WO2010034708A1 - Cassette d’expression de polynucléotide - Google Patents

Cassette d’expression de polynucléotide Download PDF

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Publication number
WO2010034708A1
WO2010034708A1 PCT/EP2009/062254 EP2009062254W WO2010034708A1 WO 2010034708 A1 WO2010034708 A1 WO 2010034708A1 EP 2009062254 W EP2009062254 W EP 2009062254W WO 2010034708 A1 WO2010034708 A1 WO 2010034708A1
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protein
hfbii
yeast host
host cell
expression cassette
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PCT/EP2009/062254
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English (en)
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John William Chapman
Nigel Malcolm Lindner
Andrew Baxter Russell
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Unilever Plc
Unilever N.V.
Hindustan Unilever Limited
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Publication of WO2010034708A1 publication Critical patent/WO2010034708A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method of synthesising HFBII protein, a polynucleotide expression cassette that can be used to synthesise H FBI I protein and a host cell comprising such a polynucleotide expression cassette.
  • the present invention also relates to a method of synthesising heterologous proteins in general.
  • Hydrophobins are a family of surface-active proteins produced naturally by filamentous fungi such as Trichoderma reesei. They are smal l protei ns (7-9 kDa) and are characterised by the presence of eight cysteine residues forming four disulphide linkages. Hydrophobins are remarkable for their high surface activity and the fact that they self- assemble at hydrophobic-hydrophilic surfaces or interfaces to form a robust, amphipathic layer. Two classes of hydrophobins have been identified and they are designated class I and class II. Class I hydrophobins (such as SC3 from Schizophyllan ses) form insoluble aggregates which can only be dissolved with strong acids.
  • Class Il hydrophobins (which include HFBI and HFBII from Trichoderma reesei) are more readily solubilised and can be dissolved in aqueous solutions to concentrations of at least 100mg/ml. It has been reported by Nakari-Setala T. et al 1997 that the amino acid similarity of HFBII to HFBI is 69%.
  • hydrophobins outlined above have enabled them to be used in applications such as coatings, emulsion stabilisation and separation technologies. It has also been determined (see Cox A et al) that the class Il hydrophobins HFBI and HFBII have surface elasticities far in excess of that known for any other protein and that, for H FB I I , th is property leads to th e stabi lisation of bu bbles to the process of disproportionation. In part, this appears to be due to the ability of HFBII to be absorbed at the air/water interface of bubbles which thereby stabilises the bubbles.
  • hydrophobins find particular application in the stabilisation of food products (e.g. frozen and chilled food products such as ice cream and mousses) which contain introduced gas in the form of bubbles of air, nitrogen and/or carbon dioxide.
  • food products e.g. frozen and chilled food products such as ice cream and mousses
  • introduced gas in the form of bubbles of air, nitrogen and/or carbon dioxide.
  • Hydrophobins are particularly suitable in this role because even relatively low levels of hydrophobins can provide excellent foam volume stability and inhibition of coarsening
  • HFBII is a useful additive for foam stabilisation
  • Trichoderma reesei produces HFBII only at low levels (Bailey et al. infra).
  • WO96/41882 reports on the expression of two class I hydrophobins, HYPA and HYPB from A. bisporus, in yeast. However, it also acknowledges the difficulties in the secretion of recombinantly expressed hydrophobins from host cells due to the extraordinary properties of these proteins.
  • the present invention has arisen because the inventors have surprisingly found that recombinantly expressing the HFBII gene, together with a suitable extracellular signal peptide, in a host cell, results in a surprisingly high level of expression of the HFBII protein and secretion thereof from the host cell.
  • a polynucleotide expression cassette comprising an extracellular signal sequence operatively associated with an oligonucleotide sequence encoding an HFBII protein.
  • the polynucleotide expression cassette is such that a yeast host cell transformed with the polynucleotide expression cassette is capable of synthesising at least 300mg/1 of the HFBII protein in a 100 hour period.
  • Yeasts are a sub-division of the kingdom of fungi. They span a number of kingdom subdivisions (phyla), but are distinguished by the fact that they are uni-cellular. By contrast, fungi which grow as multicellular organisms are known as moulds or filamentous fungi. Yeasts do not produce hydrophobins naturally, instead hydrophobins are naturally expressed exclusively by filamentous fungi such as Trichoderma reesei.
