Yeast Biotechnology Notes

Yeast

  • Brewer's yeast and Baker's yeast are common types.
    • Baker's yeast (Saccharomyces cerevisiae) is used as a leavening agent, converting sugars into carbon dioxide and ethanol.
    • Brewing yeasts ferment plant material to produce ethanol.
      • Top-cropping (Saccharomyces cerevisiae).
      • Bottom-cropping (Saccharomyces pastorianus).

Yeast in Molecular Biology

  • Expression System:
    • Easy genetic manipulation.
    • Rapid growth on inexpensive media.
    • Eukaryotic protein modifications (e.g., glycosylation).
  • Molecular Biology Tool:
    • Study protein-protein interactions.
    • Study protein-DNA interactions.
  • Model System:
    • Study protein behavior and interaction.
    • Study mutation effects.
    • Study deletion/insertion effects of genes.

Synthetic and Systems Biology Approaches

  • Synthetic Biology:
    • CAR cells, yeast biotechnology, engineered tissues/cultures.
    • Utilizes cane sugar, corn starch, ligno-cellulose, crude glycerol.
    • Focuses on pathways, biochemical reactions, proteins, and genes.
    • Deals with primary metabolites, secondary metabolites, and recombinant proteins.
  • Systems Biology:
    • Integrates experimental data with mathematical models to understand biological phenomena.
    • Combines wet (laboratory) and dry (computer simulation) experiments to test hypotheses and generate new data.
    • Experimental data includes determination of structures, functions, and interactions.

Yeast as a Protein Expression Platform

  • Saccharomyces cerevisiae: Most sophisticated eukaryotic model for recombinant DNA technology.
  • Arxula adeninivorans: Grows as budding yeast up to 42°C, filamentous form at higher temperatures.
  • Candida boidinii: Notable for growth on methanol.
  • Pichia pastoris: Efficient platform for foreign protein production (methylotrophic).
  • Yarrowia lipolytica: Dimorphic yeast with high potential for industrial applications, but no commercial recombinant products yet.

Reasons for Using Yeast as an Expression Platform

  • Simple to cultivate in inexpensive media.
  • Strains are well-characterized with detailed genetic maps.
  • Molecular biology of yeast is well-known.
  • Easy molecular manipulation.
  • Easy recombinant preparation.
  • Easier upscaling.
  • Classified as GRAS (generally recognized as safe) by FDA.

Yeast Reproduction

  • Asexual Reproduction:
    • Asexual Budding: Nucleus splits and migrates into daughter cell until separation occurs.
    • Asexual Fission: Schizosaccharomyces pombe reproduces by fission, creating two identical daughter cells.
  • Cell Reproduction Cycle:
    • Chromosome duplication (S or Synthetic phase).
    • Chromosome segregation and nuclear division (mitosis, M or Mitotic phase).
    • G1 phase (between M and S phases).
    • G2 phase (between S and M phases).
    • Fission yeast mitosis is similar to multicellular animals.
  • Sexual Reproduction (Fusion):
    • Schizosaccharomyces pombe undergoes mating when nutrients are limited.
    • Under stress, haploid cells die, but diploid cells undergo sporulation (meiosis), producing haploid spores.
    • Spores conjugate to reform diploid cells.

Global Therapeutic Protein Market

  • Market Distribution:
    • USA: 40%.
    • EU: 25%.
    • China: 7%.
    • All Other: 28%.
  • Products:
    • Growth factors, vaccines, mAbs.
    • Hormones, insulin, fusion proteins.
    • Insulin, recombinant proteins, vaccines, mAbs are key products.
  • Expression Systems:
    • Pichia, E. coli, Mammalian cells, Other systems.
    • Hormones, Fusion proteins, Recombinant vaccines, mAbs, Insulin, and Growth factors.
  • Specific Systems and Their Market Share:
    • Komagataella pastoris (31%).
    • Escherichia coli (26%).
    • Aspergillus oryzae (14%).
    • Bacillus subtilis (6%).
    • Saccharomyces cerevisiae (8.5%).
    • Hansenula polymorpha (5.5%).
    • Streptomyces lividans (3%).
    • Aspergillus awamori (3%).
    • Schizosaccharomyces pombe (3%).

