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WO2000071579A2 - Regulation de l'organite de l'homeostasie dans une production de cellule - Google Patents

Regulation de l'organite de l'homeostasie dans une production de cellule Download PDF

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Publication number
WO2000071579A2
WO2000071579A2 PCT/EP2000/004718 EP0004718W WO0071579A2 WO 2000071579 A2 WO2000071579 A2 WO 2000071579A2 EP 0004718 W EP0004718 W EP 0004718W WO 0071579 A2 WO0071579 A2 WO 0071579A2
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WIPO (PCT)
Prior art keywords
organelle
gene
homeostasis
chrysogenum
fungus
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PCT/EP2000/004718
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English (en)
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WO2000071579A3 (fr
Inventor
Roelof Ary Lans Bovenberg
Marco Alexander Van Den Berg
Maarten Nieboer
Marten Veenhuis
Jan Andries Kornelis Willem Kiel
Ida Johanna Van Der Klei
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Dsm N.V.
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Priority to EP00931260A priority Critical patent/EP1179071A2/fr
Priority to AU49249/00A priority patent/AU4924900A/en
Publication of WO2000071579A2 publication Critical patent/WO2000071579A2/fr
Publication of WO2000071579A3 publication Critical patent/WO2000071579A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/385—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Penicillium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/78—Hansenula
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium
    • C12R2001/82—Penicillium chrysogenum

