Preparing a cyanobacterial chassis for H2
production: a synthetic biology approach
Catarina Pacheco
Cell and Applied Microbiology Group
IBMC, INEB
E4. Genómica funcional e biologia sintética
Encontro Nacional de Ciência - Ciência 2009
Fundação Calouste Gulbenkian,30th July 2009
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
concepts to biology
Standardized Assembly of modules Incorporation in
parts and circuits a chassis
BioBrick™ parts assembly
BioBrick™
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 sensor modified gene
http://partsregistry.org/
Chassis
“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”
Escherichia coli
Yeast
Bacillus subtilis
FP6-2005-NEST-PATH
Jan.07- Jan.10 Contract no.: 043340
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)
BioModularH2
Design
In silico analysis
Computational design of parts
and modules
Construction
Synthesis of parts
Assembly of modules
Preparation of the chassis
Caracterization
Caracterization of parts/modules
Incorporation in the chassis
Evaluation of the final product
Final goal
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
Photoautotrophic 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 the chassis
Tuning respiration Oxygen sensing
Oxygen
consumption
Native
hydrogenase (s)
etc…
Highly-efficient
Nuclease(s) O2-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 –
generation of a hydrogenase deficient mutant
hoxY hoxH Possesses hoxYH
Sensitive to kanamycin
Resistant to sucrose
Deletion of hoxYH
Resistant to kanamycin
Sensitive to sucrose
Lacks hoxYH
Sensitive to kanamycin
Resistant to sucrose
Hydrogen Producing Device (HPD)
HydA1_Fd
Hydrogenase module
Fe-only hydrogenase fused to ferredoxin –
Chlamydomonas reinhardtii
Maturation module
HydEF + HydG – Chlamydomonas reinhardtii
Homology models based on Chang et al.
2007 (Biophys J, 93:3034-45)
Identification of neutral sites for the insertion 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-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 H2 production
Oxygen Consuming Device (OCD)
SINGLE-protein modules O2 H 2 O
- A-type flavoprotein (ATF) – Synechocystis sp. PCC 6803
- Laccase – Escherichia coli
TWO-protein module O 2 H 2 O2 ½ O2 + H 2 O
- Glucose oxidase – Penicillium amagasakiense
+
Catalase – Synechocystis sp. PCC 6803
Testing the expression of A-type flavoprotein (ATF) module
in Escherichia coli
Promoterless LuxR controled
Constructions used in the test:
Wild-type T9002 * ATF ** ATF ***
MW - + - + - + - + AHL
100 kDa
T9002 *
75 kDa
PtetR luxR PluxR gfp
ATF
50 kDa (63 kDa)
Promoterless ATF **
PtetR luxR gfp 37 kDa
ATF
LuxR controled ATF ***
PtetR luxR PluxR ATF gfp
25 kDa GFP
(27 kDa)
- A synthetic module that can be used
for the controlled expression of the
ATF was obtained.
The Cellular and Applied Microbiolgy group
Thank you for your attention