Yeast Vector Types

  • YIP (Yeast Integrating Plasmid).
  • YRP (Yeast Replicating Plasmid).
  • YEp (Yeast Episomal Plasmid).
  • YCP (Yeast Centromere Plasmid).
  • YAC (Yeast Artificial Chromosome).

YIP (Yeast Integrating Plasmid)

  • Single copy plasmid for integration into genomic locus.
  • Consists of bacterial vector components and a yeast gene with selective marker.
  • Cannot survive as free plasmid due to lack of origin of replication and centromere.
  • Site-specific integration via homologous recombination.
  • Strategies for integration:
    • Use sequences allowing chromosomal integration and linearized plasmid for homologous recombination.
    • Gene replacement by homologous recombination.

YRP (Yeast Replicating Plasmid)

  • YIP vector with ARS (autonomously replicating sequence) origin of replication.
  • Transformation efficiency is 2-3 fold higher.
  • Replicates autonomously due to ARS.
  • Contains four regions (A, B1, B2, and B3) affecting plasmid stability.
  • Rapid loss of vectors is reported.

YEp (Yeast Episomal Plasmid)

  • Self-replicating multi-copy plasmid.
  • Contains prokaryotic sequences, a selectable marker gene, and entire 2µ plasmid.
  • The 2µ plasmid has a single origin of replication and the STB locus for autonomous replication and stabilization.
  • Encodes four genes: FLP (or A), REPl (or B), REP2 (or C), and D.
  • Used for recombinant protein production and multicopy suppression studies.

YCP (Yeast Centromere Plasmid)

  • Has a replication origin and a yeast centromere sequence (CEN9 or CEN4) for mitotic and meiotic segregation.
  • As few as 125 base pairs (bp) of centromere DNA are required for proper segregation.
  • YCp vectors are true shuttle vectors.

YAC (Yeast Artificial Chromosome)

  • Typical YAC (e.g., pYAC3) contains:
    • ARS sequence for replication.
    • CEN4 sequence for centromeric function.
    • Telomeric sequences at ends for protection from exonuclease action.
    • Selectable marker genes (TRP1 and URA3).
    • SUP4, a selectable marker for DNA insert integration.
    • Required sequences from E. coli plasmid for selection and propagation.

2 Micron Plasmid

  • Exists in the nucleus at 40-60 copies.
  • Approximately 6.3 kbp long DNA with coding capacity for four proteins.
  • No specific growth phenotypes are associated with its presence or absence.
  • Copy number and transmission are carefully regulated.
  • Transmission to daughter cells requires REP1 and REP2 proteins, as well as host cell factors.
  • High copy numbers are deleterious, causing cell cycle misregulation and lethality.
  • The Rep/STB system maintains stability.
  • Genetic organization devoted to:
    • Duplication during S phase.
    • Partitioning between daughter cells.
    • Maintenance of steady state copy number.
  • Amplification occurs only when copy number decreases.
  • Regulatory complex containing Rep1p and Rep2p may control amplification by regulating FLP gene expression.

Prototroph and Auxotrophic Markers

  • Prototroph: Wild-type yeast cell that synthesizes its own nutritional requirements and grows on minimal media.
  • Auxotroph: Mutant that lost the ability to synthesize an essential nutrient due to a defective gene.
  • Auxotrophic strains require media with appropriate growth factors.
  • Strains requiring leucine (Leu 2) grow on minimal media if they have a plasmid expressing the Leu 2 gene.

Markers

  • URA3: Pyrimidine (uracil).
  • LYS2: L-lysine.
  • LEU2: L-leucine.
  • TRP1: L-tryptophan.
  • HIS3: L-histidine.
  • MET15: L-methionine.
  • ADE2: Purine (adenine).

