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WO1993007277A1 - Expression cassette for heterologous proteins in filamentous fungi - Google Patents

Expression cassette for heterologous proteins in filamentous fungi Download PDF

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Publication number
WO1993007277A1
WO1993007277A1 PCT/NL1992/000168 NL9200168W WO9307277A1 WO 1993007277 A1 WO1993007277 A1 WO 1993007277A1 NL 9200168 W NL9200168 W NL 9200168W WO 9307277 A1 WO9307277 A1 WO 9307277A1
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Prior art keywords
expression cassette
expression
oligopeptide
polypeptide
gene
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Application number
PCT/NL1992/000168
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French (fr)
Inventor
Robertus Franciscus Maria Van Gorcom
Theodorus Sonke
Cornelis Anthonius Maria Jacobus Johannus VAN DEN HONDEL
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Dsm N.V.
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Publication of WO1993007277A1 publication Critical patent/WO1993007277A1/en

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13012—Benzoate 4-monooxygenase (1.14.13.12)

Definitions

  • the invention relates to an expression cassette comprising (A) a 5' expression control system with
  • Such an expression cassette comprises the following components:
  • a region with sequences coding for the desired oligopeptide or polypeptide, and further such an expression cassette may also comprise:
  • Heterologous protein is here understood to mean a protein whose expression is not naturally controlled by the promoter present in the expression cassette. In this application, protein further stands for both an oligopeptide and a polypeptide.
  • induced expression of heterologous proteins are the production of calf chymosin in Aspergillus awamori (Ward, M. et al., Bio/Technology (1990) , 435-440), the production of human ⁇ -interferon in Aspergillus nidulans (Gwynne, D.I.
  • the present invention relates in particular to an expression cassette offering the possibility of having the growth of the host organism and the production of the heterologous protein by this host organism take place independently of each other.
  • This is called a regulated expression cassette, whereby the host organism can be cultured until the correct cell density is obtained, while the expression of the protein-encoding part of the expression cassette remains at a low level. As a consequence, there is no or hardly any production of the desired protein. After the desired cell density has been obtained, expression is subsequently induced by addition of a compound with an inducing effect.
  • a regulated expression cassette the growth of the host organism can therefore be made independent of the production of the desired protein, because the inducer, in the concentration in which it occurs in the medium for the host organism, is not a good growth substrate.
  • a major advantage of the independence of growth and production is that for instance a heterologous protein that is toxic to the host organism can be produced in the host organism.
  • This new expression cassette thus makes it possible to induce efficiently and effectively regulated expression of heterologous protein in filamentous fungi.
  • this aim has been achieved in that the expression cassette contains a 5' expression control system in which the promoter of the Aspergillus niger bphA gene is present and the sequence coding for an oligopeptide or a polypeptide does not code for the Aspergillus niger benzoate-para-hydroxylase (hereinafter to be referred to as BPH).
  • heterologous proteins or oligopeptides or polypeptides
  • promoter is understood to be a DNA fragment on which there are sequences that are involved in transcription regulation and initiation.
  • a strain with an expression cassette according to the invention can be grown on cheap, incompletely defined media such as molasses without protein production being induced.
  • the promoter ((1) (a)) present in the expression cassettes according to the subject invention is the promoter of the bphA gene (benzoate-para-hydroxylase gene) of Aspergillus niger or a functionally equivalent sequence.
  • the nucleotide sequence of this promoter is shown in Figure 1. It is known that it is not the complete sequence that is essential to the promoter activity; rather, it is only a limited number of the nucleotides, situated in certain promoter zones, that are essential to the activity of the promoter. The order of the nucleotides, and the number of nucleotides in the non-essential zones, may therefore be changed (within reasonable limits) without the promoter activity being essentially affected.
  • these sequences preferably originate from the Aspergillus niger bphA gene. This is preferred because the mRNA formed was found to be relatively stable.
  • expression cassettes are composed in which the translation control sequence originates from the heterologous gene, from which also the sequence coding for a protein originates.
  • expression cassettes are composed with the bphA promoter in the 5' expression control system ((l)(a)), the translation control sequences ((2) (a)) originating from one or several heterologous genes.
  • the 5' non-translated part of the mRNA originates at least partly from a heterologous gene whose origins differ from those of the gene coding for the protein. Though this may involve an additional step, this may be of advantage, for the translation control sequences thus introduced may influence the translation efficiency, the stability of the 5-mRNA part, etc.
  • the translation initiation site ((2)(b)), i.e. an ATG triplet may originate from either the bphA gene or the sequence whose expression is to be induced.
  • the sequence coding for a protein (B) may be a random gene portion coding for a protein, which may be of prokaryotic, eucaryotic or synthetic origins.
  • sequence coding for a protein or for an oligopeptide or polypeptide may be a sequence coding for an industrial enzyme, foodstuff protein, or a protein with pharmaceutical activity, or for proteins (precursors) which can be converted into these (in situ or not) .
  • oligopeptides that can be produced in fungi using an expression cassette according to the invention are hormones, neurotransmitters, antibiotics and peptides that can be modified to yield antibiotics.
  • polypeptides are interferon, and interleukins, blood coagulation factors, and human or animal growth hormone. Examples of peptides with other than pharmaceutical activity, industrial enzymes, foodstuff proteins, etc.
  • lipase is lipase, (gluco)amylase, cellulase, pectinase, chymosin and phylase. It is also possible to produce polypeptides that are subsequently converted into active enzymes. Chymosin, for example, is produced as prochymosin, efficiency may be improved by producing an enzyme with a protein part facilitating excretion from the fungus, or glycosylation of the peptide may be necessary.
