WO2001020007A1 - Systeme multifonctionnel de manipulation efficace de l'expression de proteines dans des champignons filamenteux, et son procede d'utilisation - Google Patents

Systeme multifonctionnel de manipulation efficace de l'expression de proteines dans des champignons filamenteux, et son procede d'utilisation Download PDF

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WO2001020007A1
WO2001020007A1 PCT/CA2000/001084 CA0001084W WO0120007A1 WO 2001020007 A1 WO2001020007 A1 WO 2001020007A1 CA 0001084 W CA0001084 W CA 0001084W WO 0120007 A1 WO0120007 A1 WO 0120007A1
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expression
protein
laca
glatt
promoter
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PCT/CA2000/001084
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English (en)
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Susan Sillaots
Amalia Martinez-Perez
Adrian Tsang
Reginald Storms
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Concordia University
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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
    • C12N15/67—General methods for enhancing the expression
    • 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

Definitions

  • the present invention relates to filamentous fungi. More particularly, the invention relates to a multifunctional system for the efficient manipulation of protein expression in filamentous fungi and method using same. The invention also relates to plasmid vectors adapted for expression in filamentous fungi. In addition, the invention relates to methods to increase the efficiency and reduce the time required for isolating strains that express proteins of interest at high levels, and to such strains.
  • Gene expression in eukaryotic cells is often studied by linking a promoter sequence to an easily detectable "reporter” gene.
  • reporter genes include firefly luciferase, and E. coli chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase and ⁇ -glucuronidase.
  • Reporter genes simplify gene expression studies by "reporting" upstream transcnptional events through the expression of an easily assayable reporter protein
  • reporter genes monitor regulatory DNA associated with promoters (cis-acting sequences) and DNA-binding proteins (trans-acting factors)
  • reporter genes are used in a variety of applications including studies of protein-protein interactions (Fields and Song, 1989), recombination events (Herzing et al , 1993), gene targeting (Vile et al , 1993), RNA processing (Huang et al , 1990), signal transduction (Medema et al , 1992 and Sakoda et al , 1992) and transformation efficiency (Takahashi et al 1991 , and Hall et al 1992)
  • a repertoire of practical reporter vectors and a robust assay would be invaluable
  • the present invention concerns a set of expression vectors for studying gene expression in filamentous fungi in general and demonstrates that these vectors can be used to isolate strains that express secreted proteins at the very high levels required for industrial-scale protein production
  • the invention pertains to the filamentous fungi A niger
  • the expression vectors constructed fall into two classes, integrating plasmids and autonomously replicating plasmids.
  • the utility of these vectors for studying gene expression has been dramatically facilitated by including a reporter gene that can be assayed easily.
  • the successful utilization of the ⁇ -galactosidase coding LacA gene from A. niger as a reporter The encoded lactase is easily assayed and efficiently secreted, making it ideal for assessing gene expression and protein secretion in A. niger.
  • the LacA reporter was successfully used in a high-throughput indicator plate-based screen to isolate strains with improved protein expression characteristics. It was also used to engineer derivatives of the
  • GlaA promoter which directed dramatically increased levels of expression.
  • the present invention also relates to an expression vector capable of enabling a systematic analysis of gene expression and/or high- throughput strain improvement screens in a filamentous fungi comprising: a) a selectable marker for said filamentous fungi; and b) a promoter operably linked to a nucleic acid sequence encoding a protein of interest or part thereof.
  • the present invention relates to a filamentous fungi strain, enabling the expression of a protein of interest to greater than about 100 times, and preferably greater than about 1000 times, as compared to the level thereof in parent strain of the filamentous fungi strain.
  • rDNA recombinant DNA
  • nucleic acid molecule refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g. genomic
  • DNA DNA, cDNA
  • RNA molecules e.g. mRNA
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized.
  • DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]).
  • recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering.
  • DNA segment is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
  • amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
  • oligos are designed to bind to a complementary sequence under selected conditions.
  • the nucleic acid e.g. DNA or RNA
  • the nucleic acid may be obtained according to well-known methods.
  • Oligonucieotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
  • the oligonucieotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
  • the oligonucieotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
  • DNA molecule or sequence (as well as sometimes the term “oligonucieotide”) refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form, and comprises or includes a "regulatory element” according to the present invention, as the term is defined herein.
