WO2002010342A2 - Yeast strains - Google Patents

Yeast strains Download PDF

Info

Publication number
WO2002010342A2
WO2002010342A2 PCT/GB2001/003457 GB0103457W WO0210342A2 WO 2002010342 A2 WO2002010342 A2 WO 2002010342A2 GB 0103457 W GB0103457 W GB 0103457W WO 0210342 A2 WO0210342 A2 WO 0210342A2
Authority
WO
WIPO (PCT)
Prior art keywords
fungal
mutated
expression
biomass
fungi
Prior art date
Application number
PCT/GB2001/003457
Other languages
French (fr)
Other versions
WO2002010342A3 (en
Inventor
John Walker
Ferdinando Palmieri
Luigi Palmieri
Original Assignee
Medical Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to AU7574101A priority Critical patent/AU7574101A/en
Publication of WO2002010342A2 publication Critical patent/WO2002010342A2/en
Publication of WO2002010342A3 publication Critical patent/WO2002010342A3/en

Links

Classifications

    • 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
    • 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
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • 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
    • C12R2001/66—Aspergillus
    • 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
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present invention relates to improved fungal strains which possess the ability to grow to a greater specific biomass and thus possess enhanced properties for both fungal production and the expression of useful gene products.
  • the invention relates to mutant yeast strains having one or more mutations in one or more yeast mitochondrial carrier genes.
  • Fungi and especially yeasts
  • yeasts are well known for their commercial applications both as expression vehicles for polypeptide production and as products in their own right (for example in the form of baker's or brewing yeasts). Techniques for culturing yeasts for such applications are well known in the art.
  • yeasts rely on carbohydrate feedstocks as energy sources for the growth of the organisms. These may be, for example, single carbohydrates or simple mixtures thereof, as commonly used in research applications, or complex natural sources such as molasses and wort, as commonly used in industrial applications. Facultative fermentative yeasts such as Saccharomyces cerevisiae can metabolise sugars either through aerobic respiration or alcoholic fermentation. alcoholic fermentation occurs to a certain degree even during aerobic cultivation. This tendency towards alcoholic fermentation limits the ability of S. cerevisiae to grow to a high specific biomass, since biomass yields are significantly reduced during fermentative growth. Fully respiratory metabolism is observed only at low specific growth rates in aerobic, sugar-limited cultures, which conditions can theoretically be met in fed-batch cultures as used in the industrial production of baker's yeast.
  • fermentation by-products not only reduces biomass yield, but also reduces the formation of biomass related products, such as heterologous polypeptides expressed in yeasts.
  • biomass related products such as heterologous polypeptides expressed in yeasts.
  • high product concentrations are desirable. Large-scale heterologous polypeptide production therefore typically occurs at high biomass densities. At high biomass densities, even low specific rates of fermentation by-product formation will rapidly lead to the build-up of toxic metabolite levels, with detrimental effects on productivity.
  • only a small fraction of the carbon and nitrogen substrates fed to heterologous protein-producing cultures is converted into the product of interest. This means that cost of feedstocks remains a major factor in the economics of biomass-directed industrial applications for yeasts.
  • fed-batch culture as referred to above may be designed to maintain nutrient concentration at a level which favours aerobic growth.
  • the aim is to avoid fermentative sugar metabolism as far as it is possible to do so.
  • Mitochondrial carrier proteins are key components in the integration of mitochondria into the metabolism of the cell through their contribution to DNA synthesis, redox balance and by participating in various biosynthetic pathways.
  • Yeast mitochondrial transport proteins have been reviewed by Walker and Runswick (Journal of Bioenergetics and Biomembranes (1993) 25:435) and more recently in Palmieri & van Ommen, (1999) in Frontiers in Cellular Bioenergetics (Papa, Guerrieri, and Tager, Eds.) pp. 489-519, Kluwer Academic/plenum Publishers, New York. 35 members of the mitochondrial carrier family are known, but the transport specificities are known for only a subset thereof.
  • a mutated fungal strain which grows to a higher specific biomass than the wild-type strain, wherein the mutation modulates the expression of at least one mitochondrial carrier protein.
  • a "mutated fungal strain” is one or more cells of a fungal strain which has been altered, with respect to the wild-type strain (see below).