Replica Plating

  • Technique developed by Esther Lederberg to compare growth of colonies on different media plates.
  • Transfers colonies to new plates in the same spot.
  • Used to determine which environments a colony can or cannot grow in.

Enhancing Vector Copy Number

  • Use marker genes with partially defective promoters (e.g., LEU2d).
  • Reduced expression of the marker gene confers a selective advantage for cells with high copy numbers.
  • Defective markers may avoid protein-burden effects from massive overexpression.
  • Both LEU2d and TRP1d markers used successfully to obtain high copy numbers.

Auxotrophic Markers for Knock-Out Mutations

  • Auxotrophic markers used for one-step gene deletion method.
  • A marker gene is equipped with short 5' and 3' sequences identical to flanking chromosomal sequences.
  • Deletion mutant becomes prototrophic for the nutrient involved.

Codon Bias and Optimization

  • Most mRNA-coding genes show bias in codon usage for particular amino acids.
  • Different genes in the same genome often have related codon preference rules.
  • Codon usage reflects the abundance of cognate tRNAs.
  • Problems:
    • Interrupted translation.
    • Frame shifting.
    • Misincorporation of amino acids (e.g., lysine for arginine due to AGA codon).
    • Inhibition of protein synthesis and cell growth.
  • Observed expression levels are often low or nonexistent.

Codon Optimization Tools

  • Web servers and applications: 'GeneDesign', 'Synthetic Gene Designer', 'Gene Designer', and 'Optimizer'.
  • Methods of Codon Optimization:
    • Complete optimization of all codons.
    • Optimization based on relative codon usage frequencies using a Monte Carlo approach.
    • Maximizing optimization with minimum changes between query and optimized sequences.

Transcription Promoters

  • Foreign Promoters:
    • Foreign promoters (e.g., Drosophila ADE8 or herpes simplex virus thymidine Kinase) are often erroneous or inactive in yeast.
    • Cognate RNA polymerase must be co-expressed for foreign promoters not recognized by yeast RNA polymerase.
    • Bacteriophage T7 RNA polymerase is highly active and used in prokaryotic and eukaryotic organisms.
    • T7 RNA polymerase with a nuclear targeting signal can be expressed from a GAL1 promoter in yeast.
  • Yeast mRNA Promoters:
    • Upstream Activation Sequences (UASs).
    • TATA elements.
    • Initiator elements.
  • Hybrid Promoters:
    • ADH2 UAS (22 bp) fused with 320 bp upstream of the GAP TATA element.
    • Achieved tightly regulated production of superoxide dismutase (SOD)-proinsulin protein.

Yeast Specific Promoters

  • Glycolytic Promoters:
    • From genes encoding abundant glycolytic enzymes (e.g., alcohol dehydrogenase I (ADHl) or glyceraldehyde-3-phosphate dehydrogenase (GAP)).
  • Galactose-Regulated Promoters:
    • GAL1, GAL7, and GAL1O are powerful, tightly-regulated promoters.
  • Phosphate-Regulated Promoters:
    • Promoter of the acid phosphatase gene, PH05, regulated by inorganic phosphate.
  • Glucose-Repressible Promoters:
    • ADH2 promoter is powerful and tightly regulated.
  • Metal Regulated Promoter:
    • Promoter of the CUPl gene (copper metallothionein).
    • Induction depends on the copper-resistance of the host strain.
  • Temperature Controlled Promoter:
    • MATαsir3 strain for secretion of hEGF using the α-factor (MFαl) promoter.
    • Switching temperature from 35°C to 25°C induces foreign protein production.

Commonly Used Promoters in Yeast System

  • ADC1 (Alcohol dehydrogenase I): Induced by fermentable carbon source.
  • PGK (Phosphoglycerate kinase): Induced by fermentable carbon source.
  • GAPDH (Glyceraldehyde-3-phosphate dehydrogenase): Induced by fermentable carbon source.
  • PHO5 (Acid phosphatase): Induced by low Pi (inorganic phosphate).
  • Gal1 GAL10 (Galactokinase): Induced by Galactose.
  • SUC2 (Invertase): Induced by Low glucose.
  • MFα1 (Mating pheromone a): Induced by MATα.
  • ADRIII (Alcohol dehydrogenase 2): Induced by Low glucose.