  • the transformed yeast host cell is capable of synthesising at least 400mg/l, at least 500mg/l or at least 600mg/l of the HFBII protein in a 100 hour period.
  • a host cell is transformed with the expression cassette and then cultured at between 20 0 C and 32°C in rich medium containing yeast extracts.
  • the culture is in a vessel of between 5 and 101 volume to permit oxygen transfer and the glucose feed is limited to the rate at which glucose is used by the yeast or is provided in a controlled feed. Suitable medium and protocols for determining HFBI I synthesis levels are disclosed in the examples herein.
  • synthesis of H FBI I by a particular yeast strain is measured using the fed batch fermentation protocol provided in Example 3. It is to be understood that the method may involve the synthesis of the threshold concentration of the HFBII protein (e.g. 300mg/l or more) in less than 100 hours as there is a tendency for the concentration of the protein to peak and then fall during culturing as foaming over takes place. However, at a time point during the 100 hour period, the concentration of HFBII reaches at least the threshold concentration.
  • the threshold concentration of the HFBII protein e.g. 300mg/l or more
  • the extracellular signal sequence is located 5' upstream of the oligonucleotide sequence encoding the HFBII protein. - A -
  • a yeast host cell transformed with the polynucleotide expression cassette and cultured in medium is capable of synthesising and secreting into the culture at least 300 mg/1 of the HFBII protein in a 100 hour period.
  • the level of synthesis is measured in terms of the amount of protein present in the whole culture.
  • the HFBII protein comprises a sequence at least 80% identical to SEQ. ID NO. 2, preferably at least 90%, 95% or 99% identical thereto.
  • a purified 1 ⁇ M sample of the HFBII protein demonstrates a surface shear elasticity of at least 0.1 Nm "1 at pH 7, 20 0 C after 10,000 seconds.
  • a purified 1 ⁇ M sample of the HFBII protein demonstrates a surface shear elasticity of at least 0.5Nm "1 at pH 7, 20 0 C after 10,000 seconds.
  • the extracellular signal sequence comprises a sequence at least 80% identical to SEQ. ID NO. 3, preferably at least 90%, 95% or 99% identical thereto.
  • the 5' ATG codon is optionally omitted.
  • the expression cassette further comprises a promoter for expression of a gene in a yeast cell.
  • the promoter is located so as to control expression of the HFBII protein.
  • a vector comprising a polynucleotide expression cassette according to the present invention.
  • the vector is a plasmid or a YAC.
  • a yeast host cell comprising a polynucleotide expression cassette according to the invention recombinantly incorporated therein.
  • the cell is a food grade yeast strain, such as a strain complying with the USFDA GRAS designation.
  • the cell is of one of the following strains: Saccharomyces cerevisiae, Kluyveromyces lactis, Schizosaccharomyces pombe, or Yarrowia lipolytica.
  • the cell is of strain CEN.PK102 or strain DXY1 rendered Hsp150 and pmt1 deficient, or a derivative thereof.
  • the polynucleotide expression cassette is integrated into the genome of the yeast host cell.
  • the expression cassette is located in the cell as an extra-genomic vector.
  • a method of synthesising HFBII protein comprising expressing a polynucleotide sequence encoding the HFBII protein in a yeast host cell such that the HFBI I protein is synthesised at a concentration of at least 300mg/1 in a 100 hour period.
  • the yeast host cell comprises a polynucleotide expression cassette according to the invention recombinantly incorporated therein.
  • the method comprises maintaining the yeast host cells in a medium and raising the pH of the medium during the method.
  • the method comprises raising the pH of the medium by at least 1 on the pH scale.
  • the method comprises raising the pH of the medium from between pH 4.5 and pH 5.5 to between pH 6.0 and pH 7.0.
  • the method comprises minimising the amount of foam in the layer above the culture such that after the 100 hour period the foam layer accounts for less than 5% of the volume of the culture, more preferably less than 1% of the volume.
  • a method for synthesising a heterologous protein in a yeast host cell that has been transformed with a polynucleotide comprising a sequence encoding the heterologous protein comprising the steps of:
  • the methods of the invention are performed using a yeast host cell of strain DXY1 rendered Hsp150 and pmt1 deficient.
  • the second pH value is at least 1 higher on the pH scale than the first pH value.
  • the first pH value is between pH 4.5 and pH 5.5 and/or the second pH value is between pH 6.0 and pH 7.0.
  • the heterologous protein is a class Il hydrophobin such as HFBII.