  • the mutation may be carried out by any one of a number of techniques, as set forth in more detail below, but the aim is to modulate the expression of at least one mitochondrial carrier protein.
  • the wild-type strain is a fungal strain upon which the mutated strain is based.
  • This may be the parental strain, in other words the strain from which the mutated strain is directly generated by mutation, or a basic strain from which the mutated strain is derived after successive modifications.
  • it is the parental strain.
  • Specific biomass refers to the biomass produced per unit volume of the fungal culture at the termination of the growth phase, when cell density reaches a plateau. As used herein, where reference is made to “cells”, it should be understood to include hyphal structures in filamentous fungi.
  • the present invention involves the modulation of the expression of mitochondrial carrier proteins in fungi.
  • Modulation refers to any change in the activity of the protein, whether achieved by alteration of that activity, or by potentiation or repression of the wild-type activity, or by an increase or decrease in the levels of expression of the wild-type protein. It includes complete or partial loss of protein activity through gene deletion and/or mutation.
  • the mutated fungal strains according to the invention are advantageously more efficient in the metabolism of at least one carbohydrate source than the wild-type strain.
  • the advantage may be specific to a single carbohydrate, or common to more than one or even substantially all carbohydrates. It is postulated that the increase in specific biomass production observed in fungi according to the invention is linked to an increase in respiratory capacity, which is associated with an increased efficiency of carbohydrate utilisation.
  • the fungal strains according to the invention are yeasts, amongst which S. cerevisiae is preferred.
  • the expression of two or more yeast carrier proteins is modulated. It has been found that simultaneous deletion of two mitochondrial carrier proteins has highly beneficial effects on fungal strains.
  • Preferred mitochondrial carrier proteins in S. cerevisiae are YMC1 (xvic2; chromosome 15, carrier 2) and YMC2 (iicl; chromosome 2, carrier 1).
  • the expression of one or both of these proteins is do nregulated.
  • the invention relates to a ymclymc2 yeast strain, in which both YMC1 and YMC2 gene loci have been disrupted or deleted so as to prevent YMC1 and YMC2 protein production.
  • the invention relates to a method for preparing a mutated fungal strain which grows to a higher specific biomass than the wild-type strain, comprising the steps of:
  • Modification of the fungi advantageously comprises gene deletion or mutation. This is preferably carried out by homologous recombination, using a selectable marker such as HIS3, which can be inserted into the yeast genome to replace or disrupt endogenous genes by known techniques.
  • a selectable marker such as HIS3
  • Yeast strains according to the invention are useful for biomass production, and for the production of heterologous polypeptides. Therefore, the invention provides a method for producing a polypeptide by recombinant expression in fungal cells, comprising the steps of transforming a mutated fungal strain according to the invention with a nucleotide sequence encoding the polypeptide under the control of suitable expression sequences, culturing the fungal cells and expressing the polypeptide therein, and isolating the polypeptide from the fungal cells.
  • the invention provides a method for producing fungal biomass, comprising the steps of culturing mutated fungal strain according to previous aspects of the invention, and optionally isolating the fungal cells from the culture medium.
  • Isolation refers to complete or partial isolation of the cells from the medium, or from at least one component of the medium.
  • isolation of cells or biomass may be achieved by any suitable technique, including sedimentation or centrifugation, drying, and the like.
  • Polypeptide isolation is likewise carried out by conventional techniques, including affinity techniques, sedimentation and metal ion chelation, as are known in the art.
  • the invention provides the use of a mutated yeast strain according to previous aspects for the production of heterologous polypeptides or biomass, as desired.
  • Mitochondrial carrier proteins may be found in any cells which comprise mitochondria, and in the context of the present invention in all fungal cells, including yeasts.
  • fungi which are in common use in commercial production of heterologous polypeptides or fungal biomass, such as for animal feed or other industrial uses, are particularly preferred for modification in accordance with the invention.
  • the invention may be applied to fungi of Aspergillus spp., such as A. niger, A. oryzae or A. aw ⁇ mori, commonly used for both endogenous enzyme production and production of heterologous polypeptides.
  • Other suitable fungi include Mucor and Endothia spp.
  • fungi used in the production of fungal rennins
  • Pennicillium citrinum nucleases