Signal Sequences

  • Classical signal sequence: Charged N-terminus, hydrophobic core, and consensus sequence for cleavage by signal peptidase in the ER.
  • Some secreted proteins, like yeast mating pheromone α-factor, have additional pro sequences.
  • Heterologous proteins may be secreted using either a foreign or yeast signal.
  • Yeast signal essential features include hydrophobic core of 6-15 amino acids, sometimes interrupted by non-hydrophobic residues.
  • Many signal peptides contain one or more basic amino acids preceding the hydrophobic core.
  • Small neutral and α-helix-disrupting amino acids are often present near the cleavage site.
  • Even with signal peptide deletion, some proteins (e.g., carboxypeptidase Y and acid phosphatase) can enter the secretory pathway.
  • Widely studied signals are from acid phosphatase, invertase, and α-factor.

Foreign Signal Sequences

  • Early attempts used protein's own signal sequence (e.g., E.coli lactamase, human α and γ interferon (IFN), mouse immunoglobulin heavy and light chain, and influenza virus haemagglutinin).
  • Expression levels were often very low, with only a proportion of the protein being secreted.
  • Some foreign proteins have been successfully secreted from yeast using their own signal.
  • A prokaryotic signal sequence may also efficiently direct secretion in yeast (e.g., Bacillus amyloliquefaciens α-amylase).
  • Using foreign leaders often results in intracellular accumulation, despite some successes.

Selection of Host Strain

  • Wild types.
  • Protease-deficient strains.
  • Auxotrophic strains.
  • Glyco-engineered strains.

Protein Secretory Pathway in Yeast

  • Genomic integration: single-/multicopy, homologous recombination, ectopic integration.
  • Promoter: constitutive, inducible.
  • Selectable marker: drug resistance, auxotrophy.
  • Intracellular protein expression.

Secretory Pathway

  • Secretion signals:
    • S.c. α-MF, P.p. PHO1.
  • Proteolytic processing, e.g.:
    • Kex2p, Ste13p.
  • Glycosylation:
    • N- or O-linked.
  • Membrane-associated proteins.
  • Surface display anchors.

Post-Translational Modification

  • Comparison of Glycosylation Patterns:
    • Human: Complex, Hybrid.
    • E. coli: No glycosylation.
    • S. cerevisiae: Hypermannosylation, High mannose.
    • High Five: Truncated, paucimannosidic, α-Gal.
    • CHO: Complex, human-like, Neu5Gc.
  • Glycoengineering.
  • Key Glycans:
    • Mannose (Man).
    • Fucose (Fuc).
    • N-Acetylneuraminic acid (Neu5Ac).
    • N-Acetylglucosamine (GlcNAc).
    • Galactose (Gal).
    • N-Glycolylneuraminic acid (Neu5Gc).

Glycosylation

  • Many therapeutic proteins are glycosylated (e.g., monoclonal antibodies, blood clotting factors, IFNs, hormones, growth factors, and viral antigens).
  • Carbohydrate side chains are involved in cell-cell recognition, hormone-receptor binding, protein targeting, host-microorganism interactions, solubility, and stability.
  • Oligosaccharides can be N-linked to asparagine or O-linked to serine or threonine residues.
  • N-linked glycosylation adds N-acetylglucosamine (GlcNAc), mannose (Man), and glucose residues.
  • Many heterologous glycoproteins have been secreted from yeast, including Aspergillus glucoamylase and Somatostatin.

Problems with Yeast Glycosylation

  • Yeast glycosylation, especially hyperglycosylation, can inhibit reactivity with antibodies or cause excessive immunogenicity.
  • O-linked oligosaccharides synthesized by yeast differ greatly from those of higher Eukaryotes (Hyper mannose).

