slr0115,

slr1037, sll0921, slr1594, slr0312, sll0039,

slr2024, sll1708, slr1909, sll0485, slr1982,

slr1041, slr0081, sll0649, sll1330, slr1594,

sll1673, sll0396, sll5059, sll1291, sll0038,

sll0044

sll1205, sll1408, slr1489, sll1392, slr0741,

slr0240, sll0998, sll1286, slr1871, slr1738,

slr1245, sll1957, sll1594, sll1872, ssl0564,

slr0895, sll0594, sll0782, slr0701, sll0030,

slr0527, slr0449, slr1860, slr1861, sll1423,

slr1529, slr0947, ssl0707, sll0690, slr0115,

sll0794, slr1666, sll0792, sll1937, sll1626,

sll1670, slr0724, sll1712, slrr0395, sll0567

slr0040,

slr0041,

slr0042,

slr0043,

slr1295,

sll0689,

sll0064,

sll0493,

slr1512,

sll0834,

sll0672,

ssr2857

sll1638,

sll1130,

sll1418,

sll0872,

sll0630,

sll1734,

slr0729,

slr1847,

ssr1853,

ssr3402,

slr1339,

sll1549,

slr0821

sll1194

sll1398

slr0172

slr1645

表：课题已构建完成的突变底盘

突变

株种

类

双组分系统反应调控蛋白 转录调控因子 运输

蛋白

未知功

能蛋白

光合

作用

相关

蛋白

数目 46 40 12 13 4

基因

ID

sll1783, slr1305, slr0687, slr1042, slr1760,

slr2041, sll1292, slr1213, slr1214, slr1837,

sll1624, slr2100, sll1879, slr1983, slr1693,

slr0322, slr0474, sll0474, sll0797, sll0789,

slr1584, slr1588, sll1544, slr6040, slr0115,

slr1037, sll0921, slr1594, slr0312, sll0039,

slr2024, sll1708, slr1909, sll0485, slr1982,

slr1041, slr0081, sll0649, sll1330, slr1594,

sll1673, sll0396, sll5059, sll1291, sll0038,

sll0044

sll1205, sll1408, slr1489, sll1392, slr0741,

slr0240, sll0998, sll1286, slr1871, slr1738,

slr1245, sll1957, sll1594, sll1872, ssl0564,

slr0895, sll0594, sll0782, slr0701, sll0030,

slr0527, slr0449, slr1860, slr1861, sll1423,

slr1529, slr0947, ssl0707, sll0690, slr0115,

sll0794, slr1666, sll0792, sll1937, sll1626,

sll1670, slr0724, sll1712, slrr0395, sll0567

slr0040,

slr0041,

slr0042,

slr0043,

slr1295,

sll0689,

sll0064,

sll0493,

slr1512,

sll0834,

sll0672,

ssr2857

sll1638,

sll1130,

sll1418,

sll0872,

sll0630,

sll1734,

slr0729,

slr1847,

ssr1853,

ssr3402,

slr1339,

sll1549,

slr0821

sll1194

sll1398

slr0172

slr1645

41 response regulators of TCS

40 transcriptional regulators

Tolerance assay

Butanol tolerance regulated by two-component

signal transduction system Slr1037

slr1037 encodes an orphan

response regulator

Butanol tolerance network regulated by

Slr1037, as revealed by iTRAQ proteomics

50s

30S

RNA

Protein synthesisSll1816, Slr0628, Ssl1784

Ssl3432, Sll1822

Sll1819, Ssr1604

eIF2BSlr0434, Slr0033, Slr0638

Slr1228, Slr1938, Sll0467

Slr1796,

Sll1382

Sll0554,

Slr0233

Electron transport PSIISll1194, Slr1739, Sll1398, Sll1769

Phycobilisome

Slr1495, Ssr3383, Slr1878

PSI

ATP synthase

CT

DNA replication

Slr0020,

Slr2058,

Slr0495

Calvin cycle

PG3

Pyruvate

PEP

F6PR5P

RuBP

Histidine

Purine

G6P

Glycogen

Glycerate

PG2

OxaloacetateSlr1124

Slr1945

Valine Leucine

Slr0229

Aspartate

Lysine

Isoleucine

Threonine

Methionine

NAD(P)

Alanine

Acetyl-CoA

TCA cycle

2-Oxoglutarate

Glutamate

Glutamine

Arginine

Pyrimidine

Proline

Purine

Sll0144 Slr0185

Sll0469, Slr0689

Slr0838,Slr0861

Sll0228

Sll0086

Sll1693

Sll0504, Sll1688Slr1022

Protein fateSll1514 Glutaredoxin

Peptide methionine

sulfoxide reductase

(PMSR)

Glucosylglycerol-

phosphate synthase

Transporters

Two orphan histidine

kinase sll1124 and

slr0222

Molecular mechanism issued by Slr1037

Up-regulated genes

Down-regulated genes

Crude

extract

without

IPTG

Crude

extract

with

IPTG

Purified

His-tagged

Slr1037

slr1037

Chen et al., 2014, Biotechnol Biofuels,7: 89.