  • the proteins e.g. HFBII
  • yeast in a medium with a pH between 4 and 8, typically between 5 and 7.
  • the percentage "identity" between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters.
  • the B L AS T a l g o r i t h m a n b e accessed on the internet using the URL http://www.ncbi.nlm.nih.gov/blast/.
  • secretion when used in relation to a protein translated in a cell means that the protein passes through the cell membrane and can be isolated from the other components of the cell without disrupting the cell membrane.
  • strain A refers to a yeast strain derived from S. cerevisiae DXY1 yeast strain (reported in Kerry-Williams S. M. et al Disruption of Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin, Yeast, 14, 161-169 (1998)) which has been rendered Hsp150 and pmt1 deficient using the techniques disclosed in WO95/033833 and US5, 714,377, respectively.
  • class Il hydrophobin is defined as an amphiphilic, surface active protein having one or more of the following features:
  • aggregates of the protein can be dissolved using aqueous dilutions of organic solvents
  • sequence of the protein has at least 29% sequence identity to the sequence in SEQ ID NO. 2, preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity thereto;
  • the protein comprises the consensus sequence of SEQ ID NO. 7;
  • the protein comprises a sequence with at least 80% identity with the consensus sequence of SEQ ID NO. 8.
  • Figure 1 is a restriction map of the HFBII synthetic gene of SEQ. ID NO. 4.
  • Figure 2 is a schematic diagram of the Saccharomyces cerevisiae multi-copy integration vector pUR8569.
  • Figure 3 is a schematic diagram of the Hydrophobin expression plasmid pUR901 1.
  • Figure 4 is a schematic diagram of plasmid pSAC35, which is a yeast expression vector described in WO2005/077042.
  • Figure 5 is a graph showing the results of HFBII production over time by CEN.PK102-
  • Figure 6 is a graph showing the results of HFBI I production over time by Strain A- pDB3268 yeast cells in accordance with another example of the present invention.
  • Figure 7 is a graph showing the results of HFBII production over time by yeast cells under the same conditions as the example whose results are shown in Figure 6 except that increased stirring of the culture was carried out to reduce the foam layer on the culture.
  • Figures 8 to 10 are graphs showing the results, in triplicate, of HFBII production over time by Strain A-pDB3268 yeast cells, in which the pH of the culture was raised during the feed phase in accordance with another example of the present invention. Brief Description of the Sequence Listing
  • SEQ. ID NO. 1 is the nucleotide sequence that encodes HFBII from Trichoderma reesei.
  • SEQ. ID NO. 2 is the protein sequence of HFBII as encoded by SEQ. ID NO. 1.
  • SEQ. ID NO. 3 is the nucleotide sequence of the SUC2 signal sequence from S. cerevisiae.
  • SEQ. ID NO. 4 is the nucleotide sequence encoding a fusion of the SUC2 signal sequence and the HFBII protein.
  • SEQ. ID NO. 5 is the protein sequence of HFBII as encoded by SEQ. ID NO. 4.
  • SEQ. ID NO. 6 is the nucleotide sequence of the plasmid pUR901 1 whose construction is described in Example 1.
  • SEQ. ID NO. 7 is an amino acid consensue sequence for class Il hydrophobins.
  • SEQ. ID NO. 8 is another amino acid consensue sequence for class Il hydrophobins.
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid SEQ. IDNO.8
  • Xaa can be any naturally occurring amino acid or no amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid or no amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid ⁇ 220>
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid or no amino acid Detailed Description
  • a polynucleotide vector which com prises a n expression cassette.
  • the expression cassette comprises an oligonucleotide sequence encoding the HFBII protein, as shown in SEQ. ID NO. 1.
  • the oligonucleotide sequence is, at its 5' end, fused to the SUC2 signal sequence, which is shown in SEQ. ID NO. 3.
  • the expression cassette also comprises a strong yeast promoter such as GALF7.
  • the polynucleotide is used to transform a host yeast cell such as a strain of Saccharomyces cerevisiae. Transformation is carried out by the lithium acetate/single- stranded carrier DNA/polyethylene glycol method (e.g. using the Sigma yeast transformation kit) for which further details may be found in Gietz RD, Woods RA Meth. Enzymol. 350: 87:96 Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Alternatively transformation may be carried out by electroporation.