Protein Folding and Transport

  • Folding of secreted proteins occurs in the ER and involves accessory proteins (e.g., BiP).
  • Nascent proteins bind to BiP and are released upon folding, assembly, and glycosylation; misfolded proteins bind permanently and are retained in the ER.
  • Correct folding, assembly, and transport are important for multimeric protein production.
  • Saturation of accessory proteins or their inability to aid heterologous protein folding can cause problems.
  • Blockage in the secretory pathway can interfere with the secretion of host proteins.
  • Membrane proteins can cause problems due to non-specific insertion into intracellular membranes.
  • Cell wall complicates the secretion process.
  • Some proteins localize mainly in the cell wall when secreted from S.cerevisiae.

Secretory Pathway

  • Synthesis, Translocation, Folding, Glycosylation in the ER.
  • ER to Golgi transport via vesicle transport.
  • Retrograde Golgi to ER transport.
  • Retrograde Golgi transport.
  • Maturation in the Golgi (Cis-, Medial-, Trans-).
  • Trans-Golgi to Lysosome/Vacuole transport.
  • Exocytosis via vesicle transport.
  • Aggregation and Degradation in the Cytoplasm.

Secretory Pathway Genes of Yeast

  • ER: SEC61, SSH1, SEC62, SEC63, SEC66/SEC71, SEC67/SEC72, SEC12, SEC65, SEC53, SEC59, SEC16, SEC20, SRP54, SRP101, SEC11, KAR2, SSA1-SSA4, SEC6, SEC22, SSS1, BET1, GOS1, SEC19, SEC21, SEC26, SEC27, SEC70, SAR1, BET4,UFE1, ARF2, PMR1, MNN9, KMA1/XMA12, KMA2, VMA2, RE1, MPD1, CNE1, PDI.
  • Golgi: BET2, RET1, BET3, RET2, SEC14,ANP1, ERD1, SEC63, SED4,SFT1, SED5, USO1, SEC24, SEC7, SEC31, SEC21, KEX2, VRG4, RET3/ARF1, KEX1, VAN1.
  • Vesicle Transport & Fusion: SNC1,SNC2, SSO1,SO2, SEC1,SEC2,SEC3, SEC4, SEC4-GDP. SEC5,SEC6, SEC8,SEC9,SEC10, SEC15, MSO1, DSS4, SMY1, SEC17, SEM1, TPM1, SEC18, SCD5, SEC19, MYO2, ACT1, VTI1, YKT6.
  • Lysosome/Vacuole: YPT7, VMA1.

Proteolytic Processing

  • Proper processing is relevant to heterologous protein secretion in yeast.
  • Correct signal peptide cleavage must occur for mature product secretion.
  • Fortuitous, undesirable processing events may occur due to cleavage by processing proteases.
  • Inefficient cleavage can cause erroneous protein folding and accumulation.
  • Aberrant protein cleavage can create fragmented protein molecules.
  • Strategies:
    • Use Yeast proteolytic sites for efficient cleaving.
    • Over-express the processing enzyme genes.
    • Eliminate or minimize internal cleavage by optimizing growth conditions and selecting the best strain.
    • Create mutants by removing internal cleavage sites.

Transformation of Yeast

  • Spheroplasts: Prepared by hydrolytic enzymes in the presence of osmotic stabilizers like 1 M sorbitol.
    • Cell wall digestion with Glusulase or Zymolyase.
    • DNA is added and co-precipitated with polyethylene glycol (PEG) and Ca^{2+}.
    • Cells are resuspended in sorbitol, mixed with molten agar, and layered on a selective plate containing sorbitol.
  • Lithium salts: Treatment with lithium acetate permeabilizes the cell wall.
    • DNA is added and co-precipitated with PEG.
    • Cells are exposed to a brief heat shock, washed free of PEG and lithium acetate, and spread on selective medium.
  • Electroporation Method: Freshly-grown yeast cultures are washed and suspended in an osmotic protectant (e.g., sorbitol).
    • DNA is added, and the cell suspension is pulsed in an electroporation device.
    • Cells are spread on selective media.
    • Efficiency can be increased using PEG, single-stranded carrier DNA, and cells in late log-phase.