Definitions

  • the present invention is concerned with a method for modulating the production of secondary metabolites in the fungus Penicillium chrysogenum by influencing the homeostasis of organelles in this organism.
  • the present invention is based on the finding that next to influencing the expression level of any protein present in said organelles, the homeostasis of organelles can also be modulated by influencing the expression of at least one gene which is directly related to organelle homeostasis.
  • the present invention provides a method for modulating the production of secondary metabolites in the fungus Penicillium chrysogenum by modulating a least one gene which is directly related to organelle homeostasis in this fungus.
  • Modulating of genes as used herein comprises influencing the level of expression and or the targeting and or the primary sequence of the genes and encompasses classical selection and screening methods, herein also identified as classical genotypic modification, and more modern genetic modification techniques varying from relatively straightforward physical or chemical mutagenic treatment followed by several rounds of screening and selection to intricate recombinant technologies that can modify the level of expression, the targeting or the primary sequence of genes directly.
  • Homeostasis herein describes the subtle balance between organelle increase or biogenesis and organelle decrease, degradation or death.
  • genes directly related to organelle homeostasis comprise genes who function essentially in regulating the biogenesis of organelles or in degradation or death of organelles.
  • the organelle homeostasis is modulated by genetically engineering at least one gene encoding a protein which is responsible for organelle biogenesis (the expression and/or activity of which is to be increased) and or at least one gene encoding a protein which responsible for organelle degeneration (the expression and/or activity of which is to be decreased).
  • the organelles of choice are the microbodies (peroxisomes, glyoxysomes).
  • Microbodies have a simple architecture and are surrounded by a single membrane. A specific feature of microbodies is that they are inducible; upon induction, the organelles multiply by growth and subsequent fission.
  • microbodies lack DNA and a protein-synthesising machinery. All peroxisomal proteins identified so far are encoded by nuclear genes and with few exceptions are synthesised on free polysomes at their mature size. Hence, the essential topogenic information resides in the structure of the polypeptide.
  • Such genetic engineering of genes related to organelle homeostasis may involve effecting either the level of expression of the instant genes or a genetic modification of the gene itself so as to result in a protein with a higher activity in organelle biogenesis or in a lower activity in organelle degradation.
  • a novel protein and the polynucleotide encoding such protein in Penicillium chrysogenum could be identified.
  • the present invention is therefor also related to a novel protein (Pex11 p) active in the biogenesis of organelles, in particular in the biogenesis of microbodies, and which is represented by the amino acid sequence SEQ ID NO: 2.
  • the present invention is also related to isolated polynucleotides encoding such protein Pex11p, and in particular to the polynucleotide pex11 which is represented by SEQ ID NO. 1.
  • Such a polynucleotide can be incorporated in an expression vector suitable for transformation of the Penicillium chrysogenum and which effects upon transformation expression of the particular polynucleotide incorporated therein.
  • the present invention further provides Penicillium chrysogenum comprising a modulated gene or functional fragment thereof related to organelle biogenesis, and more in particular Penicillium chrysogenum comprising a modulated gene encoding the protein Pex11 p as exemplified by SEQ ID NO: 1 or a functional fragment thereof.
  • the invention provides Penicillium chrysogenum comprising a modulated gene or functional fragment thereof related to organelle degradation.
  • Decreasing organelle degradation serves the same purpose as increasing organelle biogenesis; at a given moment in time, a fungal cell as provided by the invention has more organelles available by or through which a secondary metabolite can be produced, thus optimising the yield of said substance.
  • degradation can be regulated by modifying genes involved in peroxisome degradation, for example wherein said gene is a vps15 gene or a gene functionally related thereto, or wherein said gene is a pddl gene or gene functionally related thereto.
  • Microbody degradation can be regulated or influenced at various levels, for example at the level of signalling of individual cells for degradation followed by vacuolisation of the organelles destined for degradation and degradation of organelle content by vacuolar hydrolases.
  • the production of secondary metabolites by P. chrysogenum can be modulated.
  • Such secondary metabolites comprise Mactam compounds such as penem and cephem compounds. More in particular, these micro- organisms produce penicillin or core structures thereof (e.g. 6-APA, 6-amino penicillanic acid) or cephalosporin or compounds containing core structures thereof (e.g. 7-ADCA, 7- aminodesacetoxy cephalosporanic acid and 7-ACA, 7-amino cephalosporanic acid)
  • a batch of conidiospores from Penicillium strain Wisconsin54-1255 was mutagenised using nitrochinoline oxide.
  • a selected batch of mutagenised spores (survival 1-10 %) was replica- plated on a complex rich medium and on a medium containing oleic acid (0.1 %) as the sole carbon source. Mutants that were unable to utilise oleic acid were further tested for growth on other fatty acids: lauric acid, and hexanoic acid. From these tests, five mutants were selected that were generally deficient in fatty acid catabolism. These mutants were subsequently grown in a medium supporting the proliferation of peroxisomes. After 5 days of growth in this medium, samples were prepared for Electron Microscopy.
  • Peroxisome volume fraction is proportional to penicillin production.
  • Peroxisomal volume is higher in strains with increased penicillin production.