  • the expression cassettes according to the invention are preferably applied in filamentous asomcyetes, and particularly those chosen from the series of genera
  • Aspergillus Trichoderma, Penicillium or relatives. If use is made of an Aspergillus sp., preference is given to a black Aspergillus or Aspergillus nidulans. Particular preference is given to Aspergillus niger, Aspergillus tubigensis and Aspergillus awamori.
  • the invention therefore relates also to filamentous fungi, preferably of the class of the asomcyetes with an expression cassette as described above.
  • filamentous fungi preferably of the class of the asomcyetes with an expression cassette as described above.
  • the advantage of these filamentous fungi with an expression cassette according to the invention is that they can be grown in customary growth media without expression of the protein, for which the coding sequence is present in an expression cassette according to the invention, taking place.
  • the filamentous fungi with an expression cassette according to the invention can be induced by a wide range of compounds, which can be represented by the general formula: X-phenyl-(A)-Y where X is an amino or hydroxy group, or a hydrogen or halogen atom, A is a methylene group, with or without amino or hydroxy group substitution, or a carbonyl group, or is absent, while Y consists of a carboxyl group or salts, amides and esters with lower alcohols thereof, or of an aldehyde group, a hydroxy group or a hydroxymethyl group.
  • inducing compounds examples include benzoic acid, sodium benzoate, D, L-mandelic acid, phenyl glyoxylic acid , p-amino benzoic acid, benzaldehyde, m-hydroxy benzoic acid, p-hydroxy benzoic acid and phenyl acetic acid. Preference is given to p-aminobenzoic acid, for this compound gives strong induction while it is not or hardly toxic in comparison with benzoic acid.
  • inducers have the advantage that normally they do not, or only in very low concentrations, occur in the growth medium of the host organism, so that no induction takes place during growth.
  • induction of the widely used glaA promoter from Aspergillus niger is brought about inter alia by starch, glucose and maltose.
  • a disadvantage of this type of inducing compounds is that they may easily be used as carbon source by the host organism, so that growth and expression cannot be separated.
  • the expression cassettes according to the invention the growth of the host organism and the production of the heterologous protein can truly be separated, for as inducer a compound may be chosen that occurs only in such a low concentration as to render (substantial) growth on it impossible.
  • the expression cassette according to the invention after induction with p- aminobenzoic acid, yields an unexpectedly higher expression than the expression cassette with the constitutive gpdA promoter from Aspergillus niger. Consequently, the strength of the expression cassette according to the invention is at least comparable to that of the expression cassette with the glaA promoter from Aspergillus niger after induction with maltodextrin (see Punt et al.; J. Biotech (1991) 1/7 19-34). In non-induced condition the expression level of the expression cassette according to the invention is lower than 2% relative to the level after induction with p-aminobenzoic acid.
  • a cell line with an expression cassette according to the invention exhibits catabolite repression; if desired one skilled in the art can simply isolate a non-catabolite- repressive mutant.
  • the proteins (oligopeptides and/or polypeptides) that can be produced by inducing expression of the expression cassette according to the invention can be recovered, be further converted, or be used for in situ reactions. If a protein, further converted or not, is recovered as such, it is advantageous if the protein is secreted by the fungus, but this is not necessary. However, there may also be advantage in using the expression cassette according to the invention for, for instance, producing an enzyme with which a primary or secondary metabolite is made in the cell in a yield that is high for that particular cell line.
  • a modified cell line producing metabolites such as, for instance, phenyl alanine, para-hydroxy-phenyl alanine, or for instance vitamins, antibiotics, steroids or terpenoids.
  • a cell line may also be suitable for the degradation of xenobiotics such as for instance PCBs, toluene, DDT and the like, so that detoxification of, for instance, soil can take place.
  • xenobiotics such as for instance PCBs, toluene, DDT and the like.
  • the first step comprised isolating the entire bphA gene including the promoter from Aspergillus niger ATCC 1015 and introduction thereof into a vector, as described in van Gorcom et al. in Mol. Gen. Genet. (1990) 223, pp. 192-197.
  • the base sequence of the promoter, the translation control sequence and a small part of the sequence coding for the BPH protein was determined and is shown in Fig. 1.
  • an expression cassette was constructed that consisted of a promoter originating from the bphA gene, a translation control sequence of the A. nidulans gpdA gene, and the sequence coding for a protein ( ⁇ -galactosidase) of the E. coli lacZ gene.
  • the translation control part of the gpdA gene was chosen because this is known to yield a relatively stable 5'-mRNA. This transformant will hereinafter be referred to as bphA- pdA transformant.
  • strains were compared and tested for their degree of lacZ expression: the wild-type (Aspergillus niger wt N245, derived from ATCC 1015), the three transformants with the translation control part of the bphA gene up to the 1st, 2nd and 3rd ATG on the DNA coding for mRNA; the bphA-qpdA transformant and, as a reference, an A. niger transformant with an expression cassette consisting of an A ⁇ niger gpdA promoter, gpdA translation control sequences and a sequence coding for the lacZ protein.
  • This expression cassette has a constitutive promoter, i.e. a promoter that is induction-independent, so that it is always expressed.
  • the promoter of the Aspergillus niger bphA gene was cloned from the chromosomal DNA from Aspergillus niger ATCC
  • the sequence of the DNA nucleotides of the promoter region from a Sail site of the bphA gene was determined by the method described by Sanger et al., Proc. Natl. Acad. Sci. USA (1977) 1 / PP* 5463-5467. The sequence is shown in Fig. 1. Further experiments proved that at least all relevant sequences affecting the efficiency, regulation and initiation of the transcription of the bphA gene are situated at least on a fragment that starts at the Sail site located approximately 1650 base pairs upstream of the start of the bphA gene. The major transcription initiation site was determined and found to be located on nucleotide No. 1645 in Fig. 1. A few relevant restriction sites are also shown there.