Selection of Transformants

  • Complementation of Auxotrophic Mutations:
    • Strain carries a mutation in a given gene (ura3, leu2, trp1, his3).
    • Corresponding wt copy of the gene (URA3, LEU2, TRP1, HIS3) is present in the vector.
  • Dominant Selection Markers:
    • Resistance to G418, cycloheximide, formaldehyde, Cu^{2+}.
  • Red White Screening:
    • ADE1 and ADE2 encode enzymes that synthesize adenine (white colony).
    • Ade2-1 mutant (no ADE1 and ADE2) produces a red pigment.
    • SUP4 suppress the Ade2-1 mutation and create white colony.
    • Recombinant YAC vector produces red colony with mutated Ade2-1 system.
    • Non-recombinant YAC vector produces white colony with working SUP4.

Ways of Increasing Protein Secretion in Yeast

  • Isolation of super secretory mutants:
    • Several isolated, most do not affect secretion and almost all are recessive.
  • Fusion of the heterologous protein to a well secreted endogenous protein:
    • prepro α-factor, Hsp 150.
  • Deletion of a quality control gene:
    • CNE1 (Calnexin homologue) -> misfolded proteins.
  • Overexpression of er lumenal folding machinery:
    • PDI1 (protein disulfide isomerase), HAC1.
  • Overexpression of components of the secretory machinery:
    • SEB1 (ER translocon component), SSO1, SSO2 (plasma membrane t-SNARES).
  • Optimization of production conditions:
    • Rich medium, Low temperature, pH.

Strategies to Improve Secretion

  • Isolate super-secreting mutants by screening for increased secretion of a particular product.
  • Couple mutagenesis with a rapid screening assay.
  • Create a fusion protein structure using the GOI and antibiotic-resistant gene.
  • Increase antibiotic selection pressure.
  • Identify mutants resistant to the toxic effects of a foreign protein.
  • Mutations at a single locus can confer resistance to the toxic effects of IGF-I and increase its production.

Scaling Up

  • Verification and screening of mutants (3-10 ml culture).
  • Small volume reactor (5-25 ml with T flask, shaker flask).
  • Intermediate scale:
    • Small, highly controlled bioreactor (1-5 L).
  • Production scale:
    • Large reactors (20-1000 L).

Hepatitis B Vaccine Production Using Yeast

  • Hepatitis B virus DNA/RNA is used for recombinant DNA introduction into a yeast cell.
  • Recombinant yeast cell contains HB antigen producing gene.
  • Recombinant DNA: Plasmid DNA and Bacterial DNA cut with restriction enzymes.
  • Bacterium contains plasmid DNA.
  • HB antigen is produced in a fermentation tank using recombinant yeast cells.
  • Extraction and purification of HB vaccine.

Non-Conventional Yeast Species

  • Saccharomyces cerevisiae may not be optimal for large-scale production due to:
    • Lack of very strong, tightly-regulated promoters.
    • Need for fed-batch fermentation to attain high-cell densities.
    • Hyperglycosylation.
  • Alternative systems are based on commercially-important yeasts:
    • Selected for their favorable growth characteristics at industrial scale.
    • Have other favorable intrinsic properties.
  • Pichia pastoris:
    • Efficient and tightly-regulated promoter.
    • Straightforward techniques for high-biomass cultivation.
  • Hunsenula polymorphu:
    • Similar properties to P. pastoris.
    • Potential problem with toxic products due to de-repression under high-density growth conditions.
  • K. lactis and Y. lipolytica:
    • Industrial yeasts examined for their capacity for high-level secretion.