  • oligonucieotide or “DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction.
  • Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989, supra) and are commonly known in the art.
  • a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 ⁇ g/ml denatured carrier DNA (e.g. salmon sperm DNA).
  • the non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency).
  • the selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
  • Tm melting temperature
  • RNA-DNA hybrids can also be formed and detected.
  • the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al.,1989, supra).
  • Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyhbonucleic acid (DNA), and preferably of DNA. The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and
  • RNA detection Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds Other detection methods include kits containing probes on a dipstick setup and the like
  • Probes can be labeled according to numerous well-known methods (Sambrook et al , 1989, supra)
  • Non-limiting examples of labels include 3 H, 14 C, 32 P, and 35 S
  • Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies
  • Other detectable markers for use with probes which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe
  • radioactive nucleotides can be incorporated into probes of the invention by several methods Non-iimiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of£ coli in the presence of radioactive dNTP (e g uniformly labeled DNA probe using random oligonucieotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like
  • radioactive dNTP e g uniformly labeled DNA probe using random oligonucieotide primers in low-melt gels
  • SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxy
  • a "primer” defines an oligonucieotide, which is capable of annealing to a target sequence, thereby creating a double stranded region, which can serve as an initiation point for DNA synthesis under suitable conditions.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • transcription-based amplification the Q ⁇ replicase system
  • NASBA Kermuth et al., 1989, Proc. Natl. Acad. Sci.
  • amplification will be carried out using PCR.
  • PCR Polymerase chain reaction
  • U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 the disclosures of all three U.S. Patent are incorporated herein by reference.
  • PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucieotide primer for each strand of the specific sequence to be detected.
  • An extension product of each primer, which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith.
  • the extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.
  • the sample is analyzed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
  • EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
  • the term "gene” is well-known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
  • a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise to a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats, which are well- known in the art.
  • a “heterologous” e.g. a heterologous gene
  • DNA molecule is a subsegment of DNA within a larger segment that is not found in association therewith in nature.
  • the term 'heterologous can be similarly used to define two polypeptidic segments not joined together in nature.
  • heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, ⁇ -galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides.
  • vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well-known in the art.
  • expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
  • expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host
  • the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences
  • control element sequences such as promoter sequences
  • Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript
  • two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence
  • two sequences In order to be "operably linked” it is not necessary that two sequences be immediately adjacent to one another
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcnptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites
  • transcnptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites
  • Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest
  • This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (e g SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography )
  • the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies The purified protein can be used for therapeutic applications
  • the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucieotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule
  • Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence
  • the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
  • a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
  • CCAT Prokaryotic promoters contain -10 and -35 consensus sequences, which serve to initiate transcription and the transcript products contain Shine-Dalgarno sequences, which serve as nbosome binding sequences during translation initiation
  • the designation "functional derivative” denotes, in the context of a functional derivative of a sequence whether a nucleic acid or ammo acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence
  • This functional derivative or equivalent may be a natural derivative or may be prepared synthetically
  • Such derivatives include ammo acid sequences having substitutions, deletions, or additions of one or more ammo acids, provided that the biological activity of the protein is conserved
  • derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained
  • the substituting ammo acid generally has chemico-physical properties which are similar to that of the substituted ammo acid
