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Synthetic Biology in China – About the “Industrialization of Biology”

Scientific Implication Technology Innovation

Engineering Integration Industrial Application

The National Academies, Washington, DC, USA May 28, 2014

Key Laboratory of Synthetic Biology (KLSynB)

Institute of Plant Physiology and Ecology (IPPE)

Shanghai Institutes for Biological Sciences (SIBS)

Chinese Academy of Sciences (CAS)

Guo-Ping ZHAO (email: gpzhao@sibs.ac.cn)

Outlines

1. Synthetic biology in China: Foundation and Initiation – Overview

2. Historical Aspect: Starting from fermentation – Classical microbiology and biotechnology

3. Fundamental Aspect: Bridging by genomics – Life “omics” and systems biology

4. Innovation Aspect: New era of synthetic biology – Synthetic engineering of biological systems

5. Organizational Aspect: Institutional reform and organization – KLSynB

Chinese

Foundations of

SynBio

Total synthesis

of Bovine

Insulin and

Yeast tRNAAla

Genomics

“Omics”

Structure Biology

Chemistry

Biochemistry

Bioengineeting/Biotechnology/ Metabolic engineering

Computational biology

Foundations

2005: First review. Participation of iGEM 2008: Xiangshan Symposium (NSFC CAS) Key laboratory (KLSynB, CAS) 2009: Oriental Symposium (Shanghai CAE) 2010: State key program for basic research (Cell factory, chassis, minimal genome) 2011: State key program for basic research (Parts, modules, pathways, networks) Initiation

Bio- informatics

Synthetic Biology projects

funded by the 973 program

National Basic

Research

Program of China

973

C h i n a

Artificial cell factory (chemicals)

Photosynthesis and artificial leaf

Artificial systems for high-yield production

of microbial drugs

New functional biodevices (drug and food)

New pathways for biological materials

Standardization of biological components,

modules, construction of module library

Industrial, agricultural or medicine

applications

Challenge of the century: Sustainable

Development China imports: • 1/3 of “agriculture

products” • 1/2 of natural gas and oil • Economical issues

• Pollution of Air, Water and Garbage in China is probably one of the worst in the world. (Environmental issues)

• Severe problems in desertification, water and soil erosion, and loss of biodiversity. (Ecological issues)

• The Key Challenge：Imbalance b/w Population + development vs Nature resource supply + regeneration

World Population

Clothing

Housing Food

Travel Drinking

Health and medical care

for all the people

Historical Aspect: Starting from fermentation – Classical microbiology and biotechnology

• Fermentation and classical genetics

• Physiology

• Biochemistry

• Molecular genetics

A

E

B

Corn

Seed

water

EA2018 CCTCC M94061 the 721 strain, industrialized

Clostridium acetobutylicum

EA2018 CCTCC M94061

Ni and Sun, BBSRC China partner Workshop, 2009

Soil ↓ (66% butanol) (9.69 g/L solvent) ↓EMS mutation selection/screening ↓ EB20 (67.2% butanol) (13.09 g/L solvent) ↓ EMS mutation selection/screening ↓ EA2018 (69.7% butanol)

(19.163 g/L solvent)

ABE semi-continuous fermentation started in the 1950’s

ATCC 824：60% n-butanol

Shao, L. et al 2007. Cell Res

http://www.sigmaaldrich.com/sigma/general%2

0information/targetron_poster.pdf

Gene knock-out in Clostridium acetobutylicum

buk: butyrate kinase

solR: “sol operon repression gene”?

+37% +44%

pSY6-buk

pSY6-solR

pJC4BK & pDHSR: non-replicative integration plasmid

pSY6-buk & pSY6-solR: TargeTron plasmid

Comparison of DNA integration efficiency b/w non-replicative plasmid

versus Targetron

● Genomics: Genome sequencing and functional genomics Comparative and evolution genomics

● Life “Omics” and systems biology: Transcriptome and expression regulation Metabolome and metagenome Proteome and acetolome

Fundamental Aspect: Bridging by genomics – Life “omics” and systems biology

Primary Sequence

100kb 100Mb

1st ABI 37003730 MegaBase1000 4000

Comparative genomics Evolution genomics

1% HGP BAC-based sequencing

Microbial Genomes 101Mb

400Mb 102Mb

2nd First Roche/454

Solexa=Illumina

Ion Torrent

3rd PacBio

Single cell genomics Pan-genomics Meta-genomics

Comparative genomic and transcriptomic analyses revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018 Shiyuan et al. (2011)

pSY6-adc

Emr

Jiang Y. et al 2009. Metab Eng

adc knock-out

No acetone production, 80% butanol (2008)