Pichia pastoris

  • Phillips Petroleum Company developed media and protocols for growing P. pastoris on methanol in continuous culture at high cell densities.
  • Methylotrophic yeasts utilize methanol as the sole carbon and energy source using promoter from the tightly regulated AOXI gene.
  • Phenotypes:
    • Mut+ (methanol utilization plus): Grows on methanol at the wild-type rate.
    • Muts (methanol utilization slow): Disruption in the AOX1 gene.
    • Mut− (methanol utilization minus): Unable to grow on methanol due to deletion of both AOX genes; desirable for production of certain recombinant products.

Pros and Cons of Pichia pastoris Expression System

  • Advantages:
    • High expression level.
    • Simple culture conditions.
    • Low cost.
    • Relatively rapid growth.
    • Scaleable.
    • Efficient Secretion and Simple purification.
    • Choice of secreted/intracellular expression.
    • Extensive post-translational modification of proteins.
    • N-glycosylation close to higher eukaryotes.
    • Efficient protein folding.
  • Disadvantages:
    • Methanol induction is hard to control at the scale of culture.
    • Some glycosylation patterns different from mammalian cells.

Pichia pastoris

  • Gene and expression host selection.
  • PLC-Y enzyme.
  • Strain engineering.
  • Gene codon optimization.
  • Multi-copy integration.
  • Helper protein co-expression.
  • Fermentation.
  • High producing strain.
  • Industrial application.
  • Degumming.

Protein Expressed in Pichia pastoris

  • Enzymes:
    • D-alanine carboxypeptidase (0.8 g/l).
    • Alpha amylase (2.5 g/l).
    • Catalase (Aspergillus fumigatus) (2.3 g/l).
  • Proteases and Protease Inhibitors:
    • Kunitz protease inhibitor (APLP-2) (1.0 g/l).
    • Tick anticoagulant protein (TAP) (1.7 g/l).
    • Ghilanten (0.01 g/l).
    • tPA Kringle type-2 domain (0.1 g/l).
  • Membrane Proteins:
    • Human CD38 (soluble portion) (0.05 g/l).
    • Mouse serotonin receptor (0.001 g/l).
  • Antigens and Antibodies:
    • Tetanus toxin fragment C (12.0 g/l).
    • HIV-1 gp120 (intracellular) (1.25 g/l).
    • HIV-1 gp120 (secreted) (0.02 g/l).
    • Bm86 tick gut glycoprotein (1.5 g/l).
    • Murine single-chain antibody (0.25 g/l).
  • Regulatory Proteins:
    • Tumor necrosis factor (10.0 g/l).
    • Streptokinase (active) (0.08 g/l).
    • Mouse epidermal growth factor (EGF).
    • Human IFN-α2b (0.45 g/l).
    • Human IFN-a2b (0.4 g/l).

Hansenula polymorpha

  • The expression system developed in the methylotroph, H. polymorpha, is similar to that of P. pastoris.
  • The gene encoding the peroxisomally-located enzyme, methanol oxidase (MOX), has been isolated and the promoter used to express foreign genes.
  • Upon the addition of methanol into a culture medium genes of methanol metabolism are strongly induced resulting in a vast production of key enzymes like methanol oxidase (MOX) and formate dehydrogenase (FMD).
  • H. polymorpha is a thermo-tolerant organism making it most suited for the production of crystalizable proteins (TPSI promoter).
  • Transformation systems were developed using the LEU2 and URA3 genes from S. cerevisiae as selectable markers.
  • The secretion of several other foreign proteins in H. polymorpha has been reported including HSA, invertase and β-lactamase.
  • PENTAVAC PFS (Pentavalent Vaccine).

Kluyveromyces lactis

  • K. lactis has been used in the food industry for many years in the production of P- galactosidase (lactase).
  • Thus, its large scale cultivation has been extensively studied, and it is well accepted for the production of proteins for human use.
  • Transformation systems were initially developed by isolating K. lactis ARS sequences, since neither the S.cerevisiae ARSI nor 2p replicates in it.
  • A small number of promoters have been used in K. lactis expression vectors. The best- characterized K. lactis promoter is that of the LAC4 gene, encoding P-galactosidase, which is induced up to 100- fold by lactose or galactose.
  • The secretion of interleukin 1 p (IL- 10) by K. lactis has also been reported, using the toxin a-subunit signal, either with or without the pro region derived from HSA LACTAPROQ is a food-grade lactase produced by fermentation of a strain of Kluyveromyces lactis.

