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WO1993007277A1 - Cassette d'expression pour proteines heterologues dans les champignons filamenteux - Google Patents

Cassette d'expression pour proteines heterologues dans les champignons filamenteux 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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PCT/NL1992/000168
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English (en)
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/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Cassette d'expression comportant: (A) un système de régulation d'expression 5' possédant (1a) un promoteur muni de séquences de contrôle de transcription; (1b) un site initiateur de transcription; (2a) des séquences de contrôle de traduction du côté 3' du site initiateur de transcription; (2b) un site initiateur de traduction; et (B) un séquence codant pour un oligopeptide ou un polypeptide. Ladite cassette d'expression est apte à provoquer l'expression d'un oligopeptide ou d'un polypeptide chez les champignons filamenteux, et elle contient un système de régulation d'expression dans lequel le promoteur du gène bphA de Aspergillus niger, ou une séquence fonctionnellement équivalente, est présent, et la séquence codant pour un oligopeptide ou un polypeptide ne code pas pour la benzoate-para-hydroxylase de Aspergillus niger. On décrit également les champignons filamenteux obtenus à l'aide de cette cassette d'expression.
PCT/NL1992/000168 1991-10-01 1992-09-30 Cassette d'expression pour proteines heterologues dans les champignons filamenteux WO1993007277A1 (fr)

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BE9100903A BE1005432A4 (nl) 1991-10-01 1991-10-01 Expressiecassette voor heterologe eiwitten.
BE9100903 1991-10-01

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1562417A2 (fr) * 2002-11-12 2005-08-17 Purdue Research Foundation Promoteurs inductibles par du benzoate
WO2011161207A1 (fr) * 2010-06-25 2011-12-29 Novozymes A/S Polynucléotides présentant une activité de promoteur
WO2011161208A1 (fr) * 2010-06-25 2011-12-29 Novozymes A/S Polynucléotides présentant une activité de promoteur
US8268585B2 (en) 1998-10-06 2012-09-18 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
EP2505651A2 (fr) 2006-12-10 2012-10-03 Dyadic International, Inc. Isolat de champignon avec activité protéase réduite
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
WO2018226900A2 (fr) 2017-06-06 2018-12-13 Zymergen Inc. Plate-forme d'ingénierie génomique htp permettant d'améliorer les souches fongiques
US11028401B2 (en) 2018-06-06 2021-06-08 Zymergen Inc. Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production
US11479779B2 (en) 2020-07-31 2022-10-25 Zymergen Inc. Systems and methods for high-throughput automated strain generation for non-sporulating fungi

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0439997A1 (fr) * 1990-01-29 1991-08-07 Ciba-Geigy Ag Système d'expression de champignons

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0439997A1 (fr) * 1990-01-29 1991-08-07 Ciba-Geigy Ag Système d'expression de champignons

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND GENERAL GENETICS vol. 223, 1990, BERLIN pages 192 - 197 R. VAN GORCOM ET AL 'Isolation and molecular characterization of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: A member of a new gene family of the cytochrome P450 superfamily' cited in the application *
NUCLEIC ACIDS RESEARCH. vol. 16, no. 18, 1988, ARLINGTON, VIRGINIA US page 9052 R. VAN GORCOM AND C. VAN DEN HONDEL 'Expression analysis vectors for Aspergillus niger' cited in the application *
TRENDS IN BIOTECHNOLOGY. vol. 7, no. 10, October 1989, CAMBRIDGE GB pages 283 - 287 G. SAUNDERS ET AL 'Heterologous gene expression in filamentous fungi' *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8916363B2 (en) 1996-10-10 2014-12-23 Dyadic International (Usa), Inc. Construction of Highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8268585B2 (en) 1998-10-06 2012-09-18 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
EP1562417A4 (fr) * 2002-11-12 2007-08-29 Purdue Research Foundation Promoteurs inductibles par du benzoate
EP1562417A2 (fr) * 2002-11-12 2005-08-17 Purdue Research Foundation Promoteurs inductibles par du benzoate
US7705203B2 (en) * 2002-11-12 2010-04-27 Purdue Research Foundation Benzoate inductible promoters
EP2505651A2 (fr) 2006-12-10 2012-10-03 Dyadic International, Inc. Isolat de champignon avec activité protéase réduite
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
US8802412B2 (en) 2010-06-25 2014-08-12 Novozymes A/S Polynucleotides having promoter activity
CN103068989A (zh) * 2010-06-25 2013-04-24 诺维信公司 具有启动子活性的多核苷酸
CN103068988A (zh) * 2010-06-25 2013-04-24 诺维信公司 具有启动子活性的多核苷酸
WO2011161207A1 (fr) * 2010-06-25 2011-12-29 Novozymes A/S Polynucléotides présentant une activité de promoteur
WO2011161208A1 (fr) * 2010-06-25 2011-12-29 Novozymes A/S Polynucléotides présentant une activité de promoteur
CN103068989B (zh) * 2010-06-25 2015-09-09 诺维信公司 具有启动子活性的多核苷酸
WO2018226900A2 (fr) 2017-06-06 2018-12-13 Zymergen Inc. Plate-forme d'ingénierie génomique htp permettant d'améliorer les souches fongiques
US10954511B2 (en) 2017-06-06 2021-03-23 Zymergen Inc. HTP genomic engineering platform for improving fungal strains
US11180753B2 (en) 2017-06-06 2021-11-23 Zymergen Inc. HTP genomic engineering platform for improving fungal strains
US11242524B2 (en) 2017-06-06 2022-02-08 Zymergen Inc. HTP genomic engineering platform for improving fungal strains
US11028401B2 (en) 2018-06-06 2021-06-08 Zymergen Inc. Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production
US11299741B2 (en) 2018-06-06 2022-04-12 Zymergen Inc. Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production
US11479779B2 (en) 2020-07-31 2022-10-25 Zymergen Inc. Systems and methods for high-throughput automated strain generation for non-sporulating fungi

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