Strain medium

Butanol

%

2018p P2 71±1

2018p P2/CaCO3 72±0.6

2018p P2/CaCO3/methyl viologen 71.6±0.1

2018adc P2 80±0

2018adc P2/CaCO3 74.3±0.4

2018adc P2/CaCO3/methyl viologen 82±2

adc：acetoacetate decarboxylase

adc

802 Strain

Innovation Aspect: New era of synthetic biology – Synthetic engineering for biological systems

• Systems biology and synthetic biology

• Innovation in enabling technology

• Towards engineering:

Parts, modules, circuits, chassis cells

• Towards application:

Serving social/economical developments

Synthetic biology: Discovery → Innovation

Core of Molecular Biology

Systems Biology

Synthetic Biology

Genome Metabalome Proteome Transcriptome

1990 2000 Initiation

Reverse engineering

Bottom-up strategy Forward engineering

DNA mRNA Protein Metabolites

Transcription Translation Catalysis

Top-down strategy

Parts

Devices

Modules

Pathway

Network

Regulation

Discovery phase

Innovation phase

Core of “Omics”

Enabling Technology

• Quantitative biology technology:

Measuring and quantitation

• Bioinformatics technology：

Analysis and design

• Bioengineering technology：

Standardized registry

• DNA synthesis+recombination technology：

High efficiency and accuracy

• Systems biology technology：

Tuning and optimization

Jiang Y. unpublished

Reconstruction of butanol pathway: 95% butanol (2010)

EA2018

901 Strain

Feedstock dominates the variable cost for solvent production

Distribution of variable cost for solvent production (data from NCPC)

Butanol price (RMB/t) oil price $/barrel

12 biobutanol plants restored since 2006

• Not to use grains for the production of commodity

• Not to use gasoline for the production of food

Gu Y. et al 2010. BMC Genomics

Xylose metabolism of Clostridium acetobutylicum

木糖利用

Identification of xylose-catabolic genes and reconstruction of

xylose-metabolic pathway in C. acetobutylicum

pIMP1-TAL

5758 bp

bla

mslR

tal

ori

ptb promotor

BamHI

Sma I

xylA xylB tal tklxylA xylB tal tkl

0

5

10

15

20

25

wild type wild type mutant mutant

g/L

Significant improvement in

xylose utilization

Y. Gu, et al. BMC Genomics, 2010

Bioinformatic analysis and identification

of genes involved in xylose metabolism

Corn stover

Steam

Continuous Sterilization

Water

Fermentation Pretreatment

acid

hydrolysis

strain

cellulase

Distillatio

n

4-5% sugar

0.275

0.34

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

8052 8052xylRxylT

Solv

en

t yi

eld

82% of the theoretical yield

WO2012159571

CIBTS0795*

corncob

n-Butanol from Corn Stover Hydrolysate/corncob by engineered Clostridium

C5(pentose) yeast for lignocellulosic ethanol

ETHANOL

Hexose Pentose

Inhibitors

2G Ethanol Pilot of COFCO in Zhaodong

Metabolic engineering enables Saccharomyces cerevisiae to utilize xylose

Xylose (in)

Xylitol Xylulose

D-Xylulose-5-P

Glyceraldehyde-3-P

Xylose

Pentose phosphate pathway

non-oxidative enzymes

(RPE1, RKI1, TAL1, TKL1)

Ethanol (in)

XI

GXF1

XKS1

Ethanol

Na+

Formate

CO2 + NADH

FDH

HMF & Furfural

Organic acids

Phenols

Salts

Gly

coly

sis

Inhibitors

Na+

SO42-

Diao BMC Biotechnol in revision

……

Evolutionary engineering significantly enhance xylose consumption rate in rich medium

Xy

lose

co

nc.

g/l

Evolution time, h

Consumed Residual

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

190 352 430 573 712 905 1074 1175 1340 1508 1681 1840 1944 2057 2116 2183 2223 2248 2272

Diao BMC Biotechnol 2014

Evolutionary engineering significantly enhance xylose consumption rate in the presence of inhibitors