Chen et al., 2014, Mol BioSystems,10:1765-74

0 12 24 36 48 60 72

0.03125

0.0625

0.125

0.25

0.5 WT

WT+B0.25

WT.pTX-slr1037

WT.pTX-slr1037+B0.25

OD

63

0

Time (h)

Overexpression

on pTX vector

Overexpression of slr1037 leads to

high tolerance

Ethanol tolerance regulated by transcriptional

regulator Sll0794

Wang et al., 2012, Biotechnol Biofuels. 7:89.

Electrophoretic Mobility Shift Assays (EMSAs)

reveals direct gene targets of Sll0794

Putative sodium-

dependent bicarbonate

transporter(SbtA)

Small heat shock

protein, molecular

chaperon

Carbon dioxide

concentrating

mechanism (CcmK)

Purified

Sll0794

Song et al., 2014, Mol Cell Proteomics,13(12)：Chen et al., 2014, J Proteomics,103:87-102

1 2 3 4 M (kDa)

9572

55

34

43

26

0 1.5 3.0 0 1.5 3.0 0 1.5 3.0 0 1.5 3.0

slr1512 sll1514 slr1838 ssr2061

His6-Sll0794 (μM)

Protein-DNA

complex

Free DNA

Negative

control

Unique tolerance

mechanisms?

C48 C72

C24

B48

B24

B72

PCA analysis for sRNA deep-sequencing data

(A) (B)

Non-coding small RNA involved in tolerance

Small RNA involved in butanol stress

Control

Overexpression (nc255+)

Suppression (nc255-)

Wild type

Km PpsbA nc255

Km PpsbA

nc255

nc255(+) tended to be sensitive under 0.3% butanol (v/v) stress

while nc255(-) was the same as wide type.

Small RNA nc250 involved in butanol

tolerance

Secondary

structure

Sun et al., 2014, Scientific Reports,submitted

Small RNA nc250 involved in butanol

tolerance

Secondary structure and target gene prediction for nc255

(B) Predicted interaction regions ( CopraRNA)(A)

Predicted secondary

structure

1 3.40E-08 slr0847 phosphopantetheine adenylyltransferase

2 0.0004699 sll0821 Phytochrome-like protein cph2 (Bacteriophytochrome cph2)

3 0.00125 sll1770 ABC1-like protein kinase

4 0.002376 slr1619 hypothetical protein

5 0.00328 sll1601 hypothetical protein

Research hypothesis -3 ?

How complex the tolerance regulatory

network in cyanobacteria?

A) B)

C)

0 12 24 36 48 60 72

Time (h)

0.25

0.125

0.0625

0.03125

0.5

OD

63

0n

m

0.25

0.125

0.0625

0.03125

0.5

OD

630

nm

0 12 24 36 48 60 72

Time (h)

0.25

0.125

0.0625

0.03125

0.5

OD

630

nm

0 12 24 36 48 60 72

Time (h)

WT

△ sll1392

WT – 1.9% Ethanol

△ sll1392 – 1.9% Ethanol

WT

△ sll1712

WT – 1.9% Ethanol

△ sll1712 – 1.9% Ethanol

WT

△ slr1860

WT – 1.9% Ethanol

△ slr1860 – 1.9% Ethanol

sll1392: Transcriptional regulator sll1712: Transcriptional regulator

slr1860: Eukaryotic-like protein phosphatases

Ethanol tolerance by three

different regulators

48 h A) B)

72 h

T[2

] 4

2

0

-2

-4

-8 -6 -4 -2 0 2 4 6 8

T[1]

48 h

72 h

T[2

]