Yarrowia lipolytica

  • Y. lipolytica has been investigated for use in a number of industrial processes, including the production of a various metabolites.
  • The inherent capacity for high-level secretion, plus the ability to grow to high cell density at industrial scale, have prompted the investigation of Y. lipolytica as a host for heterologous gene expression.
  • Y. lipolytica is a dimorphic yeast, being unicellular in minimal medium containing glucose or n-hexadecane, forming mycelia in minimal medium containing olive oil or casein, and giving a mixture of both forms in complex medium.
  • Mutants which form smooth colonies have been isolated but the molecular events involved in the regulation of growth morphology are uncharacterized.
  • LEU2, LYS2, ADE1,HIS1 and URA3 are used as selection marker.
  • The strong and constitutive promoters from TEF and RPS7 genes, are used.
  • Among the inducible promoters, metallothioneins gene, isocitrate lyase (ICL1), 3-oxo-acyl-CoA thiolase (POT1), and acyl-CoA oxidases (POX1, POX2 and POX5) are being tested.

Advantage of using Yeast expression system

  • P. pastoris can produce expressed foreign proteins either intracellularly or extracellularly. Extracellular production of foreign proteins is most desirable in order to avoid the usual first steps of purification.
  • Pichia also secrete very low levels of native proteins, thus making it easier to recover the foreign secreted protein from the fermentation fluid by simple removal of whole cells by filtration or centrifugation.
  • Moreover, secretion signals can be attached to the protein of interest, causing it to be exported out of the cell.
  • The P. pastoris expression system has been successfully used to produce proteins that are highly disulphide-bonded. Prokaryotic systems have been generally unsuccessful in achieving this, due to the reducing environment of the cytoplasm, resulting in the necessity to refold disulphide-bonded proteins from inclusion bodies, or to secrete the proteins into the periplasmic space
  • Pichia, like other yeasts and fungi, add O-oligosaccharides to the hydroxyl groups of serine and threonine of secreted proteins. It is possible that Pichia will glycosylate heterologous proteins, even when those proteins are not normally glycosylated by the native host; and even when the protein is glycosylated in the native host, Pichia may not glycosylate it on the same serine and threonine residues.

Proteins Expressed in Yeast

  • Humans:
    • μ-Opioid receptor (HUMOR): G-protein coupled receptor (GPCR).
    • Angiostatin: Used in long-term therapy in suppression of metastases.
    • Anti-HBs Fab fragment: Prevention and treatment of hepatits B virus.
    • DNA topoisomerase I (hTopo I): Role in DNA replication, transcription, recombination, chromosome assembly.
    • Granulocyte-macrophage colony-stimulating factor (hGM-CSF): Regulation of activity of mature, differentiated granulocytes and macrophages.
    • Serine protease inhibitor SERpin CI-Inhibitor (CI-Inh): Plasma glycoprotein, inhibitor of inflammatory reactions.
    • Serum albumin: Binding and transport, colloid osmotic pressure.
    • Serum amyloid P (SAP): G-protein, inhibition of DNA auto-antibody formation.
    • Transforming growth factor-β type II receptor extracellular domain (SoIRII): TGF-B antagonist, regulates cell proliferation and differentiation.
  • Plants:
    • Amaryllidaceae snowdrop agglutinin: Binding of α-D-mannose groups.
    • Arabidopsis thaliana core a 1,3-fucosyltransferase: Biosynthesis of N- and O-linked oligosaccharides.
    • Gsp (Panax ginseng C, medicinal peptide): Potential use as drug against diabetes.
    • Maize cysteine proteinase (Mirl):