  • the expression vector comprises rDNA sequences for the targeted integration of the expression vector into the host cell genome but in other embodiments, the oligonucleotide vector contains the necessary elements (such as an origin of replication and stability determining genes of the native 2 micron plasmid or centromere sequences) for stable extra-genomic reproduction.
  • the transformed cell After transformation of the yeast host cell, the transformed cell is cultured in medium such that expression of the HFBII protein takes place and the protein is secreted into the cell culture medium.
  • the SUC2 signal sequence is cleaved off from the nascent protein during the expression and synthesis process.
  • the HFBII protein is then extracted from the culture medium by filtration and purified.
  • a concentration of at least 300 mg/l of the HFBII protein is secreted into the culture after 100 hours by the transformed cell culture. The level of secretion of the protein is measured in the whole culture, although the protein is predominantly present in the fermentation supernatant.
  • This level of synthesis is achieved under optimal conditions including fermentation in a container of 101 volume, to permit a high oxygen transfer rate, and using separate batch and feed phases.
  • yeast growth is optimised using an excess of glucose.
  • yeast growth and protein synthesis are balanced and optimised by limiting the feed of glucose.
  • This level of expression in a yeast host cell such as S. cerevisiae is surprising because hydrophobic proteins such as HFBII are not normally well expressed in yeast. Because the protein is secreted into the cell culture medium, it is possible to recover the protein without disrupting the plasma membranes of the host cells. This avoids the need for an additional step to separate the protein from other cell components.
  • around 10% of synthesised hydrophobin becomes attached to the external cell wall of the hosts cells. However, this hydrophobin is still regarded as being "secreted” because it can be recovered without disrupting the cell membranes, for example by washing the attached hydrophobin from the cell wall with ethanol.
  • an oligonucleotide encoding the HFBII protein, in accordance with SEQ. ID NO. 1.
  • the oligonucleotide sequence is varied without significantly affecting the properties of the resulting HFBII protein.
  • any oligonucleotide sequence which encodes the HFBI I protein as set out in SEQ. ID NO. 2 may also be used in alternative embodiments.
  • various additions, deletions and substitutions of amino acid residues of the HFBII protein sequence that is encoded may be made without significantly affecting the properties of the HFBII protein.
  • the HFBII protein must have at least 80% sequence identify to SEQ. ID NO.
  • variant proteins demonstrate a surface shear elasticity of at least 0.1 Nm "1 at pH 7, 2O 0 C after 10,000 seconds when at a concentration of at least 1 ⁇ M, this being the principal functional characterisation of the HFBII protein.
  • the variant protein demonstrates a surface shear elasticity of at least 0.5 Nm "1 under the same conditions.
  • These thresholds are derived from Cox A. et al. 2007 in which it is reported that the surface shear elasticity of HFBII protein is "far in excess" of proteins such as sodium caseinate, ⁇ -casein and ⁇ -lactoglobulin.
  • Cox A. et al is hereby incorporated by reference, particularly as regards the methodology for determining surface shear elasticity of a protein.
  • amino acid residue number 216 of SEQ. I D NO: 1 i.e. the C-terminal phenylalanine residue
  • a different extracellular signal sequence from SUC2 may be used, such as a sequence with at least 80% sequence identity to SEQ. ID NO. 3.
  • one of the 5' ATG codons of the sequence may be omitted.
  • an entirely different extracellular signal sequence may be used, for example the pre-pro signal sequence of yeast mating factor alpha. What is important is that the signal sequence results in the HFBII protein being secreted into the yeast cell growth medium rather than being retained within the cell.
  • the polynucleotide vector is a plasmid.
  • the vector is of a different type such as a yeast artificial chromosome (YAC).
  • YAC comprises the minimum elements required for independent reproduction, namely a telomere, centromere and replication of origin sequence elements.
  • the yeast host cell is a strain of Saccharomyces cerevisiae.
  • other yeast strains are used, in particular, food grade yeast strains.
  • food grade yeast strains are those complying with the GRAS (Generally Regarded As Safe) designation given by the USA Food and Drug Administration.
  • preferred food grade yeast strains include: Kluyveromyces lactis, Schizosaccharomyces pombe, and Yarrowia lypolytica.