Inh

ibit

or

con

c. %

SO42-

012.52537.55062.57587.5100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Na+

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xylo

secon

c. g/l

3 8 35 55 59 69 73 75 91 97 103 109 113 119 141 147 168 172 178

No. of transfer (generations)

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xy

lose

con

c. g

/l

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Residual Consumed

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Xy

lose

con

c. g

/l

Plants of biobutanol and 2G ethanol in China

COFCO (Zhaodong)

500 t/y

25-31

COFCO (Anhui)

5,000 t/y

Shandong Longlive

50,000 t/y

Jinan Shengquan

20,000 t/y

Shandong Zesheng

3,000 t/y

Henan Tianguan

30,000 t/y

10,000 t/y

Laihe-Rockley

100,000 t/y

Guangxi Jinyuan

50,000 t/y

Jiangsu Lianhai

50,000 t/y

Jiangsu Jinmaoyuan

30,000 t/y

Cathay (Jilin)

30,000 t/y

10000 t/y

Tangshan Jidong

5,000 t/y

Heilongjiang Haohua

5,000 t/y

Lianyungang Lianhua

40,000 t/y

Hebei Jizhou

3,000 t/y

Jilin Zhonghai

5,000 t/y

Jilin Fuel Ethanol

3,000 t/y, planned

Zhongketianyuan

Engineered strains ready for pilot scale lignocellulosic biofuel production

Xylose

Ethanol

XylT

D-Glucose-P

FBP

HprK/P

Hpr-P-His

Hpr

Hpr-P-Ser

ATP

ADP

Xylose

XylA

XylB

Xylulose-5-P

L-Arabinose

AraR

PPP

XylR

cre gene

CcpA

Xylose

n-Butanol

Hexose Pentose

Inhibitors Furfural

Organic acids

Phenolics

Salts

Acetone

Na+

SO42-

Clostridia

HMF

Xylose (in)

Xylitol Xylulose

D-Xylulose-5-P

Glyceraldehyde-3-P

Pentose phosphate pathway

non-oxidative enzymes

(RPE1, RKI1, TAL1, TKL1)

Ethanol (in)

XI

GXF1

XKS1

Na+

Formate

CO2 + NADH

FDH

Gly

coly

sis

Organizational Aspect： From the West Bund to the East Bund of Shanghai: Life science research and biotechnology development

SIBS

IPPE

NGRC

IBC/CB

ION

Inst. Nutrition

Inst. Health

Science

Shanghai

Pasteur

Proteomics Center

Bioinformatics Center

Fudan Univ.

Zhang-Jiang HiTech Park ( E. Bund )

BioTech Centers

Experimental Animal

Facilities

Shang JiaoTong

Univ. IMM (East Bund)

Institute of Computational Biol.

The West Bund

The East Bund: Vision of Zhang Jiang Biopharmaceutical Valley

Mechanism of Disease/Drugs

Biological Targets Biological Targets

Purification Structure of

Bio-macromolecules

Synchrotron Synchrotron - X-Ray, NMR, E/M, etc

Lead Compounds

Biological Screening

Structure Based

Drug Design

Nature Products

CombiChem

CHGCS BioChip Center

Drug Screening Center

Inst. Material Medica

TCM Research Center

Shanghai Univ Chinese Medicine

Optimization （Structure/Function)

Drug Candidates

Clinical Trials

SFDA Approval New Drug

Drug Safety Evaluation

Center of Model Animals

Clinical Research

（ （ Hospital/GLP ） ）

Bioinformatics Center

Biological Screening

CAS-Key Laboratory of Synthetic Biology (KLSynB)

Industry Biotechnology

Center Tech R&D

IPPE-Lab for Photosynthesis Environmental Biology

State Key

Lab for

Plant Mol Genetics

Energy, Environment, Chemical industry, Pharmaceutics, Medicine, Health……

SIBS Inst. Organic Chem CUST, Fudan, SJTU

Res Institutions＋Tech Platforms Multidisciplinary research

CHGC-SBC

“Omics” centers

“Omics” Research Platforms

Theoretical/Technological Research

CAS-Key

Lab for

Insect Science

KL SynB

KL SysB

Bioinformatics

technology

center

Bio- medicine

Translational Research

Center

Cloud

Core

Route Applied Research

Translational Research