  • the nucleotide sequence downstream of the transcription initiation site (the translation control sequence of the bphA gene) is also indicated in Fig. 1.
  • the first two ATG initiation codons belong to open reading frames that can lead to 2 oligopeptides of 18 and 5 amino acids, respectively.
  • the third one is the initiation codon of the open reading frame coding for the BPH sequence.
  • a vector was selected in which the translation direction of the BPH part and the translation direction of the lacZ- ⁇ c part present in ml3mpll were identical.
  • This vector was called mAB8-81; it is shown in Fig. 2 (cont.).
  • Kunkel Proc. Natl. Acad. Sci. USA (1985) 82_, pp.
  • BamHI adaptors (sequence, see fig. 6). This gave rise to in- frame fusion of the start codon of the bphA-gpdA-lacZ fusion gene to the complete lacZ gene present in the expression analysis vectors. The result was the vector pAB94-86 (Fig. 6 (cont.)). The relevant sequence in the fusion region was reverified.
  • Aspergillus niger N245 (ATCC 1015, csp, met, pyrG) was transformed with the constructed vectors described above (pAB94-83, -84, -85 and -86).
  • PyrG + transformants were screened for the presence of transformants in which a single copy of the relevant plasmid is integrated at the pyrG locus of the chromosomal DNA of the strain (according to the method described by van Hartingsveldt et al.; Mol. Gen. Genet. 206 (1986), pp. 71- 75). For each plasmid two independent single-copy transformants were isolated this way.
  • spore suspensions that were not older than one week. All experiments were conducted in a New Brunswick G25 air incubator (rotary shaker) at 30°C and 300 rpm. Further, in all cases identical 500 ml Erlenmeyer flasks were used.
  • the preculture phase comprised inoculation of 2xl0 7 spores in 200 ml complete medium (CM; see below), + 1 mg/ml methionine (+ met), + lOmM uridine (+ uri). After 24 hours these cultures were filtered using Miracloth (Calbiochem) and washed with 1* DSM medium without sugars (1* DSM-S; see below), + met, + uri.
  • the mycelium was again filtered over Miracloth and washed with 1* DSM-S, + met, + uri and subsequently with 50 mM Na phosphate, pH 7, 1 mM MgCl 2 , 20 /JM PMSF; 0.3 g of these samples was frozen in liquid nitrogen for further analysis. All mycelium samples were ground in a mortar in liquid nitrogen and the mycelium powder was suspended in 1 ml 50 mM Na phosphate buffer, pH 7, 1 mM MgCl 2 , 20 ⁇ m PMSF (van Gorcom et al.; Gene, (1985) 40, 99-106).
  • compositions of the media used Complete medium (CM) per litre AD:
  • DSM medium without sugars per litre of tap water: 0.25 g MgS0 4 . 7H 2 0, 1.04 g NaCl, 5.24 g K 2 HP0 4 , 0.22 g urea.

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Abstract

The invention relates to an expression cassette comprising (A) a 5' expression control system with (1a) a promoter with transcription control sequences; (1b) a transcription initiation site; (2a) translation control sequences on the 3' side of the transcription initiation site; (2b) a translation initiation site; (B) a sequence coding for an oligopeptide or a polypeptide, which expression cassette is suitable for inducing the expression of an oligopeptide or a polypeptide in filamentous fungi, the expression cassette containing a 5' expression control system in which the promoter of the Aspergillus niger bpHA gene or a functionally equivalent sequence is present and the sequence coding for an oligopeptide or a polypeptide does not code for the Aspergillus niger benzoate-para-hydroxylase. The invention also relates to filamentous fungi with such an expression cassette.

Description

EXPRESSION CASSETTE FOR HETEROLOGOUS PROTEINS IN FILAMENTOUS FUNGI
The invention relates to an expression cassette comprising (A) a 5' expression control system with
(1) (a) a promoter with transcription control sequences; (b) a transcription initiation site;
(2) (a) translation control sequences on the 3' side of the transcription initiation site; (b) a translation initiation site;
(B) a sequence coding for an oligopeptide or a polypeptide, which expression cassette is suitable for inducing the expression of an oligopeptide or a polypeptide in filamentous fungi. Such an expression cassette is described by .R.M.
Berka and CC. Barnett in an article entitled "The Development of Gene Expression Systems for Filamentous Fungi", Bio-tech. Adv. (1989), ]_, pp. 127-154, and in WO-A- 86/06097. Such an expression cassette comprises the following components:
(1) a region (the promoter) with 5' transcription control sequences, and the transcription initiation site;
(2) a region for translation initiation, including the translation initiation site;
(3) a region with sequences coding for the desired oligopeptide or polypeptide, and further such an expression cassette may also comprise:
(4) if extracellular production is to take place, a region with secretion-facilitating sequences;
(5) a region for transcription termination regulation;
(6) a region for polyadenylation regulation. An expression cassette as described above can be used to induce expression of heterologous proteins in filamentous fungi. Heterologous protein is here understood to mean a protein whose expression is not naturally controlled by the promoter present in the expression cassette. In this application, protein further stands for both an oligopeptide and a polypeptide. Some examples of induced expression of heterologous proteins are the production of calf chymosin in Aspergillus awamori (Ward, M. et al., Bio/Technology (1990) , 435-440), the production of human α-interferon in Aspergillus nidulans (Gwynne, D.I. et al., Bio/Technology (1987) 5_, 713-719), and the production of a lipase from Humicola lanuqinosus in Aspergillus oryzae (EP-A-0305216) .
In spite of the fact that the state of the art makes it possible to induce the expression of heterologous proteins in fungi, there are only few good expression cassettes. The demand for new, improved expression cassettes therefore is very great, with good expression regulation being of special importance. The expression cassettes described, for instance, in WO-A-86/06097 are induced, inter alia, by compounds which can be utilized by the organism as well-metabolizable nutrients (C sources), such as threonine or ethanol. Consequently, such an inducer is consumed.