  • the similar chemico-physical properties include, similarities in charge, bulk ess, hydrophobicity, hydrophy city and the like
  • the term "functional derivatives" is intended to include "fragments",
  • variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention
  • the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology All these methods are well-known in the art
  • “chemical derivatives” is meant to cover additional chemical moieties not normally part of the subject matter of the invention Such moieties could affect the physico-chemical characteristic of the derivative (e g solubility, absorption, half life, decrease of toxicity and the like) Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980) Methods of coupling these chemical-physical moieties to a polypeptide or nucleic acid sequence are well-known in the art
  • allele defines an alternative form of a gene which occupies a given locus on a chromosome
  • a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell
  • a mutation can be, for example, a detectable change in one or more deoxy bonucleotide
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position Spontaneous mutations and experimentally induced mutations exist
  • a mutant polypeptide can be encoded from this mutant nucleic acid molecule
  • the term "purified” refers to a molecule having been separated from a cellular component
  • a “purified protein” has been purified to a level not found in nature
  • a "substantially pure” molecule is a molecule that is lacking in most other cellular components
  • a host cell or indicator cell has been 'transfected” by exogenous or heterologous DNA (e g a DNA construct) when such DNA has been introduced inside the cell
  • the transfectmg DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell In prokaryotes, yeast, and mammalian cells for example, the transfectmg DNA may be maintained on a episomal element such as a plasmid
  • Figure 1 shows A niger shuttle vectors constructed for this study
  • the various modules are indicated in the figure as follows, pUC18 sequences (pUC), glucoamylase transciption terminator (GlaTt), glucoamylase promoter region (GlaPr), ⁇ -galactosidase coding region (LacA), the selectable marker (PyrG) and the sequence to support autonomous replication (AMA)
  • pUC18 sequences pUC
  • GaTt glucoamylase transciption terminator
  • GaPr glucoamylase promoter region
  • LacA the selectable marker
  • AMA sequence to support autonomous replication
  • AMA sequence to support autonomous replication
  • the present invention had at least three objectives. The first was to develop a modular set of cassettes and vectors for studying gene expression and protein secretion in filamentous fungi. The second was to develop a rational and efficient high-throughput reporter gene-based screen that could be used to isolate strains that express proteins of commercial importance at high levels. The third was to demonstrate that these expression vectors in combination with the high-throughput screen could be used to generate strains that express commercially valuable proteins at the high levels required for industrial applications.
  • pGlaPr-GlaTt is an integrating vector which can be used to express any protein coding sequence. It contains unique cloning sites for the insertion of protein coding information flanked by regulatory information for transcription initiation and transcription termination derived from the A. niger GlaA gene.
  • GlaA gene was used as the source of these control signals because it is quite well characterized (Hata et al. 1992; Verdoes et al., 1993; Fowler et al., 1990; Santerre et al., 1999) and because it has been used to direct high levels of both homologous (Withers et al , 1998) and heterologous (Carrez et al , 1990, Jeenes et al , 1994, and Jeens et al 1993) protein expression Analysis of the sequenced GlaA promoter region (Genbank accession # K02465) and its upstream sequences revealed a potential gene that coded for a protein with 42% identity to a Candida rugosa t ⁇ acylglycerol lipase precursor (Genbank accession # AF044078) The start and stop codons for this putative lipase gene begin and end respectively 2840 and 660 base pairs upstream of the GlaA coding region Thus A niger expression vectors utilizing GlaA
  • LacA encodes a reporter suitable for studying both gene expression and protein secretion in A. niger
  • the A. niger LacA gene codes for a secreted ⁇ -galactosidase (Kumar et al., 1992).
  • X eLacA coding region was cloned into the backbone of plasmid pGlaPr-GlaTt prepared by Nhe ⁇ and Fsel digestion.
  • the resulting plasmid was designated pGlaPr-LacA-GlaTt.
  • a second plasmid, pPkiPr-LacA-GlaTt, with LacA expressed from the PkiA promoter was also constructed.
  • Lactase expression from both pGlaPr-LacA- GlaTt and pPkiPr-LacA-GlaTt was compared. Cotransformation was performed with 1 ⁇ g of plasmid pPyrG and 10 ⁇ g of pGlaPr-LacA-GlaTt or pPkiPr-LacA- GlaTt. Transformants harbouring both plasmids were obtained by screening for blue colonies on selective minimal medium indicator plates containing X-gal. Blue PyrG * colonies therefore identify transformants harbouring both integrating plasmids.
  • pGlaPr-LacA-GlaTt and 6 pPkiPr-LacA-GlaTt dark blue colonies were selected and grown in 30 ml liquid cultures and assayed for ⁇ -galactosidase activity.
  • the mean activity obtained for the pGlaPr-LacA-GlaTt and pPkiPr-LacA-GlaTt transformants was 4.3 (+/- 2.2) and 5.3 (+/- 3.1 ) units as compared to the less than 0.01 units expressed by transformants harbouring only pPyrG (Table 1).
  • a Co-transformations were performed with 1 ⁇ g pPyrG and 10 ⁇ g of the indicated plasmids.
  • pGlaPr-LacA-GlaTt-PyrG transformation was performed with 1 ⁇ g of plasmid DNA.