4

2

0

-2

-4

6

-6

-8 -6 -4 -2 0 2 4 6 8

T[1] WT-C

Δsll1392-C

Δslr1860-C

Δsll1712-C

WT-E

Δsll1712-E

Δslr1860-E

Δsll1392-E

0.0 -1.0 1.0

WT

C -

1

WT

C -

2

WT

C -

3

s

ll13

92C

- 1

s

ll1

392

C -

2

s

ll1

39

2C

- 3

s

lr1

860

C -

1

s

lr18

60C

- 2

s

lr1

860

C -

3

s

ll17

12C

- 1

s

ll17

12C

- 2

s

ll1

712

C -

3

WT

E -

1

WT

E -

2

WT

E -

3

s

ll13

92E

- 1

s

ll13

92E

- 2

s

ll13

92E

- 3

s

lr18

60E

- 1

s

lr1

86

0E

- 2

s

lr1

86

0E

- 3

s

ll1

71

2E

- 1

s

ll1

71

2E

- 2

s

ll1

71

2E

- 3

UDP-Glucose

ATP

PEP 3PG FBP DHAP F6P G6P R5P NADH AcCOA NAD ADP-GCS NADPH NADP RiBP AKG GAP

ADP

COA AMP GLU OXA FUM

WT

C -

1

WT

C -

2

WT

C -

3

s

ll13

92C

- 1

s

ll1

392

C -

2

s

ll1

39

2C

- 3

s

lr1

860

C -

1

s

lr18

60C

- 2

s

lr18

60C

- 3

s

ll17

12C

- 1

s

ll17

12C

- 2

s

ll1

71

2C

- 3

WT

E -

1

WT

E -

2

WT

E -

3

s

ll13

92E

- 1

s

ll13

92E

- 2

s

ll13

92E

- 3

s

lr18

60E

- 1

s

lr1

860

E -

2

s

lr1

860

E -

3

s

ll17

12E

- 1

s

ll17

12E

- 2

s

ll1

712

E -

3

-1.0 0.0 1.0

3PG PEP RiBP G6P NAD F6P AMP R5P AcCOA

NADH FUM OXA NADPH NADP ADP-GCS UDP-Glucose ATP ADP COA FBP GAP

AKG GLU

DHAP

LC-MS metabolomics showed

different physiological status

glyceric acidtaloseD-glucose-6-phosphate

3-

hydroxypyridine squalene

2-amino-1-phenylethanolglycolic acid

L-threoninebenzoic acidporphinespermidinecaprylic acid

dioctyl-phthalatearachidic acidD-malic acidpalmitoleic acid

2-hydroxypyridineureabenzene-1,2,4-triolmethyl palmitatemethyl stearatemethyl-beta-D-galactopyranosidepyruvic acid

L-serinestearic acidpalmitic acid

L-(+)-lactic acidsucroseglycerol 1-phosphate

D-(+)-trehaloseoleic acidlinoleic acidphytolglycine

adenosineL-glutamic acidL-pyroglutamic acidcapric acid

maleic acidsuccinic acid

heptadecanoic acidlauric acid

phosphoric acid

glycerol

M1

ethanol stress

r = -0.54, p = 0.006

48h

ethanol stress

r = 0.64, p =7e-04△sll1712

r= 0.5, p = 0.001

M2

ethanol stress

r = 0.63, p = 0.001

△sll1712

r = 0.53, p = 0.008

M3

myristic acid

L-(+)-lactic acidpalmitoleic acid

methyl-beta-D-galactopyranosideglycolic acid3-hydroxypyridine

maleic acidL-serine

benzene-1,2,4-triolphosphoric acid

D-glucose-6-phosphate

urea

adenosinephytollinoleic acidglycerol 1-phosphatearachidic acid

benzoic acidoleic acidstearic acidpalmitic acid

heptadecanoic acidglycine

squalene

porphine

D-malic acidsuccinic acidsucrosemethyl palmitatemethyl stearate2-amino-1-phenylethanolpyruvic acid

glyceric acid

caprylic acid

D-(+)-trehalosespermidine

capric acidglycerol talosemyristic acid

lauric acidL-glutamic acidL-pyroglutamic aciddioctyl-phthalate2-hydroxypyridine

L-threonine

72h

M5

ethanol stress

r=-0.78, p=7e-06

M7

△slr1860

r=0.53, p=0.008

ethanol stress

r=0.73, p=5e-05

△sll1712

r=0.58, p=0.003

M6

ethanol stress

r=0.65, p=6e-04

△slr1860

r=0.53, p=0.007

M4

A)

B)

Zhu et al., 2014, Mol Biosyst,Accepted

Mutant-specific

metabolic

modules found!

WGCNA Metabolic

networks constructed

Low productivity of biofuels in cyanobacterial may be at least

partially due the high toxicity of biofuel products to the

cyanobacterial hosts.

Systems biology based approach used to uncover the unique

tolerance mechanisms of Synechocystis to several biofuels products.

Possible tolerance regulatory mechanism involved two-

component signal transduction systems and transcriptional regulators

identified

The discoveries used to guide chassis engineering.

Summary

Acknowledgments

Laboratory of Synthetic Microbiology

Tianjin University

National “973 Program”

and “863 program”

National Science

Foundation of China

Tianjin University “985”

Program”

Thanks!

Weiwen Zhang, Prof. Dr.

Laboratory of Synthetic Microbiology

School of Chemical Engineering & Technology

Tianjin University

wwzhang8@tju.edu.cn

Office: 022-2740-6394