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Yeast in synthetic biology
Stefan HohmannCell and Molecular Biology, Göteborg UniversityBox 462, 40530 Göteborg, [email protected]
Outline
• Yeast biology• Yeast research – brief history• Yeast resources• Gene cloning in yeast• Engineering yeast – targeted integration• Using yeast genetics• Examples of yeast in synthetic biology
Yeast
• Saccharomyces cerevisiae, baker’s or brewer’s yeast• There are many other yeasts ! (Kluyveromyces lactis,
Schizosaccharomyces pombe, Candida albicans, Pichia pastoris)• Single-celled eukaryote, fungus• Free-living in Nature, flowers and fruits• Domesticated: Wine, beer, bread, alcohol, proteins, research• Cells are about 5 micrometer long• Grows in liquid culture and as colonies on agar plates• Devides by budding generating a mother and a daughter cell• Generation time about 90min• In Nature and industry usually diploid, but stable haploids in the
laboratory
Well characterised
12,156,678 bp of DNA on 16 chromosomes4,619 verified ORFs
1,175 uncharacterised ORFs815 dubious ORFsTotal 6,609 (5,794)
ca 1,400 other genetic elements (e.g. RNA genes, transposons etc)Mitochondrial genome
Sequence and annotation constantly updatedSources: genome comparison, strain sequencing, tiling arrays
Categories of gene productsComponentProcess
Well conserved
• Cellular organisation is conserved (yeast has a cell wall)• Basic cellular processes are highly conserved from yeast
to human• Metabolic and signalling pathways are conserved• A high proportion of yeast genes have human orthologues• Many human disease genes have human orthologues• Yeast lacks multi-cellularity, still has features like cell type
determination, ageing and apoptosis
At the forefront of developments
• Genetics established since the 1950ies, genetic maps with more than 1,000 loci• Gene cloning and transformation in the 1970ies, gene libraries• Targeted gene deletion in the 1980ies – reverse genetics• Model organism: e.g. cell cycle and vesicular transport – wide coverage• First eukaryotic genome sequenced in 1996 (community effort)• Functional genomics: first genome-wide studies (microarray, two-hybrid protein
interaction), genome-wide strains collections• Bioinformatics developed on yeast data• Comparative genomics, e.g. Genolevure; genome evolution• Systems biology: first large scale networks (expression, transcription factor
binding, protein interaction, genetic interaction), reconstruction of metabolism• Systems biology: data-rich dynamic models
Yeast resources
• An open and interactive community of 1,000 labs• Saccharomyces Genome Database www.yeastgenome.org• Yeast GRID thebiogrid.org• Yeast Resource Center depts.washington.edu/yeastrc/• Yeast resource collections (deletion strains, tagged ORFs,
cloned ORFs etc)www.openbiosystems.com/GeneExpression/Yeast/web.uni-frankfurt.de/fb15/mikro/euroscarf/
• Yeast parts for synthetic biology parts.mit.edu
Gene cloning in yeast
• Yeast – E.coli shuttle vectors (e.g. pRS series)• Single copy CEN vectors versus multi-copy 2micron vectors• Selection markers for plasmids: nutritional requirements
LEU2, HIS3, ADE2, TRP1, LYS2, URA3 (URA3 can be selected for or against)
• Regulatable promoters: GAL1, Tet, MET25, many more• Yeast can maintain multiple different plasmids• Episomal plasmid versus genome integration
Targeted integration by homologous recombination
Amplification of cassette containing marker, element of interest
Transformation of yeast
Homologous recombination with high fidelity and efficiency
Less than 100bp overlap sufficient
Selection of transformants and confirmation
Using targeted integration
• Gene replacement (precise gene deletion)• Gene tagging (XFP, TAP etc)• Promoter-reporter fusions (XFP, beta-Gal etc)• Simple integration• Gap repair cloning
• Virtues: precise, fast, effective
Gap repair
X
X
gapped plasmid
repair fragment
YFG1
Transformation of a gapped plasmid
Cotransformation of an amplified fragment with homologous overhang
Successful transformation requires repair via recombination
Useful to construct for instance mutant libraries of a specific gene
Yeast can be haploid or diploid
In Nature yeast is homothallic (switches mating type) and therefore commonly diploidLaboratory yeast are HO-mutants and therefore heterothallic with stable haploids
Using haploids and diploids
Haploids express recessive genetic changes
Diploids allow for promoter-reporter fusions of one copy while the second copy expresses the wild type gene
Haploids can be crossed to diploids which are then sporulated to haploids again: simple combination of different genetic changes
Haploid transformations can be crossed to diploids that carry two plasmid
Diploids are more vigorous and more robust
Haploid-diploid phases are the basis for sophisticated genetic analysis and genetic interaction studies (even in high throughput)
Metabolic engineering• Jay Keasling’s lab engineers yeast to