  • Number of PyrG * LacA * transformants was determined by counting the number of blue PyrG * colonies present of minimal medium with X-gal.
  • the lactase producing transformants characterized above were obtained by cotransformation with plasmid pPyrG and either pGlaPr-LacA- GlaTt or pPkiPr-LacA-GlaTt. Cotransformation was required because the reporter gene expression cassettes were present on plasmids lacking a selectable marker. To simplify the transformation procedure and the characterization of transformants a vector that included a selectable marker was constructed. This plasmid, pGlaPr-LacA-GlaTt-PyrG, has theE. nidulans PyrG gene (prepared by PCR as described herein).
  • a system that can be used for rapid high-throughput indicator plate screening that also facilitates the isolation and characterization of mutations that improve protein expression was developed This system utilizes the AMA replicator sequence that supports autonomous replication Adding a replicator to the expression vectors facilitates the identification of vector versus host mutations that alter reporter gene expression
  • An AMA based module was constructed and cloned into the unique Not ⁇ site of plasmid pGlaPr-GlaTt-PyrG Reporter gene expression and the transformation frequency obtained with a LacA expressing derivative, pGlaPr-LacA-GlaTt-PyrG- AMA, were compared with the values obtained with the integrating plasmid pGlaPr-LacA-GlaTt-PyrG (Table 2) Levels of expression from both vectors were essentially the same, however, including the AMA replicator increased the transformation frequency 35-fold (Table 2) to 100-fold (data not shown)
  • the AMA module supports, high transformation frequencies, autonomous replication and high levels of lactase expression
  • Transformations and ⁇ -galactosidase activity assays were determined as described for Table 1.
  • the cultures used were 30 ml cultures of transformants that were selected for expressing relatively high levels of LacAp (dark blue colonies) as described in the Examples.
  • the transformants obtained with the self-replicating vector could be cured of the plasmid and the plasmid could be retrieved into E. coli for further analysis. Plasmid retrieval was attempted with 9 independent pGlaPr-LacA-GlaTt- PyrG-AMA transformants. The data in Table 3 show that 8 of the ⁇ transformants could be cured of their plasmids and that the plasmids could be readily retrieved into E. coli. Table 3- pGlaPr-LacA-GlaTt-PyrG-AMA transformants of A. niger are unstable and the plasmid can be readily retrieved into E. coli
  • GlaA promoter deletion set was constructed (Fig 2).
  • the plasmids pGla-670Pr-LacA-GlaTt-PyrG-AMA through pGla-89Pr-LacA- GlaTt-PyrG-AMA were transformed into strain N593 and ⁇ -galactosidase expression by the transformants in MM with maltose and in MM with glucose as the carbon source was determined.
  • Two of the seven regions defined bythese promoter deletions harbour upstream information important for the regulation of GlaA (Table 4). Sequentially deleting these two important upstream regions, overlapping sequences between -483 and -454 bp, and -454 and -369 bp, reduced expression from 2.5 units to 0.7 units and less than 0.01 units respectively.
  • the autonomously replicating vector pGlaPr-LacA-GlaTt-PyrG-AMA and its derivatives can therefore be used to characterize plasmid-based sequences that affect expression levels and/or the efficiency of protein secretion.
  • GlaA promoter Each promoter construct extends from the identical 3' nucleotide
  • the modular expression system of the present invention was tested to assess whether it could be used for high-throughput strain improvement screening to obtain mutants that secreted much higher amounts of ⁇ - galactosidase than the 1.5 units per ml typically expressed by pGlaPr-LacA- GlaTt-PyrG-AMA transformants.
  • pGlaPr-LacA-GlaTt-PyrG-AMA transformant was subjected to three rounds of mutagenesis where each round was followed by high-throughput screening for mutants that expressed increased levels of lactase. Each round of mutagenesis was performed as follows.
  • Conidia were mutagenized with nitrosoguanidine (15 ⁇ g/ml for 40 minutes) to give a final survival of 0.2%. Following mutagenesis the conidia were plated on minimal medium plates with 0.1% Triton 100 and X-gal. For each round, 10,000 survivors were screened for mutants that produced significantly higher levels of lactase (dark blue colonies). Sixty of the highest lactase producing colonies were isolated and grown in 5 ml MM medium cultures. Following growth for 6 days levels of lactase in the medium were determined. The five highest producing isolates were then grown in 30 ml cultures and assayed for lactase production.