The present invention relates in particular to an expression cassette offering the possibility of having the growth of the host organism and the production of the heterologous protein by this host organism take place independently of each other. This is called a regulated expression cassette, whereby the host organism can be cultured until the correct cell density is obtained, while the expression of the protein-encoding part of the expression cassette remains at a low level. As a consequence, there is no or hardly any production of the desired protein. After the desired cell density has been obtained, expression is subsequently induced by addition of a compound with an inducing effect. By the use of such a regulated expression cassette the growth of the host organism can therefore be made independent of the production of the desired protein, because the inducer, in the concentration in which it occurs in the medium for the host organism, is not a good growth substrate.
A major advantage of the independence of growth and production is that for instance a heterologous protein that is toxic to the host organism can be produced in the host organism.
Surprisingly, new expression cassettes have now been composed by which good expression of heterologous protein is obtained while, in addition, growth of the host organism can be made independent of expression of heterologous protein.
This new expression cassette thus makes it possible to induce efficiently and effectively regulated expression of heterologous protein in filamentous fungi. According to the invention this aim has been achieved in that the expression cassette contains a 5' expression control system in which the promoter of the Aspergillus niger bphA gene is present and the sequence coding for an oligopeptide or a polypeptide does not code for the Aspergillus niger benzoate-para-hydroxylase (hereinafter to be referred to as BPH).
In the context of this patent application the term heterologous proteins (or oligopeptides or polypeptides) is understood to mean all proteins other than Aspergillus niger BPH. The term promoter is understood to be a DNA fragment on which there are sequences that are involved in transcription regulation and initiation.
For an industrial application of the invention, for instance, it is a major advantage that a strain with an expression cassette according to the invention can be grown on cheap, incompletely defined media such as molasses without protein production being induced.
The promoter ((1) (a)) present in the expression cassettes according to the subject invention is the promoter of the bphA gene (benzoate-para-hydroxylase gene) of Aspergillus niger or a functionally equivalent sequence. The nucleotide sequence of this promoter is shown in Figure 1. It is known that it is not the complete sequence that is essential to the promoter activity; rather, it is only a limited number of the nucleotides, situated in certain promoter zones, that are essential to the activity of the promoter. The order of the nucleotides, and the number of nucleotides in the non-essential zones, may therefore be changed (within reasonable limits) without the promoter activity being essentially affected. In addition, it is known that certain parts of the sequence in the essential zones may be replaced with equivalent sequences, with no or little effect on the activity. It will be clear that this type of new promoters with essential zones of the Aspergillus niger bphA promoter or corresponding sequences can also be used in the new expression cassettes. Preferably the transcription initiation site ((1)
(b) ) also originates from the above-mentioned bphA gene.
As regards the translation control sequences ((2) (a)), in a first embodiment these sequences preferably originate from the Aspergillus niger bphA gene. This is preferred because the mRNA formed was found to be relatively stable.
From the nucleotide sequence of the promoter of the bphA gene (see Figure 1) it appears that the DNA sequence coding for the mRNA contains three ATG codons, which are situated at positions 5, 48 and 287, calculated from the main transcription initiation site.
For the sake of comparison reference is made to research on the trpC gene of Aspergillus niger, which proved that the main transcription initiation sites are located at positions -5 and -6 from the ATG initiation codon (Kos, T. et al.; Curr. Genet. (1988), L3, 137-144). The presence of such a short, non-coding 5' nucleotide sequence was demonstrated also on the trpC mRNA from Aspergillus nidulans, for here the major transcription initiation sites were found to be located at position -9 or -10 from the AUG initiation codon (Mullaney, E.J. et al.; Mol. Genet. (1985) 199, 37-45). In addition, for eucaryotes it is known that it is mainly the first AUG codon on the mRNA that is used as translation initiation site. According to the current theory, fusion of a sequence coding for a heterologous protein to the transcription and translation control sequences of the bphA gene therefore had best take place at the position of the first ATG codon.
Most remarkably, however, it has been found that such a fusion of a sequence coding for a heterologous protein to the transcription and translation control sequences of the bphA gene yields no or hardly any production of heterologous protein. Surprisingly, it has been found that fusions of a sequence coding for a heterologous protein to the bphA transcription and translation control sequences at the position of the second as well as the third ATG codon leads to excellent expression properties.
In a second embodiment relating to the translation control sequence ((2) (a)), expression cassettes are composed in which the translation control sequence originates from the heterologous gene, from which also the sequence coding for a protein originates.
In a third embodiment of the invention relating to the translation control sequence ((2) (a)), expression cassettes are composed with the bphA promoter in the 5' expression control system ((l)(a)), the translation control sequences ((2) (a)) originating from one or several heterologous genes. In that situation the 5' non-translated part of the mRNA originates at least partly from a heterologous gene whose origins differ from those of the gene coding for the protein. Though this may involve an additional step, this may be of advantage, for the translation control sequences thus introduced may influence the translation efficiency, the stability of the 5-mRNA part, etc. The translation initiation site ((2)(b)), i.e. an ATG triplet, may originate from either the bphA gene or the sequence whose expression is to be induced.
The sequence coding for a protein (B) may be a random gene portion coding for a protein, which may be of prokaryotic, eucaryotic or synthetic origins.