  • the yeast strain In order for the H FBI I protein to be used in the applications described above, it is desirable, or even essential, for the protein to be non-glycosylated. Accordingly, it is advantageous for the yeast strain to have a reduced level of glycosylation of secreted proteins, for example by being deficient in expression of the pmt1 gene. For this reason, particularly preferred strains of Saccharomyces cerevisiae are CEN.PK102-3 ⁇ ga/7 ⁇ pmt1 , Strain A, and derivatives thereof that, when transformed with an expression cassette of the invention, are capable of synthesising at least 300mg/L of the HFBII protein in a 100 hour period.
  • yeast strains in particular Strain A which is disclosed herein
  • HFBII heterologous proteins
  • maintaining yeast strains at such pH levels permits contaminating bacteria (whose presence in batch fermentations is difficult to avoid) rapidly to outgrow the yeast strains and therefore limit effective yeast multiplication and protein production.
  • maintaining yeast host cells in growth medium at a relatively low pH value e.g. of around pH 5
  • a relatively low pH value e.g. of around pH 5
  • the pH of the growth medium in which the cells are located is raised to around pH 6.5 (or, alternatively, the growth medium is replaced with a new growth medium with a pH of around 6.5).
  • the higher pH level of the growth medium results in a greater level of production of the heterologous protein (e.g. HFBII) and the high quantity of the yeast biomass is, by this stage in the fermentation process, sufficient to ensure that it is not overgrown by any remaining bacterial contaminants.
  • the heterologous protein e.g. HFBII
  • HFBII protein in solution otherwise migrates into the foam, thereby reducing the yield of the protein that can be recovered from the culture.
  • the fermentation is stirred at multiple (e.g. 3) levels within the fermentation vessel.
  • an anti-foaming agent such as Struktol J647 is provided.
  • An anti-foaming agent may be provided in combination with one of the stirring techniques previously described.
  • An expression cassette for the Trichoderma reesei HFBII protein was constructed as follows.
  • a synthetic gene (SEQ. ID NO. 4) was designed encoding the HFBII sequence (SEQ. ID NO. 1 ) fused to the Saccharomyces cerevisiae SUC2 signal sequence (SEQ. ID NO. 3), based on the mature protein sequence of HFBII (Genbank accession number P79073, Nakari-Setala,T., Aro,N., llmen.M., Munoz,G., Kalkkinen,N. and Penttila.M (1997).
  • the gene was synthesised commercially at Baseclear (Einsteinweg 5, P.O. Box 1336, 2333 CC Leiden, The Netherlands) and cloned in the cloning vector pGEMTteasy (Promega).
  • the resulting plasmid was digested with Sacl and Hin ⁇ ⁇ ⁇ and the 303bp fragment containi ng the expression cassette extracted and purified after agarose gel electrophoresis.
  • This fragment was ligated with the 8914bp Sac ⁇ /Hin ⁇ fragment of the vector pUR8569 (a schematic diagram of which is shown in Figure 2), designed to integrate at the rDNA locus as described by Lopes et al (Lopes TS, Klootwijk J, Veenstra AE, van der Aar PC, van Heerikhuizen H, Ra ⁇ e HA and Planta RJ. 1989 High-copy- number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression Gene 15;79: 199-206). The ligation mix was used to transform Escherichia coli TG1 (Sambrook, J., Fritsch, E. F.
  • pUR901 1 was digested with Hpa ⁇ and the 6559bp fragment isolated and purified after agarose gel electrophoresis. This fragment lacks the E. coli and ampicillin resistance gene DNA but contains the expression cassette for HFBII; rDNA sequences for the targeted integration of this fragment in the S. cerevisiae genome; the Ieu2d gene which acts as a selection marker for amplification of the cassette in let/2 auxotrophic mutants and a region of S.
  • This 6559bp fragment was used to transform the S. cerevisiae strain CEN.PK102-3 ⁇ ga/1 ⁇ gal1:URA3, Ieu2, ura3) to leucine prototrophy (Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method. Methods in Molecular Biology:313:107-120 (2005)).
  • the resulting transformants were screened for expression of HFBII by growth for 48 hours in YNB minimal medium (Difco Yeast Nitrogen Base, 2% glucose) followed by 48 hours growth in inducing medium (1% yeast extract, 2% peptone, 2%glucose, 0.5% galactose).
  • the culture was extracted by addition of ethanol to 60% vol/vol and the cells removed by filtration through a 2 ⁇ m milipore filter.
  • the presence of HFBII was assessed by HPLC analysis using the assay conditions shown at the end of this Example.
  • the results are shown in Table 1.