  • a A niger transformed with plasmid pGlaPr-LacA-PyrG-AMA was subjected to four rounds of mutagenesis followed by screening for high lactase production on minimal medium X-gal plates. Mutagenesis was performed with nitrosoguanidine as described in the Materials and Methods. 1 X 1Cf viable conidia were screened for each mutagenesis.
  • Lactase expression levels given are the averages obtained with the isolates from each mutagenesis (3 from the first round, 9 from the second round and 27 from the third round) that produced the highest levels of LacAp in liquid cultures.
  • Lactase is widely used in the milk industry, both for the hydrolysis of lactose in milk products prepared for people with lactose intolerance and for treating milk industry byproducts such as the whey produced during the manufacture of cheese.
  • the GlaA region between nucleotides -483 and -369 was amplified by PCR as described in the Materials and Methods.
  • the resulting PCR product was cloned in various copy numbers into the unique Mlu ⁇ site of pGla-369Pr-LacA-GlaTt- PyrG-AMA.
  • this region (bp -483 to -369) included upstream regulatory sequences (URS) important for GlaA expression
  • URS upstream regulatory sequences
  • a PCR-based strategy was used to generate a library of randomly mutated GlaA URS sequences.
  • a library of mutated elements, cloned into the unique Mlu ⁇ site of plasmid pGla-369Pr-LacA-GlaTt-PyrG-AMA were screened using the high- throughput indicator plate screen.
  • a total of 33 mutant URSs that directed significantly increased levels of expression and 72 mutants that directed significantly reduced levels of expression were identified and retrieved intoE coli for further analysis. Initially these mutant elements will be subject to DNA sequencing to identify residues critical fortranscriptional regulation by the GlaA URS region.
  • the present invention provides the means to modulate the activity of the promoter and/or the expression levels of proteins operably linked to such promoters. Lactase expression with the GlaPr-LacA-GlaTt gene replacement cassette
  • this cassette consists of three elements. First, it includes a derivative of IheGlaA promoter region that directs expression at levels that are nine-fold higher than levels obtained with the original wild-type promoter. The improvements engineered into the promoter module are based on mutations introduced into the autonomously replicating vectors obtained during high-throughput screening (see above sections). The second element is the lactase-encoding portion of LacA It encodes a commercially valuable product but can easily be replaced by any other gene. Finally the cassette includes 1.1 kbp derived from genomic sequences immediately downstream of the GlaA coding region. It includes the information for transcription termination and polyadenylation.
  • This GlaPr-LacA-GlaTt cassette can be used to replace the wild type GlaA locus.
  • the utility of this system for engineering strains is evident in the levels of lactase expression obtained using this cassette (Table 7), since expression of lactase in amounts of 1 gram per liter make the resulting strain suitable for the production of lactase for the milk fluids industry and the treatment of lactose intolerance.
  • ⁇ xpression levels are for the transformant (identified by Southern analysis) that gave the highest level of expression
  • the system provides two advantages over existing systems for engineering strains that express heterologous proteins at high levels First the heterologous protein does not require its own signal sequences for secretion, because ⁇ -galactosidase, an efficiently secreted protein, provides this information Second, since ⁇ -galactosidase is not inactivated by fusing protein sequences to its carboxyl end, simple end-point or indicator plate assays performed to determine levels of ⁇ -galactosidase expression also estimate expression levels of the protein fused to its carboxyl terminus Vectors expressing a protein of interest as a fusion with ⁇ -galactosidase can therefore be subjected
  • Strain N814 (cspA1 ;fwnA1 ;pyrG5;nicB5) and N593 (cspA1 ;pyrG6) were provided by CJ. Bos (Wageningen Agricultural University), and strain A742 was obtained from the Fungal Genetics Stock Centre (FGSC). All the above strains are derived from strain N402, a UV induced cspA mutant derived from strain ATTC 9029 (Bos et al, 1988). The strains used are listed in Table 8. Unless otherwise stated all liquid cultures of A niger used for protein expression studies were grown at 30°C and 200 rpm in an air incubator shaker for six days. Agar media plates were incubated at 30°C.