More particularly, the sequence coding for a protein or for an oligopeptide or polypeptide may be a sequence coding for an industrial enzyme, foodstuff protein, or a protein with pharmaceutical activity, or for proteins (precursors) which can be converted into these (in situ or not) . Examples of oligopeptides that can be produced in fungi using an expression cassette according to the invention are hormones, neurotransmitters, antibiotics and peptides that can be modified to yield antibiotics. Examples of polypeptides are interferon, and interleukins, blood coagulation factors, and human or animal growth hormone. Examples of peptides with other than pharmaceutical activity, industrial enzymes, foodstuff proteins, etc. are lipase, (gluco)amylase, cellulase, pectinase, chymosin and phylase. It is also possible to produce polypeptides that are subsequently converted into active enzymes. Chymosin, for example, is produced as prochymosin, efficiency may be improved by producing an enzyme with a protein part facilitating excretion from the fungus, or glycosylation of the peptide may be necessary.
Likewise, it is possible to induce expression of a protein affecting the metabolism in the cell.
The expression cassettes according to the invention are preferably applied in filamentous asomcyetes, and particularly those chosen from the series of genera
Aspergillus, Trichoderma, Penicillium or relatives. If use is made of an Aspergillus sp., preference is given to a black Aspergillus or Aspergillus nidulans. Particular preference is given to Aspergillus niger, Aspergillus tubigensis and Aspergillus awamori.
The invention therefore relates also to filamentous fungi, preferably of the class of the asomcyetes with an expression cassette as described above. The advantage of these filamentous fungi with an expression cassette according to the invention is that they can be grown in customary growth media without expression of the protein, for which the coding sequence is present in an expression cassette according to the invention, taking place.
The filamentous fungi with an expression cassette according to the invention can be induced by a wide range of compounds, which can be represented by the general formula: X-phenyl-(A)-Y where X is an amino or hydroxy group, or a hydrogen or halogen atom, A is a methylene group, with or without amino or hydroxy group substitution, or a carbonyl group, or is absent, while Y consists of a carboxyl group or salts, amides and esters with lower alcohols thereof, or of an aldehyde group, a hydroxy group or a hydroxymethyl group. Examples of suitable inducing compounds are benzoic acid, sodium benzoate, D, L-mandelic acid, phenyl glyoxylic acid , p-amino benzoic acid, benzaldehyde, m-hydroxy benzoic acid, p-hydroxy benzoic acid and phenyl acetic acid. Preference is given to p-aminobenzoic acid, for this compound gives strong induction while it is not or hardly toxic in comparison with benzoic acid.
All these inducers have the advantage that normally they do not, or only in very low concentrations, occur in the growth medium of the host organism, so that no induction takes place during growth. By way of comparison it is noted that induction of the widely used glaA promoter from Aspergillus niger (Fowler et al.; Curr. Genet. (1990) J 537-545) is brought about inter alia by starch, glucose and maltose. A disadvantage of this type of inducing compounds, however, is that they may easily be used as carbon source by the host organism, so that growth and expression cannot be separated. By means of the expression cassettes according to the invention the growth of the host organism and the production of the heterologous protein can truly be separated, for as inducer a compound may be chosen that occurs only in such a low concentration as to render (substantial) growth on it impossible.
The examples given below show that the expression cassette according to the invention, after induction with p- aminobenzoic acid, yields an unexpectedly higher expression than the expression cassette with the constitutive gpdA promoter from Aspergillus niger. Consequently, the strength of the expression cassette according to the invention is at least comparable to that of the expression cassette with the glaA promoter from Aspergillus niger after induction with maltodextrin (see Punt et al.; J. Biotech (1991) 1/7 19-34). In non-induced condition the expression level of the expression cassette according to the invention is lower than 2% relative to the level after induction with p-aminobenzoic acid.
Furthermore, it is known that the quantity of protein produced can be increased strongly by using multicopy transformants. It will be clear that there is also advantage in using the expression cassette according to the invention in a host organism in a multicopy situation.
A cell line with an expression cassette according to the invention exhibits catabolite repression; if desired one skilled in the art can simply isolate a non-catabolite- repressive mutant.
The proteins (oligopeptides and/or polypeptides) that can be produced by inducing expression of the expression cassette according to the invention can be recovered, be further converted, or be used for in situ reactions. If a protein, further converted or not, is recovered as such, it is advantageous if the protein is secreted by the fungus, but this is not necessary. However, there may also be advantage in using the expression cassette according to the invention for, for instance, producing an enzyme with which a primary or secondary metabolite is made in the cell in a yield that is high for that particular cell line. This, therefore, is more a case of a modified cell line producing metabolites such as, for instance, phenyl alanine, para-hydroxy-phenyl alanine, or for instance vitamins, antibiotics, steroids or terpenoids. Such a cell line may also be suitable for the degradation of xenobiotics such as for instance PCBs, toluene, DDT and the like, so that detoxification of, for instance, soil can take place. The invention will be elucidated on the basis of the following, non-limiting examples. In these examples the figures will also be elucidated.
Examples
General
The following strategy was adopted to obtain and test the expression cassette according to the invention:
The first step comprised isolating the entire bphA gene including the promoter from Aspergillus niger ATCC 1015 and introduction thereof into a vector, as described in van Gorcom et al. in Mol. Gen. Genet. (1990) 223, pp. 192-197. The base sequence of the promoter, the translation control sequence and a small part of the sequence coding for the BPH protein was determined and is shown in Fig. 1.
Subsequently a number of different experiments were conducted in which the promoter, optionally with a part of the translation control sequences, was tested. To this end a part of the bphA gene was cut from the above-mentioned vector and introduced into a second vector, which also contained the nucleotide sequence at the start of the E.coli lacZ gene. By in vitro mutagenesis with specially synthesized oligonucleotides, the desired part of the bphA gene was coupled to the lacZ gene. Then the relevant piece of DNA was cut from the vector and introduced into a plasmid. This plasmid possessed the complete lacZ gene and also, as selection marker, a mutant pyrG gene from A. niger. The plasmid was then introduced into an Aspergillus niger strain which was a pyrG- mutant strain. By screening for pyrG prototrophy, transformants could be obtained.