overproduce artemisinic acid• Expression of the heterologous enzyme
ADS on a plasmid using GAL1 promoter• GAL1-driven overexpression of truncated
HMGR• Stimulated production of several enzymes
by introducing into the genome a hyperactive version of the trancription factor Upc2
• Downregulation of ERG9 expression by replacement with the repressible MET3promoter
• Heterologous expression of both CYP71AVIand CPR using GAL1 promoter
• Combination of all alterations resulted in best yield of artemisinic acid
Metabolic engineering in yeast• Suitable promoters for heterologous expression• Suitable sources for relevant enzymes• Codon optimisation• Plasmids with suitable selection markers, co-transformation• Plasmids with multiple expression constructs• Altering expression of genes using yeast regulators• Altering expression using suitable promoters• Truncated genes for eliminating regulation• Combining different engineering steps using repeated
transformation and re-usable markers plus genetic crossing
Yeast MAP kinase pathways
Crosstalk
Diverting signalling• Wendell Lim’s lab generated diverters that redirect
signalling• Generating new signalling devices• All constructs were made on single-copy plasmids with
endogenous promoters
Drug screening
• Pheromone receptor can be replaced by human GPCRs
• Sometimes co-expression of G-alpha is needed
• Eliminating feedback loops and cell cycle arest
• Employ suitable reporters for pathway output (growth, beta-Gal, XFP)
Cell communication systems
Redesigning yeast for communication• Ron Weiss’ lab designed a sender cell and a receiver cell, each with suitable
reporter systems• System requires heterologous expressions, reporter constructs and gene
overexpression• Moreover, deletion of the essential gene SLN1 and keeping strain alive with
overexpression plasmid with pGAL1-PTP2• All constructs were done on plasmids, i.e. strains carry three plasmids
employing different selection markers
Conclusions
• S. cerevisiae is a versatile system for Synthetic Biology• Its genetics and molecular cell biology are extremely
well developed• Sophisticated genetics and engineering tools allow
ambitious projects to be performed• This concerns both redesign of cellular pathways and
heterologous introduction, and combination thereoff• Combine with genetic screens and forced evolution• S. cerevisiae has direct biotechnological use and a
long-standing experience in industry
Hohmann lab, research vision
• Understanding at the molecular level cellular control mechanisms by employing S. cerevisiae as a model system
• Advancing system-level understanding by combining experimentation and mathematical/computational reconstruction
The Hohmann lab, summer 2007Prof. Stefan Hohmann
Peter Dahl (lab manager)Takako Furukawa (technician)Abraham Ericsson (PhD student, bioinformatician)Dr. Marcus Krantz (returning post-doc from Japan)
MAPK SIGNALLINGOSMOREGULATION
Dr. Bodil Nordlander
Dr. Carl TigerDr. Kentaro FurukawaElzbieta PetelenzDoryaneh AhmadpourJimmy Kjellén
ARSENITESIGNALLING
Dr. Markus Tamás
Dr. Julia IlinaDr. Jenny VeideMichael Thorsen
NUTRIENT REGULATION
Dr. Karin Elbing
Dr. Gemma BeltranDr. Raul SalcedoDr. Dominik MojzitaDaniel BoschTian YeIvan Pirkov
AQUAPORINSSPECIFICITY
Dr. Karin Lindkvist
Dr. Kentaro FurukawaCecilia GeijerMadelene PalmgrenSylwia Zoltowska
Present collaborators and funding• The QUASI EC Project (2007): F Posas, M Peter, G Ammerer, E Klipp, M Grøtli, P Sunnerhagen
• The MalariaPorin EC Project (2007): E Beitz, P Agre, S Flitsch, H Grubmüller
• The Sleeping Beauty EC Project (2008): E Lubzens, M Clark, R Reinhard, J Cerda, J Nielsen
• The Systems Biology Early Stage Training EC project (2008): R van Driel, E Klipp, R Heinrich
• The Yeast Systems Biology Network (2008) with about 20 groups in Europe (EC-funded Coordination Action) and 40 groups world-wide
• The Sweden-Japan Vinnova project (2009): H Kitano
• The AMPKIN EC Project (2009): D Carling, J Nielsen, O Wolkenhauer, Biovitrum/Arexis AB
• The Aqua(glycero)porin RTN EC Project (2010): S Flitsch, H Grubmüller, P Deen, A Engel, S Nielsen, R Neutze, J Cerda, Z Vajda, E Klipp
• The CELLCOMPUT NEST Project (2011): F Posas, R Solé, M , E Klipp, M Grøtli• Funding from the Swedish Research Council (2007)
• Ingvar grant from SSF (2010) to Karin Lindqvist
• Funding from the Swedish Research Council (2007) to Markus Tamás (position and project)
• Faculty platforms in Quantitative Biology and Chemical Biology (2009/11) with groups in in physics (D Hanstorp), chemistry (M Grøtli), computational biology (M Jirstrand, O Nerman, B Wennberg), structural biology (R Neutze) and biology (T Nyström, A Blomberg, P Sunnerhagen)
Synthetic Biology Meeting in Göteborg Aug 27/28
http://www.chalmers.se/biocenter/EN/Calendar/functional-genomics-2007
Ron Weiss, Jay Keasling, Bengt Nordén, Hiroki Ueda, Chris Voigt, Michael Katze, Kobi Benenson, Jef Boeke, Jörg Stelling, Daisuke Umeno....
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