Yeast in synthetic biology

Stefan HohmannCell and Molecular Biology, Göteborg UniversityBox 462, 40530 Göteborg, Swedenstefan.hohmann@gu.se

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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