In an analogous manner an expression cassette was constructed that consisted of a promoter originating from the bphA gene, a translation control sequence of the A. nidulans gpdA gene, and the sequence coding for a protein (β-galactosidase) of the E. coli lacZ gene. The translation control part of the gpdA gene was chosen because this is known to yield a relatively stable 5'-mRNA. This transformant will hereinafter be referred to as bphA- pdA transformant.
Eventually 6 strains were compared and tested for their degree of lacZ expression: the wild-type (Aspergillus niger wt N245, derived from ATCC 1015), the three transformants with the translation control part of the bphA gene up to the 1st, 2nd and 3rd ATG on the DNA coding for mRNA; the bphA-qpdA transformant and, as a reference, an A. niger transformant with an expression cassette consisting of an A^ niger gpdA promoter, gpdA translation control sequences and a sequence coding for the lacZ protein. This expression cassette has a constitutive promoter, i.e. a promoter that is induction-independent, so that it is always expressed.
1) Cloning of the bphA gene with the promoter and the characterization of the promoter and of the 5' part of the bphA gene
The promoter of the Aspergillus niger bphA gene was cloned from the chromosomal DNA from Aspergillus niger ATCC
1015 on the 9.8 kb EcoRI fragment that is present in pAB8-2 plasmid as described in van Gorcom et al. in Mol. Gen.
Genet. (1990) 223, pp. 192-197.
The sequence of the DNA nucleotides of the promoter region from a Sail site of the bphA gene was determined by the method described by Sanger et al., Proc. Natl. Acad. Sci. USA (1977) 1/ PP* 5463-5467. The sequence is shown in Fig. 1. Further experiments proved that at least all relevant sequences affecting the efficiency, regulation and initiation of the transcription of the bphA gene are situated at least on a fragment that starts at the Sail site located approximately 1650 base pairs upstream of the start of the bphA gene. The major transcription initiation site was determined and found to be located on nucleotide No. 1645 in Fig. 1. A few relevant restriction sites are also shown there.
The nucleotide sequence downstream of the transcription initiation site (the translation control sequence of the bphA gene) is also indicated in Fig. 1. In this part of the gene three ATG codons were found to be present, located at 1650, 1693 and 1932, respectively. The first two ATG initiation codons belong to open reading frames that can lead to 2 oligopeptides of 18 and 5 amino acids, respectively. The third one is the initiation codon of the open reading frame coding for the BPH sequence.
2) Promoter testing using a reporter gene
The efficiency of the promoter of the bphA gene was tested using an expression analysis system developed for fungi by van Gorcom and van den Hondel (Nucleic Acids Res. (1988) _?_, p. 9052. Generally speaking, this system determines the promoter efficiency by placing the promoter upstream of a so-called reporter gene (in this case the @- galactosidase gene (lacZ) of E. coli) . These so-called fusion genes are tested, in a single copy, on a fixed place in the fungus genome, viz. the pyrG locus of Aspergillus niger. The plasmids (expression analysis vectors) started from are described in the above-mentioned article. Example I
Preparation of a plasmid with an expression cassette with the 5' part of the bphA gene up to the 3rd ATG as promoter and translation control sequence (bp 1932, Fig. 11)
The approximately 2.1 kb Sail fragment, inter alia containing the promoter of the bphA gene (Fig. 1), was cloned at the Sail site of vector ml3mpll (this vector is described by Messing in Methods Enzymol. 1983, 101, pp. 20- 78). A vector was selected in which the translation direction of the BPH part and the translation direction of the lacZ-<c part present in ml3mpll were identical. This vector was called mAB8-81; it is shown in Fig. 2 (cont.). Using this vector, in vitro mutagenesis experiments were carried out according to the method of Kunkel (Proc. Natl. Acad. Sci. USA (1985) 82_, pp. 488-492) using the oligonucleotide shown in Fig. 2 (cont.) (left-hand bottom), to construct a new vector (mAB8-83, Fig. 3), in which the translation initiation codon of the BPH (3rd ATG) was placed in frame upstream of the code of the lacZ-« part of ml3mpll. The fusion brought about by in vitro mutagenesis was confirmed by means of DNA sequence analysis.
The promoter of the bphA gene and the first part of the fusion gene, located on a Sail fragment, was cloned at the unique BamHI site of the expression analysis vector pAB94-12 (van Gorcom and van den Hondel; Nucleic Acids Res. (1988) JL6_, p. 9052) by means of Sail-BamHI adaptors (sequence see Fig. 3, right-hand). For further research use was made of a plasmid with an expression cassette according to the invention, designated pAB94-83 (Fig. 3 (cont.)), in which the bphA promoter, an -in frame- translation fusion of the start codon of BPH (3rd ATG) and the complete lacZ gene were present. The relevant sequence in the fusion region was reverified. Example II and Comparative Experiment A
Preparation of a plasmid with an expression cassette with the 5' part of the bphA gene up to the 1st and 2nd ATG codons, respectively (bp 1650 and 1693, respectively, in Fig. 1) as promoter and translation control sequences
Starting from vector mAB8-81 and using in vitro mutagenesis techniques according to the method of Kunkel (Proc. Natl. Acad. Sci. USA (1985) 2_, pp. 488-492) two vectors were constructed with oligonucleotides as shown in Fig. 2 (cont.), centre and right-hand bottom. These experiments resulted in the vectors mAB8-84 (1st ATG fusion; comparative experiment A) and mAB-85 (2nd ATG fusion; example II). The fusions brought about by in vitro mutagenesis were checked by means of DNA sequence analysis.