  • Penicillium strain Wisconsin54-1255 and a strain with a increased penicillin production were cultivated in a medium containing lactose and phenoxy acetic acid. After 5 days of growth at 25 degrees Celsius these cultures were harvested. Part of the samples were prepared for electron microscopy, while the remaining were used to determine the penicillin production rate. The slides for EM were labelled with AT antibody to stain the peroxisomes. Several individual cells were scored to calculate the peroxisomal volume fraction. The cells of strain DS04825 contained a substantial higher volume fraction of peroxisomes, which coincided with an increased penicillin production rate (Figure 7). This supports the conclusion that in order to obtain strains with higher penicillin production Penicillium chrysogenum can be modified so as to produce higher volumes of peroxisomes.
  • Penicillium chrysogenum was cultivated in shake flasks on minimal medium containing lactose as carbon source to create /-Mactam producing conditions. The culture was incubated for seven days at 25 °C and 280 rpm. Each day a sample was analysed for penicillin production and carbon consumption to calculate the specific -Mactam production rate. At the same time points samples were fixated in KMnO 4 and labelled with AT antibody which is exclusively localised in peroxisomes (M ⁇ ller, 1991) to monitor the peroxisomes. By screening a lot of individual cells using electron microscopy it was obvious that during the latter stages of the fermentation, when the production rate is decreasing, the vacuoles of P.
  • Penicillium chrysogenum strain e.g. DS04825 or P2
  • penicillin production medium Theilgaard et al., 1997 1
  • Escherichia coli DH5a (Sambrook et al., 1989 2 ) was grown in LB medium supplemented with the appropriate antibiotics.
  • P. chrysogenum hyphe were protoplasted by incubation for 1.5 h in KC buffer (6 % KCI, 0.2 % citric acid) containing 2 mg/ml Novozym.
  • Protoplasts were harvested, washed with a 1 :1 mixture of KC buffer and buffer A (1.2 mol/l sorbitol, 5 mmol/l MES, 1 mmol/l MgCI 2 , 1 mmol/l EDTA, pH 5.5) followed by a wash with buffer A.
  • Protoplasts were homogenised in buffer A supplemented with 1 mM PMSF.
  • a dominant 23 kDa protein band gave an N-terminal sequence that was similar to the peroxisomal membrane protein Pex11 p from Saccharomyces cerevisiae (Erdmann and Blobel, 1995 3 ; Marshall et al., 1995 4 ) and Candida boidinii (Sakai et al., 1995 5 ). (Fig. 1).
  • cDNAs were cloned as 5' Sal ⁇ -Not ⁇ 3' fragments between the Sal ⁇ and Not ⁇ sites in the pCMV-SPORT4 vector.
  • PCR was performed with the pexl 1- specific primers and the M13/pUC universal and reverse sequencing primers.
  • PCR with the PEX11-F1/universal primer combination resulted in the synthesis of a highly specific fragment of approximately 1 kb. This fragment was digested with Not ⁇ , partly filled-in and cloned between the Not ⁇ and Smal sites of pBluescript II SK+ (Stratagene, San Diego, CA, USA), and sequenced.
  • the gene is for example placed under the control of the P. chrysogenum IPNS promoter.
  • the expression vector pGBRH2 was constructed (Fig. 4): Two PCR fragments containing the P. chrysogenum IPNS promoter and P. chrysogenum -AT terminator regions were isolated by PCR using the primer combinations IPNS #1 (SEQ ID NO: 5) plus IPNS#2 (SEQ ID NO: 6) and AT #1 (SEQ ID NO: 3) plus AT#2 (SEQ ID NO: 4), respectively.
  • the A T terminator fragment was digested with Smal and Not ⁇ and cloned between the Smal and Not ⁇ sites of pBluescript II KS+ (Stratagene). Subsequently, the IPNS promoter fragment, digested with Asp7 .8 and Hind ⁇ , was cloned between the Asp718 and Hindlll sites of the polylinker of the resulting plasmid.
  • the P. chrysogenum pex11 gene is inserted as a Sa/l(blunted)-£coRI fragment between the SamH1 (blunted) and EcoRI sites of the polylinker of pGBRH2, resulting in plasmid pGBRH2-PcPEX11.
  • the expression plasmid is digested with Not ⁇ to release the P IP N S -PCPEX77-T AT cassette. This fragment is co-transformed with a DNA fragment carrying the Aspergillus nidulans AMDS gene as a selection marker into various P. chrysogenum strains. Transformants were selected on acetamide agar and were screened for overproduction of Pex11 p using Western blotting with polyclonal antibodies.
  • Figure 8 shows a clear example of the ultrastructural changes in the cell upon introduction of additional Pex11 p protein; a huge proliferation of the amount of peroxisomes is observed. All these peroxisomes were shown to be functional; e.g. the contain acyltransferase (AT). The total amount of AT was equal to the parent strain (data not shown); therefore
  • pexl 1 cDNA of P. chrysogenum as a probe allows to isolate the genomic region containing pex11. This allows the construction of mutants lacking pex11 (so-called p ex17 strains) by disruption (single crossing-over) or deletion of (part of) the gene (double crossing-over). pex77strains are analysed using ultrastructural (quantification of number/volume fraction of peroxisomes) and biochemical (production of penicillin) techniques.
  • Fig. 1 Sequence similarity between the N-terminal amino acid sequence of the dominant 23 kDa P. chrysogenum peroxisomal membrane protein and Pexl 1 p from Candida boidinii (Cb-Pex11 p) and Saccharomyces cerevisiae (Sc-Pex11 p). Identical residues are indicated by an asterisk, conservative replacements by a dot. Question marks indicate amino acids that could not be assigned with 100 % certainty during sequencing.
  • Fig. 2 Nucleotide sequence of clone 12 containing P. chrysogenum pex11 cDNA and the deduced amino acid sequence of Pexl 1 p.
  • the linker used to clone the cDNAs in pCMV- SPORT4 as well as the PEX11-STOP primer used to isolate the cDNA are indicated.
  • Fig. 3 Multiple sequence alignment between the amino acid sequences of Pexl 1 p of P. chrysogenum (PCPEX11P; 238 residues), Candida boidinii (CBPEX11P; 256 residues) and Saccharomyces cerevisiae (SCPEX11 P; 236 residues).
  • PCPEX11P Pexl 1 p of P. chrysogenum
  • CBDP Candida boidinii
  • SCPEX11 P Saccharomyces cerevisiae
  • Penicillin production rate is seriously hampered in microbody-deficient strains mut 1 , 2 or 3, as compared to parent strain Wisconsin54-1255.
  • Fig. 8 Strong proliferation of microbodies in a Pexl 1p overproducing Penicillium chrysogenum strain as compared to a wild type strain (Wisconsin54-255) is shown.
  • Fig. 9 Electron microscopic observation of fusion of the vacuoles of P. chrysogenum fuse with peroxisomes during the latter stages of the fermentation, when the production rate is decreasing.