  • the level of protein secretion is measured in terms of the amount in the whole culture.
  • LCMS l iq u id chromatography mass spectroscopy
  • Example 2 - Expression of HFBII in Yeast strain with impaired O-glycosylation WO94/04687 describes the preparation of S. cerevisiae deficient in 0-mannosylation activity.
  • the 6559bp fragment of pUR901 1 was used to transform the S. cerevisiae strain CEN.PK102-3 ⁇ ga/1 ⁇ pmt1 (gal1:ura3, Ieu2, ura3, pmt1:URA3) to leucine prototrophy .
  • the resulting transformants were screened for HFBII production as described above.
  • Example 3 Production of HFBII in small scale fermentation using strain CEN.PK102- 3 ⁇ al1 ⁇ pmti -DURSO'I 1#2. Inoculum Preparation
  • a shake flask containing 50 ml YNB medium was inoculated with a 1.4 ml glycerol stock of the strain and incubated for 48 hours at 30 degrees C. at 120 rpm. Subsequently, the inoculum was transferred to a shake flask containing 500 ml medium consisting of 10 g/l Yeast extract (Difco), 20 g/l Bacto Peptone (Difco) and 20 g/l glucose and incubated for 24 hours, 30degrees C at 120 rpm.
  • Fed Batch Fermentations 10 g/l Yeast extract (Difco), 20 g/l Bacto Peptone (Difco) and 20 g/l glucose and incubated for 24 hours, 30degrees C at 120 rpm.
  • the 5.5L batch medium consisted of 22 g/kg glucose, 10 g/kg yeast extract KatG (OhIy),
  • the 4L feed medium contained 440 g/kg glucose, 3 g/l galactose (Duchefa), 25 g/kg yeast extract, 12 g/kg KH 2 PO 4 , 2.5 g/kg MgSO 4 JH 2 O, 0.8 g/kg, Struktol J647, 20 g/kg EgIi trace metals, 2 g/kg EgIi vitamins.
  • the fed batch fermentations were performed in standard bioreactors with a working volume of 10 litres.
  • Dissolved oxygen (DO 2 ) was measured with an lngold DO 2 electrode (Mettler-Toledo) and controlled by automatic adjustment of the speed of the 6-bladed Rushton impeller to a maximum of 1000 rpm.
  • the pH was measured with an lngold lnpro 3100 gel electrode (Mettler-Toledo) and controlled using 3 M phosphoric acid (Baker) and 12.5% v/v ammonia (Merck).
  • Temperature was measured by a PT100 electrode and controlled via a cooling jacket and cooling and heating fingers.
  • the batch phase was started by transferring 500 ml of full grown inoculum to the batch medium.
  • the temperature was maintained at 30 degrees C and airflow at 2 l/min. DO 2 was controlled above 30% and the pH at 5.0.
  • the feed phase was started.
  • the airflow was set to 6 l/min.
  • the feed rate was applied according to an exponential profile required to maintain a growth rate of 0.06 l/h. The exponential feed continued until the DO 2 level in the fermenter decreased below 15% after which a linear feed rate was maintained.
  • Example 4 Expression of HFBII in a yeast with improved protein secretion properties It is possible to increase the secretion capacity of yeast by selecting specifically for strains that have a higher capacity to secrete heterologous proteins (GB2239315 and Sleep D, Belfield GP, Ballance DJ, Steven J, Jones S, Evans LR, Moir PD and Goodey AR. (1991 ) Saccharomyces cerevisiae strains that over express heterologous proteins, Biotechnology N.Y. 9: 183-187).
  • WO95/023857 shows how deletion of the protease YAP3 and/or KEX2 proteases also increases heterologous expression.
  • WO99/000504 describes how deletion of UBC4 and/or UBC5 genes increases plasmid copy number of the yeast 2 ⁇ plasmid and vectors based on the yeast 2 ⁇ plasmid.
  • WO95/33833 describes how deletion of hsp150 can also improve heterologous protein production by removal of this specific contaminating yeast protein.
  • the S. cerevisiae DXY1 yeast strain (reported in Kerry-Williams S. M. et al Disruption of Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin, Yeast, 14, 161-169 (1998)) was rendered Hsp150 and pmt1 deficient using the techniques disclosed in WO95/033833 and US5, 714,377, respectively, in order to generate new Strain A.
  • Strain A has the following genotype: Ieu2, pra1, ubc4, yap3, hsp150, pmt1.