From the resulting vectors mAB8-84 and -85 the BamHI fragment with promoter and the first part of the fusion gene was isolated, following which this was cloned in the largest fragment formed after partial BamHI digestion of vector PAB94-83; the BamHI sites used in PAB94-83 are marked with an arrow (Fig. 3 (cont.)). Thus, in pAB94-83 the BamHI fragment with the largest part of the promoter and translation control sequences were replaced with the corresponding BamHI fragments from mAB8-84 and mAB8-85. This resulted in the vectors pAB94-84 (comparative experiment A) and pAB94-85 (Example II) (Fig. 4 (cont.)). The relevant sequences in the fusion region were reverified.
Example III
Preparation of a plasmid with an expression cassette with the promoter of the bphA gene, and as translation control sequence a part of a gpdA gene up to the ATG initiation codon of the gpdA open reading frame The promoter of the bphA gene (Fig. 1, up to and including position 1644) up to the transcription start was fused to an existing construct in which the Aspergillus nidulans gpdA gene had been fused to the lacZ gene. To do so, a PvuII-BamHI fragment containing the relevant sequences (from upstream of the transcription start to downstream of the translation start) of this fusion (described by Punt et al. in Gene (1990) 3_, 101-109) was inserted between the Smal and EcoRI sites of the intermediate vector mAB8-81 (using a BamHI-EcoRI adaptor; see Fig. 5). This resulted in the vector mAB8-82.
By means of in vitro mutagenesis according to Kunkel the transcription initiation sites of the bphA gene and the gpdA-lacZ fusion gene, respectively, were coupled. The oligonucleotide used is given in Fig. 5 (cont.). In this manner the mAB8-86 vector was created. The fusion brought about by in vitro mutagenesis was checked by means of DNA sequence analysis.
The promoter of the bphA gene and the first part of the fusion gene from mAB8-86, located on a Sail fragment, was cloned at the unique BamHI site of the expression analysis vector pAB94-13 (van Gorcom and van den Hondel; Nucleic Acids Res. (1988) 16_, p. 9052) by means of Sall-
BamHI adaptors (sequence, see fig. 6). This gave rise to in- frame fusion of the start codon of the bphA-gpdA-lacZ fusion gene to the complete lacZ gene present in the expression analysis vectors. The result was the vector pAB94-86 (Fig. 6 (cont.)). The relevant sequence in the fusion region was reverified.
Of the four vectors the verified sequences in the fusion region are shown in Fig. 7.
Examples IV-XV and Comparative Experiments B-N
Transformation of Aspergillus niger N245 with the four constructed expression analysis vectors pAB94-83, -84, -85 and -86:
Aspergillus niger N245 (ATCC 1015, csp, met, pyrG) was transformed with the constructed vectors described above (pAB94-83, -84, -85 and -86). Using Southern blotting, PyrG+ transformants were screened for the presence of transformants in which a single copy of the relevant plasmid is integrated at the pyrG locus of the chromosomal DNA of the strain (according to the method described by van Hartingsveldt et al.; Mol. Gen. Genet. 206 (1986), pp. 71- 75). For each plasmid two independent single-copy transformants were isolated this way.
Analysis of the β-galactosidase expression in wild-type Aspergillus niger, in the above-mentioned isolated sinqle- copy transformants and in Aspergillus niger with a constitutively expressed gpdA-lacZ fusion gene:
Two independent single-copy transformants obtained with the plasmids pAB94-83, -84, -85 and -86 were grown (and induced) in several media and, after harvesting, analyzed for their β-galactosidase activity. For the sake of comparison, the original N245 strain and an Aspergillus niger transformant containing a single copy of an Aspergillus niger gpdA-lacZ fusion gene at the pyrG locus were included (strain used:. Aspergillus niger AB4.1 (van Hartingsveldt et al.; see above); plasmid used pAB94-53 (Fig. 8). This plasmid has been registered under No. CBS 515.91. In each experiment use was made of spore suspensions that were not older than one week. All experiments were conducted in a New Brunswick G25 air incubator (rotary shaker) at 30°C and 300 rpm. Further, in all cases identical 500 ml Erlenmeyer flasks were used. The preculture phase comprised inoculation of 2xl07 spores in 200 ml complete medium (CM; see below), + 1 mg/ml methionine (+ met), + lOmM uridine (+ uri). After 24 hours these cultures were filtered using Miracloth (Calbiochem) and washed with 1* DSM medium without sugars (1* DSM-S; see below), + met, + uri. After washing with 50 ml 1* DSM-S, + met, + uri, and subsequently with 50 mM Na phosphate, pH 7, 1 mM MgCl2 , 20 μM PMSF (protease inhibitor), 0.3 g was frozen in liquid nitrogen for analysis for comparative experiments B-D and examples IV-VI. Furthermore, 1 g was added to 125 ml 1* DSM-S, + met, + uri, +, respectively, 0.3% para-amino-benzoic acid (paba) or 0.1% para-hydroxy benzoic acid (phb) or 0.1% benzoic acid (benz.) (see below). These cultures were incubated for 4 hours at 30 °C, 300 rpm. After this induction the mycelium was again filtered over Miracloth and washed with 1* DSM-S, + met, + uri and subsequently with 50 mM Na phosphate, pH 7, 1 mM MgCl2 , 20 /JM PMSF; 0.3 g of these samples was frozen in liquid nitrogen for further analysis. All mycelium samples were ground in a mortar in liquid nitrogen and the mycelium powder was suspended in 1 ml 50 mM Na phosphate buffer, pH 7, 1 mM MgCl2 , 20 μm PMSF (van Gorcom et al.; Gene, (1985) 40, 99-106). After thawing and vortexing this suspension was centrifuged at 4 °C for 15' (Eppendorf centrifuge, 12,000 rpm) . Of the supernatant the @-galactosidase activity (Miller, in 'Experiments in Molecular Genetics', Cold Spring Harbor Laboratory, 1972) and the protein concentration were determined by the Bradford method, as described in Anal. Bioche . (1976) 72., 248-254. The results of these experiments are presented in the following table.