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Abstract

L'invention concerne une méthode permettant de moduler la production de métabolites secondaires dans le champignon Penicillium chrysogenum par influence des organites de l'homéostasie par modulation d'au moins un gène directement lié à l'organite de l'homéostasie dans ce champignon. De préférence, l'organite de l'homéostasie est modulé par organisation génétique d'au moins un gène codant pour cette protéine responsable de la biogenèse de l'organite (protéine dont l'expression et/ou l'activité doit être augmentée) et/ou au moins un gène codant pour une protéine responsable de la dégénérescence de l'organite (protéine dont l'expression et/ou l'activité doit être réduite). Des gènes intervenant dans la biosynthèse de l'organite dans le P. chrysogenum sont illustrés par le gène dénommé pex11. Des gènes intervenant dans la dégradation de l'organite sont illustrés par les gènes dénommés vps15 et pdd1.
PCT/EP2000/004718 1999-05-21 2000-05-22 Regulation de l'organite de l'homeostasie dans une production de cellule WO2000071579A2 (fr)

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EP00931260A EP1179071A2 (fr) 1999-05-21 2000-05-22 Regulation de l'organite de l'homeostasie dans une production de cellule
AU49249/00A AU4924900A (en) 1999-05-21 2000-05-22 Regulating organelle homeostasis in a cell factory

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EP99201630 1999-05-21
EP99201630.3 1999-05-21

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014003555A1 (fr) * 2012-06-27 2014-01-03 Rijksuniversiteit Groningen Production améliorée de pénicilline

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0448180A2 (fr) * 1990-03-23 1991-09-25 Gist-Brocades N.V. Méthode pour moduler la production des métabolites secondaires
WO1994024289A1 (fr) * 1993-04-19 1994-10-27 Eurolysine Procede d'adressage de proteines dans les peroxysomes de levures
WO1997038107A1 (fr) * 1996-04-04 1997-10-16 Gist-Brocades B.V. Enzyme possedant une forte activite d'adipoyl-coenzyme a synthetase et utilisations de celle-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0448180A2 (fr) * 1990-03-23 1991-09-25 Gist-Brocades N.V. Méthode pour moduler la production des métabolites secondaires
WO1994024289A1 (fr) * 1993-04-19 1994-10-27 Eurolysine Procede d'adressage de proteines dans les peroxysomes de levures
WO1997038107A1 (fr) * 1996-04-04 1997-10-16 Gist-Brocades B.V. Enzyme possedant une forte activite d'adipoyl-coenzyme a synthetase et utilisations de celle-ci

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MARSHALL P ET AL: "Pmp27 promotes peroxisomal proliferation" J. CELL BIOL., vol. 129, no. 2, April 1995 (1995-04), pages 345-355, XP002120786 cited in the application *
ROGGENKAMP R ET AL: "Formation of irregular giant peroxisomes by overproduction of the crystalloid core protein methanol oxidase in the methylotrophic yeast Hansenula polymorpha" MOL CELL BIOL., vol. 9, no. 3, March 1989 (1989-03), pages 988-994, XP000037387 *
SAKAI Y ET AL: "The Candida boidinii peroxisomal membrane protein Pmp30 has a role in peroxisomal proliferation and is functionally homologous to Pmp27 from Saccharomyces cerevisiae" J BACTERIOL., vol. 177, December 1995 (1995-12), pages 6773-6781, XP002120787 cited in the application *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014003555A1 (fr) * 2012-06-27 2014-01-03 Rijksuniversiteit Groningen Production améliorée de pénicilline

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