  • WO88/00802 describes a stable 2 ⁇ based yeast expression vector suitable for gene expression in such strains.
  • a derivative of this vector, pSAC35 (a schematic diagram of which is shown in Figure 4) is described in WO2005/077042 and by Sleep et al (1991 ).
  • An expression cassette for the synthetic HFBII gene was constructed.
  • the expression cassette may be constructed in a similar manner to that described in WO2005/061718.
  • the expression cassette contained the S. cerevisiae PREfI promoter, the invertase signal sequence linked to the HFBII structural gene and the S. cerevisiae ADM terminator sequence.
  • a Not ⁇ fragment carrying this expression cassette was ligated into pSAC35.
  • the resulting plasmid, pDB3268 was introduced into S. cerevisiae strain Strain A using the LiAc technique. It is to be noted that the SUC2 signal sequence encoded within this plasmid was modified and contains only a single N-terminal methionine.
  • One of the resulting transformants was grown for 98h at 3O 0 C in a shake flask culture containing YEPS (Yeast extract, 1 g/l, peptone 2 g/l, sucrose, 2 g/l) and was shown to produce 102 mg/l hydrophobin as determined by HPLC assay.
  • YEPS Yeast extract, 1 g/l, peptone 2 g/l, sucrose, 2 g/l
  • Strain A-pDB3268 was grown for 48 hours in BMMS (Difco yeast nitrogen base, without amino acids, without NH 4 SO 4 , 1.7 g/l, NH 4 SO 4 5 g/l, citric acid monohydrate 6.09 g/l, Na 2 HPO 4 .2H 2 O 2.527 g/l, sucrose 20g/l) and 60 mg of cell dry weight used to inoculate a fermenter containing 6 I batch medium.
  • the temperature was maintained at 30 degrees C and airflow at 2 l/min. DO 2 was controlled above 30% and the pH at 6.5.
  • the feed phase was started. In the feed phase the airflow was set to 6 l/min.
  • the feed rate was applied according to an exponential profile required to maintain a growth rate of 0.06 l/h. The exponential feed continued until the DO 2 level in the fermenter decreased below 15% after which a linear feed rate was maintained.
  • a reference batch was prepared as follows. Strain A-pDB3268 was grown for 48 hours in BMMS (see above) and 100 ml was used to inoculate the batch medium. 8 test batches were prepared as follows. Strain A-pDB3268 was grown for 48 hours in BMMS (see above) and 100 ml was used to inoculate 500ml YPD and grown for 24 hours to give a batch time of 30 hours.
  • YPD was yeast-extract, pepton and dextrose broth medium comprising 2% yeast extract, 1 % peptone and 2% glucose. To prepare YPD, all ingredients were dissolved in demi-water and sterilised at 121 ° C for 15 minutes. The results and details of other culturing parameters are shown in Table 10. It can be seen that all batches produced a concentration of at least 300mg/l of HFBII, or would have done had fermentation continued for 100 hours.
  • AF Anti-foaming agent (concentration shown is relative to Reference Batch 2008003)
  • Remark 3 SS was not forced at min 800 rpm, so that the foam was out of the control.
  • Remark 4 after 76 h: airflow 4L/min with 02 20%.

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Abstract

La présente invention concerne une cassette d’expression de polynucléotide comprenant une séquence de signal extracellulaire fonctionnellement associée à une séquence d’oligonucléotide codant pour la protéine HFBII. La cassette d’expression de polynucléotide est telle que lorsqu’une cellule hôte de levure est transformée avec la cassette d’expression, la cellule est capable de synthétiser au moins 300 mg/l de HFBII en une durée de 100 heures.
