Preparing a cyanobacterial Preparing a cyanobacterial chassischassis for H for H22

production: a synthetic biology approachproduction: a synthetic biology approach

Catarina PachecoCatarina Pacheco

Cell and Applied Microbiology Cell and Applied Microbiology

GroupGroup

IBMC, INEBIBMC, INEB

E4. Genómica funcional e biologia sintética

Encontro Nacional de Ciência - Ciência 2009

Fundação Calouste Gulbenkian,30th July 2009

Synthetic Biology is...Synthetic Biology is...

... the design and construction of new biological

parts, devices and systems and the re-design of

existing, natural biological systems for useful

purposes.

Synthetic Biology is the application of engineering Synthetic Biology is the application of engineering

concepts to biologyconcepts to biology

StandardizedStandardizedpartsparts

Assembly of modulesAssembly of modulesand circuitsand circuits

Incorporation inIncorporation ina a chassischassis

BioBrick™ parts assemblyBioBrick™ parts assembly

BioBrickBioBrick™Standardized DNA fragment designed for a specific purpose and that can be easily assembled with other bricks to generate modules and devices.

e.g.

promoter sensormodified

gene

http://partsregistry.org/

“The candidates for chassis should be well studied organisms with high throughput genomic and proteomic data available, minimalist in terms of the subset of genes that will allow retaining viability, and easy to engineer with the available molecular tools, becoming a versatile platform for multiple purpose applications”

ChassiChassiss

Escherichia coliEscherichia coli

Bacillus subtilisBacillus subtilis

YeastYeast

Consortium members:Instituto de Biologia Molecular e Celular (Portugal)

École Polytechnique (France)

Universidad Politécnica de Valencia (Spain)

Uppsala Universitet (Sweden)

University of Sheffield (UK)

Weizmann Institute of Science (Israel)

FP6-2005-NEST-PATHContract no.: 043340Jan.07- Jan.10

In silico analysisComputational design of parts and modules

Synthesis of partsAssembly of modulesPreparation of the chassis

Caracterization of parts/modulesIncorporation in the chassisEvaluation of the final product

BioModularHBioModularH

22Design

Construction

Caracterization

Final Final goalgoal

A cyanobacterial chassis that

together with the designed

devices will harvest solar

energy for H2 production.

The synthetic parts and

modules will be available for

other biotechnological

applications

PhotoautotrophicPhotoautotrophic chassis - chassis - Synechocystis sp. PCC 6803

the most studied cyanobacteria

unicellular and non-N2-fixing

simple nutritional requirements

naturally transformable

molecular tools for manipulation available

small genome comprising a 3.6 Mb genome

and 7 plasmids (1st cyano genome

sequenced)

Preparation of thePreparation of the chassischassis

Tuning respiration

Nativehydrogenase (s)

Nuclease(s)

etc…

Oxygenconsumption

Oxygen sensing

Highly-efficientO2-tolerant hydrogenase

• Reduce constraints, e.g. enhance transformation efficiency

• Remove redundant genes / parts

• Minimize O2 production / maximize O2 consumption H2ases are very sensitive

to O2

Deletion of redundant parts – Deletion of redundant parts – generation of a hydrogenase deficient generation of a hydrogenase deficient mutantmutant

Possesses hoxYHSensitive to kanamycinResistant to sucrose

hoxY hoxH

Deletion of hoxYHResistant to kanamycinSensitive to sucrose

Lacks hoxYHSensitive to kanamycinResistant to sucrose

HydA1_Fd

Hydrogen Producing Device Hydrogen Producing Device (HPD)(HPD)

Homology models based on Chang et al. 2007 (Biophys J, 93:3034-45)

Hydrogenase module

Fe-only hydrogenase fused to ferredoxin – Chlamydomonas reinhardtii

Maturation module

HydEF + HydG – Chlamydomonas

reinhardtii

Identification of neutral sites for the Identification of neutral sites for the insertion of synthetic modulesinsertion of synthetic modules

Genes encoding proteins:

- unknown or hypothetical

- with maximum length of 300 a.a.

- without predicted transmembrane domains (TMHMM Server v. 2.0)

- primary or secondary structure without relevant homologues

- that do not interact with other proteins in two-hybrid system

(CyanoBase data)

16 potential neutral sites identified

Analysis of gene expression by RT-PCRAnalysis of gene expression by RT-PCR

Generation of deletion mutants in the ORFs corresponding to the neutral sites N5, N7, N8, N10, N15 and N16.

Mutant analysis will reveal the true neutral sites that can be used for the integration of synthetic modules and devices.

Design and characterization of parts for HDesign and characterization of parts for H22 production productionOxygen Consuming Device (OCD)Oxygen Consuming Device (OCD)

- A-type flavoprotein (ATF) – Synechocystis sp. PCC 6803

- Laccase – Escherichia coli

SINGLE-protein modules O2 H2O

TWO-protein module O2 H2O2 ½ O2 + H2O

- Glucose oxidase – Penicillium

amagasakiense

+

Catalase – Synechocystis sp. PCC 6803

Testing the expression of A-type flavoprotein (ATF) module Testing the expression of A-type flavoprotein (ATF) module in in Escherichia coliEscherichia coli

100 kDa

AHL

75 kDa

50 kDa

37 kDa

25 kDa

+ + + +- - - -MW

Wild-type T9002 *Promoterless

ATF **LuxR controled

ATF ***

ATF(63 kDa)

GFP(27 kDa)

100 kDa

AHL

75 kDa

50 kDa

37 kDa

25 kDa

+ + + +- - - -MW

Wild-type T9002 *Promoterless

ATF **LuxR controled

ATF ***

100 kDa

AHL

75 kDa

50 kDa

37 kDa

25 kDa

+ + + +- - - -MW

Wild-type T9002 *Promoterless

ATF **LuxR controled

ATF ***

100 kDa

AHL

75 kDa

50 kDa

37 kDa

25 kDa

+ + + +- - - -MW

Wild-type T9002 *Promoterless

ATF **LuxR controled

ATF ***

ATF(63 kDa)

GFP(27 kDa)

- A synthetic module that can be

used for the controlled expression

of the ATF was obtained.

PtetR luxR gfpPluxR ATF

LuxR controled ATF ***

T9002 *

PtetR luxR gfpPluxR

Promoterless ATF **

PtetR luxR gfpATF

PtetR luxR gfpPluxR ATF

LuxR controled ATF ***

PtetR luxR gfpPluxR ATFPtetR luxR gfpPluxR ATFPtetR luxR gfpPluxR ATF

LuxR controled ATF ***

T9002 *

PtetR luxR gfpPluxR

T9002 *

PtetR luxR gfpPluxR

Promoterless ATF **

PtetR luxR gfpATF

Promoterless ATF **

PtetR luxR gfpATF

Constructions used in the

test:

Thank you for your attention

The Cellular and Applied Microbiolgy group