  • MM Minimal media (MM) as described previously (Kafer et al., 1977) with 15% carbohydrate was used for liquid cultures. Agar (final concentration of 1.5%) was added for plates. When using strain N814, nicotinic acid was added (1 ⁇ g/ml final concentration). Ampicillin (100 ⁇ g/ml of culture) to prevent bacterial growth was added to all 30- ml cultures. Complete medium (CM) (Kafer et al., 1977) was used when growing strains for protoplast generation as well as for the isolation of genomic DNA . E coli strain DH ⁇ alpha (Woodcock et al., 1989) was used for the maintenance and production of plasmid DNAs.
  • CM Complete medium
  • ⁇ -galactosidase assays were initiated by adding 2 ⁇ l of media to 100 ⁇ l of reaction buffer, 50mM sodium acetate pH 4.1 , 2 mg/ml ofortho- nitrophenyl- ⁇ -D-galactoside (ONPG) and incubated for 10 minutes at 37C Reactions were stopped by adding 900 ⁇ l of 0.1 M sodium carbonate.
  • E. coli transformations and the isolation of plasmid DNAs from E. coli were performed as described previously (Sambrook et al., 1989, and Williams et al. 1996). Genomic DNA was isolated using a modification of the method of E. Kafer (Oza and Kafer, 1990). A niger cultures were grown overnight in CM at 30°C and 150 rpm. Mycelia were harvested by passing the culture through Miracloth, rinsed with distilled water and blotted dry. The mycelial mass was then placed in a mortar containing liquid nitrogen and ground to a fine powder with a pestle.
  • the powdered extract was then added to a solution of lOmMTrisHCI, 100mM EDTA pH 8.0 (1 g mycelial wet weight/5ml). Next 20% Sarkosyl (0.5ml/1g mycelial wet weight) followed by 0.2ml of RNaseA (10mg/ml in 50mM sodium acetate) were added. After incubation at 65°C for 30 minutes, the extract was centrifuged at room temperature for 30 minutes at 13,000 rpm in a Beckman JA17 centrifuge rotor.
  • the supernatant after transfer to a new centrifuge tube, was extracted two times with an equal volume of phenokchloroform and once with chloroform before DNA precipitation with 0.6 volumes of isopropanol.
  • the precipitated DNA was pelleted by centrifugation in a Beckman JA17 rotor at 15,000 rpm for 30 minutes, rinsed with 70% ethanol, and resuspended in water.
  • PCR amplifications using A niger genomic DNA templates were performed using a Perkin Elmer GeneAmp 2500 thermal cyclerTM.
  • the oligonucleotides used are presented with their pertinent features in Table 9.
  • Protoplasts of A niger were prepared essentially as described previously (Debets and Bos, 1986). Complete medium was inoculated with conidia of A. niger at a concentration of 10 6 conidia/ml, and grown overnight (16 hours) at 30°C and 150 rpm in a New Brunswick model G76waterbath shaker. Mycelia (10 g wet weight), harvested by filtration through Miracloth (Calbiochem) and rinsed with distilled water, were added to 20 ml oflytic solution (0.7M sodium chloride, 0.2M calcium chloride pH 5.8, 10mg/ml Novozyme 234) . Digestion was allowed to proceed for 1-2 hours at 30°C, with shaking at 150rpm.
  • the final yield of protoplasts varied between 1 X 10 7 and 4 x 10 7 protoplasts per gram of mycelia.
  • Protoplasts were harvested by centrifugation in a clinical centrifuge at 2,800 rpm. Three washes were carried out, one by suspension in 0.7 MKCI and two by suspension in 1M sorbitol/50mM calcium chloride (S/C). Each wash was followed by harvesting at 2,800 rpm in a clinical centrifuge at room temperature. After the final wash the protoplasts were suspended in S/C (1 x 10 8 protoplasts/ml) and used for transformations performed essentially as described previously (Werner et al., 1987).
  • the protoplasts (200 ⁇ l containing 2 x 10 7 cells) were incubated on ice with 1-1 O ⁇ g of transforming DNA (maximum volume 10 ⁇ l) and 50 ⁇ l of PEG solution (25% polyethyleneglycol 8000 in 50mM CaCI 2 , 10mM Tris-HCI pH 7.5), then 2 ml of PEG solution and 4 ml of S/C were added.