PCT/EP2009/062254 2008-09-23 2009-09-22 Cassette d’expression de polynucléotide WO2010034708A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015001049A1 (fr) 2013-07-04 2015-01-08 Novartis Ag Cellules fongiques filamenteuses déficientes en o-mannosyltransférase et leurs procédés d'utilisation
US20170327774A1 (en) * 2016-05-10 2017-11-16 The Procter & Gamble Company Cleaning composition
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041882A1 (fr) * 1995-06-12 1996-12-27 Proefstation Voor De Champignoncultuur Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression
US20030143191A1 (en) * 2001-05-25 2003-07-31 Adam Bell Chemokine beta-1 fusion proteins
WO2005061718A1 (fr) * 2003-12-23 2005-07-07 Delta Biotechnology Limited Technique d'expression genique
EP1623631A1 (fr) * 2004-07-27 2006-02-08 Unilever Plc Produits alimentaires aérés, contenant de l'hydrophobine
US20070026013A1 (en) * 2001-04-12 2007-02-01 Human Genome Sciences, Inc. Albumin fusion proteins

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041882A1 (fr) * 1995-06-12 1996-12-27 Proefstation Voor De Champignoncultuur Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression
US20070026013A1 (en) * 2001-04-12 2007-02-01 Human Genome Sciences, Inc. Albumin fusion proteins
US20030143191A1 (en) * 2001-05-25 2003-07-31 Adam Bell Chemokine beta-1 fusion proteins
WO2005061718A1 (fr) * 2003-12-23 2005-07-07 Delta Biotechnology Limited Technique d'expression genique
EP1623631A1 (fr) * 2004-07-27 2006-02-08 Unilever Plc Produits alimentaires aérés, contenant de l'hydrophobine

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BAE C S ET AL: "Improved process for production of recombinant yeast-derived monomeric human G-CSF", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 52, no. 3, September 1999 (1999-09-01), pages 338 - 344, XP002515608, ISSN: 0175-7598 *
BAILEY M J ET AL: "Process Technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 58, 1 January 2002 (2002-01-01), pages 721 - 727, XP003015238, ISSN: 0175-7598 *
CALADO CECILIA R C ET AL: "Optimisation of culture conditions and characterisation of cutinase produced by recombinant Saccharomyces cerevisiae", ENZYME AND MICROBIAL TECHNOLOGY, vol. 31, no. 1-2, 1 July 2002 (2002-07-01), pages 161 - 170, XP002515609, ISSN: 0141-0229 *
ERIK BÖER ET AL: "Yeast expression platforms", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 77, no. 3, 9 October 2007 (2007-10-09), pages 513 - 523, XP019560739, ISSN: 1432-0614 *
KIM SE HOON ET AL: "Culture method to enhance the productivity of hepatitis B surface antigen (pre S2 + S-Ag) with recombinant Saccharomyces cerevisiae", BIOTECHNOLOGY TECHNIQUES, CHAPMAN & HALL, vol. 10, no. 4, 1 April 1996 (1996-04-01), pages 233 - 238, XP009112452, ISSN: 0951-208X *
KUBICEK CHRISTIAN P ET AL: "Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea", BMC EVOLUTIONARY BIOLOGY, BIOMED CENTRAL LTD., LONDON, GB, vol. 8, no. 1, 10 January 2008 (2008-01-10), pages 4, XP021032522, ISSN: 1471-2148 *
LINDER ET AL: "Hydrophobins: the protein-amphiphiles of filamentous fungi", FEMS MICROBIOLOGY REVIEWS, ELSEVIER, AMSTERDAM, NL, vol. 29, no. 5, 1 November 2005 (2005-11-01), pages 877 - 896, XP005107912, ISSN: 0168-6445 *
NAKARI-SETALA T ET AL: "DIFFERENTIAL EXPRESSION OF THE VEGETATIVE AND SPORE-BOUND HYDROPHOBINS OF TRICHODERMA REESEI CLONING AND CHARACTERIZATION OF THE HFB2 GENE", EUROPEAN JOURNAL OF BIOCHEMISTRY, BERLIN, vol. 248, 1 January 1997 (1997-01-01), pages 415 - 423, XP002949768, ISSN: 0014-2956 *
WHITEFORD JAMES R ET AL: "Hydrophobins and the interactions between fungi and plants", MOLECULAR PLANT PATHOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 3, no. 5, 1 September 2002 (2002-09-01), pages 391 - 400, XP009125460, ISSN: 1464-6722, [retrieved on 20020830] *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015001049A1 (fr) 2013-07-04 2015-01-08 Novartis Ag Cellules fongiques filamenteuses déficientes en o-mannosyltransférase et leurs procédés d'utilisation
US10724013B2 (en) 2013-07-04 2020-07-28 Glykos Finland Oy O-mannosyltransferase deficient filamentous fungal cells and methods of use thereof
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
US20170327774A1 (en) * 2016-05-10 2017-11-16 The Procter & Gamble Company Cleaning composition
US10640737B2 (en) * 2016-05-10 2020-05-05 The Procter & Gamble Company Cleaning composition

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