TABLE
Figure imgf000019_0001
* values are an average of two measurements, and therefore less reliable. In experiments B-D and IV-VI the six different strains were tested without inducer. Except for the constitutive strain (D), all types yielded a very low @- galactosidase activity.
In a second series of experiments the 6 strains were induced with para-hydroxy-benzoic acid. The Aspergillus niger wild-type (exp. E), which did not contain an expression cassette, and the non-functional expression cassette (fusion on the first ATG codon, exp. F) exhibited no induction at all in comparison with the constitutive strain (exp. G). Examples VII, VIII and IX clearly show that β-galactosidase expression is higher than in E or F. From the third series an analogous picture emerges, but now the activity of the para-aminobenzoic acid-induced filamentous asomcyetes according to the invention even is clearly higher than that of the fungi in which constitutive expression of the lacZ gene by means of the gpdA promoter has taken place (exp. K).
From the fourth series it is apparent that benzoic acid is also a very suitable inducer.
Compositions of the media used: Complete medium (CM) per litre AD:
2 g neopeptone, 1 g yeast extract, 20 ml 50*AspA (300 g NaN03 , 26 g KCl, 76 g KH2P04 per litre; adjust to pH 5.5 using 10 N KOH), 2 ml 1 M MgS04 , 1 ml 1000*spores (2.2 g ZnS04 . 7H20, 1.1 g H3B03, 0.5 g MnCl2 . 4H20, 5 g FeS04 . 7H20, 0.17 g CoCl2 . 6 H20, 0.16 g CuS04 . 5H20, 0.15 g
Na2Mo04 . 2H20, 5 g EDTA in 100 ml AD; after sterilization, adjust to pH 6.5 using 10 N KOH), 10 ml 50% glucose, 2 ml vitamin solution (100 mg thiamine, 1000 mg riboflavine, 100 mg para-aminobenzoic acid, 1000 mg nicotinamide, 500 mg pyridoxine, 100 mg'pantothenic acid, 2 mg biotin per litre), 10 ml 10% casamino acids.
DSM medium without sugars (DSM-S), per litre of tap water: 0.25 g MgS04 . 7H20, 1.04 g NaCl, 5.24 g K2HP04, 0.22 g urea. A. niger N245 and plasmid pAB94-53 (in E. coli DH 5<=) were registered with the CBS in Baarn, the Netherlands, on September 26.

Claims

C L A I M S
1. An expression cassette comprising
(A) a 5' expression control system with
(la) a promoter with transcription control sequences; (lb) a transcription initiation site; (2a) translation control sequences on the 3' side of the transcription initiation site;
(2b) a translation initiation site;
(B) a sequence coding for an oligopeptide or a polypeptide, which expression cassette is suitable for inducing the expression of an oligopeptide or polypeptide in filamentous fungi, characterized in that the expression cassette contains a 5' expression control system in which the promoter of the Aspergillus niger bphA gene or a functionally equivalent sequence is present and the sequence coding for an oligopeptide or a polypeptide does not code for the Aspergillus niger benzoate-para- hydroxylase.
2. An expression cassette according to claim 1, in which fusion of the sequence coding for an oligopeptide or polypeptide has taken place to the 2nd or 3rd ATG codon on the part of the bphA gene coding for mRNA.
3. An expression cassette according to claim 1 in which the translation control sequences originate from a heterologous gene.
4. An expression cassette according to any one of claims 1- 3, characterized in that the sequence coding for an oligopeptide or a polypeptide codes for an industrial enzyme, feedstuff protein or a peptide with pharmaceutical activity, or for precursors thereof.
5. Filamentous fungus with one or more expression cassettes according to any one of the claims 1-4.
6. Filamentous fungus according to claim 5, characterized in that it is chosen from the series of genera Aspergillus, Penicillium, Trichoderma or relatives.
7. Filamentous fungus according to claim 6, characterized in that it is an Aspergillus niger, tubigensis, awamori or nidulans.
8. An expression cassette or filamentous fungus according to any one of claims 1-7, characterized in that it can be induced by compounds of formula I
X-phenyl-(A)-Y (I)
where X is an amino group or a hydroxy group, or a hydrogen or halogen atom, A is a methylene group, with or without amino or hydroxy group substitution, or a carbonyl group, or is absent, and Y consists of a carboxyl group or salts, amides and esters with lower alcohols thereof, or of an aldehyde group, a hydroxy group or a hydroxymethyl group.
9. An expression cassette or fungus according to claim 8, characterized in that the inducing compound is para- aminobenzoic acid.
10. Process for the preparation of an oligopeptide or polypeptide by adding a compound according to formula I to a filamentous fungus according to any one of claims 5-7 and recovering and optionally purifying the resulting oligopeptide or polypeptide.
11. Process for the preparation of a metabolite by adding a compound according to formula I to a filamentous fungus according to any one of claims 5-7 and recovering and optionally purifying the metabolite formed by the action of the oligopeptide or polypeptide whose expression has been induced.
12. Process according to either of claims 10-11, characterized in that the filamentous fungus is grown virtually without an inducer for the expression cassette according to any one of claims 1-4, and in that at a desired cell density there is hardly any further growth and an inducer is added.
13. Expression cassette, filamentous fungus and/or process as substantially described in the specification and the examples.
PCT/NL1992/000168 1991-10-01 1992-09-30 Expression cassette for heterologous proteins in filamentous fungi WO1993007277A1 (en)

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WO2011161207A1 (en) * 2010-06-25 2011-12-29 Novozymes A/S Polynucleotides having promoter activity
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EP2505651A2 (en) 2006-12-10 2012-10-03 Dyadic International, Inc. Isolated fungus with reduced protease activity
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