  • the 1057 bp GlaA transcription termination and polyA signal module was generated by PCR amplification using A niger genomic DNA template and oligonucleotides PGIaTt ⁇ ' (complementary to nucleotides +2136 to +2155 where numbering throughout is relative to the first nucleotide of the relevant start codon unless stated otherwise) and PGIaTt3' (complementary to nucleotides +3179 to +3198).
  • PGIaTt ⁇ ' has an 19 nucleotide extension that includes EcoRI and Fsel sites and PGIaTt3' had an 10 nucleotide 5' extension that includes an Sspl site.
  • the promoter region module (GlaPr), which includes bp -667 to bp -1 of the GlaA gene region, was generated by PCR amplification using A niger genomic DNA and oligonucleotides PGIaPr ⁇ ' (complementary to nucleotides -667 to -649) and PGIaPr3'.
  • PGIaPr3' is complementary to the GlaA coding strand from bp -1 to -24 except for the replacement of residues -7 to -5 (CTC) with GCT. These changes introduce an Nhe ⁇ site extending from bp -7 to -2 into the PCR product.
  • PGIaPr3' has a 5' extension that includes Fsel and Xba ⁇ sites.
  • PGIaPr ⁇ ' has a 5' extension that includes ⁇ /ofl and AatW sites.
  • the GlaTt and GlaPr modules were sequentially cloned into pUC18 as follows. First the PCR-generated GlaTt region was digested with EcoRI and Sspl and cloned into a pUC18 backbone prepared by EcoRI and Sspl digestion. The resulting intermediate plasmid, pGlaTt, was sequentially digested with Sapl, end-filled with Klenow polymerase and digested with Fsel. The PCR- generated promoter module (GlaPr) after Fsel digestion was inserted into the pGlaTt backbone to generate pGlaPr-GlaTt.
  • pGlaPr-GlaTt-PyrG ( Figure 1), an integrating plasmid with the A nidulans PyrG gene as a selectable marker, was constructed as follows. Primers PPyr5' (complementary to nucleotides -476 to -459 of the noncoding strand) and PPyr3' (complementary to nucleotides +903 to +921 of the coding strand) were used to PCR amplify PyrG using plamid pPyrG as template The resulting PCR product, which has Not ⁇ and Aafll sites at its 5' and 3' termini, was digested with ⁇ /ofl and Aafll and cloned into the backbone of pGlaPr-GlaTt prepared by ⁇ /ofl and Aafll digestion
  • the LacA reporter module was prepared by PCR amplification using primers PLacA ⁇ ' and PLacA3' and A niger genomic DNA
  • the resulting PCR product contains LacA sequences from base pair -13 to +100 relative to the start and stop translation codons respectively
  • the PCR product includes Nhe ⁇ and Fsel sites (encoded in the two PCR oligonucleotides) at its 5' and 3' ends respectively
  • the resulting LacA fragment was digested with Fsel and Nhe ⁇ and cloned into the backbones of pGlaPr-GlaTt, pGlaPr-GlaTt-PyrG and pGlaPr-GlaTt-PyrG-AMA generated by Fsel and Nhe ⁇ digestion
  • the resulting integrating and autonomously replicating plasmids designated pGlaPr-LacA-GlaTt, pGlaPr-LacA-GlaTt-
  • pGlaPr-LacA-GlaTt-PyrG- AMA were prepared and inserted into the backbone of pGlaPr-LacA-GlaTt-PyrG- AMA as follows.
  • the DNA for each promoter deletion derivative was prepared by PCR amplification using vector pGlaPr-LacA-GlaTt-PyrG as template.
  • the downstream or 3' oligonucieotide used to generate the six promoter regions was 0 PGIaPr3', the same oligonucieotide used to prepare the GlaA promoter region present in construct pGlaPr-GlaTt.
  • the six PCR products were individually digested with Mlu ⁇ and Nhe ⁇ and cloned into the large fragment of pGlaPr-LacA-GlaTt-PyrG- AMA prepared by Mlu ⁇ and Nhe ⁇ digestion, phosphatase treatment and isolation 0 following agarose gel fractionation.
  • the 829 base-pair (bp) PkiA promoter fragment was prepared by PCR-amplification from A niger genomic DNA using oligos PPki ⁇ ' and PPki3'. ⁇ Proceeding from its 3' end PPki ⁇ ' has 20 nucleotides that anneal to nucleotides
  • PPki3' has 21 nucleotides matching bases -1 to -21 of the noncoding strand at its 3' end followed by 12 nucleotides encoding adjacent Nhe ⁇ and Xba ⁇ sites and finally four filler nucleotides.
  • the PCR- generated PkiA promoter fragment was cloned into the backbone of pGlaPr- LacA-GlaTt-PyrG-AMA prepared as described for the GlaA promoter deletion set. ⁇ It will be recognized that other fusion proteins could be designed in accordance with the present invention.
  • fusions comprising domains enabling affinity purification (or otherwise) linked to the protein sequence of interest by a protease site, as commonly known, could be used.
  • fusion proteins include hemaglutinin fusions and Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions.
  • GST Gluthione-S-transferase
  • MBP Maltose binding protein
  • protease cleavage sites between two heterologously ⁇ fused polypeptides are well-known in the art.

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Abstract

L'invention porte sur des modules d'expression pouvant servir à assembler des vecteurs d'intégration et de réplication autonomes utilisables avec des champignons filamenteux. Les vecteurs élaborés à l'aide de ces modules se basent tous sur la structure du plasmide pUC18 du E. Coli. On peut donc les utiliser pour exécuter la plupart des manipulations usuelles sur l'ADN à l'intérieur d'un même plasmide qu'on réintroduit dans le champignon. Ce système de vecteurs peut servir à l'étude de séquences promotrices, à l'expression de protéines hétérologues et homologues et à l'identification des mutations des souches des vecteurs et hôtes altérant l'expression des protéines. Une sous-population de ces vecteurs, qui comporte la région codante de l'Aspergillus niger LacA comme gène reporter, a été utilisée pour mettre au point un crible indicateur à plaque à fort débit permettant d'identifier les mutants des hôtes et des vecteurs augmentant l'expression de la protéine reporter sécrétée, la LacAp. Finalement les vecteurs et souches mis au point peuvent servir à élaborer des souches susceptibles d'exprimer la région codante essentiellement de toute protéine hétérologues et homologues, à des niveaux élevés.
PCT/CA2000/001084 1999-09-13 2000-09-13 Systeme multifonctionnel de manipulation efficace de l'expression de proteines dans des champignons filamenteux, et son procede d'utilisation WO2001020007A1 (fr)

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Cited By (1)

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WO1989001969A1 (fr) * 1987-09-04 1989-03-09 Novo-Nordisk A/S Procede de production de proteines dans aspergillus et promoteurs destines a exprimer ce champignon
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EP0625577A1 (fr) * 1985-08-29 1994-11-23 Genencor International, Inc. Polypeptides hétérologues exprimés dans les champignons filamenteux, leur procédé de préparation ainsi que vecteurs pour leur préparation
EP0778348A1 (fr) * 1989-07-07 1997-06-11 Unilever N.V. Procédé de préparation d'une protéine à partir d'un champignon transformé par intégration multicopie d'un vecteur d'expression

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EP0625577A1 (fr) * 1985-08-29 1994-11-23 Genencor International, Inc. Polypeptides hétérologues exprimés dans les champignons filamenteux, leur procédé de préparation ainsi que vecteurs pour leur préparation
EP0225078A2 (fr) * 1985-11-20 1987-06-10 Panlabs, Inc. Cassettes d'expression composées pour la transformation de moisissures
WO1989001969A1 (fr) * 1987-09-04 1989-03-09 Novo-Nordisk A/S Procede de production de proteines dans aspergillus et promoteurs destines a exprimer ce champignon
US5252726A (en) * 1987-09-04 1993-10-12 Novo Nordisk A/S Promoters for use in aspergillus
EP0778348A1 (fr) * 1989-07-07 1997-06-11 Unilever N.V. Procédé de préparation d'une protéine à partir d'un champignon transformé par intégration multicopie d'un vecteur d'expression

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006051418A3 (fr) * 2004-11-11 2006-08-24 Danisco Facteurs de transcription
EP2210899A1 (fr) * 2004-11-11 2010-07-28 Danisco A/S Facteur de transcription pntr
US8017341B2 (en) 2004-11-11 2011-09-13 Danisco A/S Transcription factors

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