  • Pennicilium chrysogenum glucose oxidase
  • Kluyveromyces lactase
  • Trychoderma cellulase
  • suitable fungi are mentioned below in connection with recombinant polypeptide production. It will be understood that they need not necessarily be used for such purposes, but may be equally siotable for biomass or endogenous polypeptide production.
  • Biomass production, in the form of single-cell protein (SCP) is commonly based on yeasts such as Candida, Khiyveromyces, Endomycopsis or Pichia.
  • Saccharomyces spp. is highly important in commercial applications and is a preferred organism for use in the present invention.
  • S. cerevisiae is commonly used, but other varieties, such as S. carlsbergensis, S. ellipsoideus and Schizosaccharomyces axe also used.
  • Saccharomyces and especially S. cerevisiae, are particularly preferred in the context of the present invention.
  • 35 mitochondrial carriers are known, and are present in mitochondria from all eukaryotic species, including fungi and mammals.
  • mitochondrial carriers known to be highly similar to yeast mitochondrial carriers are known and can be identified in GenBank (4 April 2000):
  • NP_009662 mitochondrial carrier protein; Ymc2p [Saccharomyces cerevisiae] gi
  • CAA18388 (AL022299) mitochondrial carrier protein [Schizosaccharomyces pombe] gi
  • the fungal equivalents of known mitochondrial carriers, and any other proteins which are discovered to possess evolutionary homology with known mitochondrial carriers, may be targets for modification in accordance with the present invention.
  • yeast mitochondrial carriers YMC 1 and YMC2, and fungal homologues thereof are particularly preferred.
  • such carrier proteins are downregulated in the mutated fungal strains, and are preferably entirely deleted by genetic manipulation.
  • the polypeptide sequences of YMCl and YMC 2 are set forth herein as SEQ. ID. No. 1 and SEQ. ID. No. 2, respectively.
  • the nucleic acid sequence of YMCl is SEQ. ID. No. 3.
  • modulation of the activity of fungal mitochondrial carriers may be achieved in any one of a number of ways.
  • modulation refers to the alteration, upregulation or downregulation of the activity of a wild-type mitochondrial carrier, and can encompass deletion of the activity through genetic manipulation.
  • a number of strategies for modulation of mitochondrial carriers is set forth below; the examples are non-limiting and should be seen as illustrative of the types of techniques which may be used in the present invention.
  • Antisense nucleic acids preferably 10 to 20 base pair oligonucleotides capable of specifically binding to the expression control sequences or mRNA of the target mitochondrial carrier are introduced into cells (e.g., by transformation with a nucleic acid vector or by transfer of naked DNA).
  • the antisense nucleic acid binds to the target sequence in the fungal cell and prevents transcription or translation of the target sequence.
  • the antisense molecule recruits cellular nucleases which cleave the antisense: sense complex.
  • Phosphothioate and methylphosphate antisense oligonucleotides are more stable and are specifically contemplated for use in the invention.
  • the antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end.
  • fungal cells are transformed with vectors which express the required antisense oligonucleotides, such that expression of the target mitochondrial carrier protein is inhibited permanently in the fungal cell.
  • fungal cells may be subjected to entirely random mutagenesis, for example using mutagenic chemical agents or radiation, and selected according to their ability to achieve high biomass after a period of growth.
  • assays may be automated, by determining biomass according to, for example, optical density (such as OD 60 o) and conducting the growth experiments in multiwell plates, or other test systems suitable for high-throughput automated analysis.
  • the mutagenesis may be directed, using site-directed mutagenesis procedures as known in the art, and or mutagenesis kits available from a variety of manufacturers. Such procedures can be conducted in vitro or in vivo, and may be randomised to a greater or lesser degree.
  • Such mutation events may result in the potentiation or inhibition of the activity of a mitochondrial carrier, or in a qualitative change in its activity.
  • Advantageous mutations are generally selected by screening of numerous mutations, some of which may be neutral or disadvantageous in the context of the present invention.
  • modulation of mitochondrial carrier activity may be achieved by disruption or deletion of the gene encoding the carrier protein by homologous recombination.
  • flanking sequences which are complementary to sequences flanking the gene which it is desired to delete.
  • flanking sequences may be obtained by PCR amplification from fungal genomic nucleic acid, or by synthesis.
  • PCR-based approaches are preferred, and an exemplary approach may be based largely on the method described in Baudin et al., 1993, NAR 21: 3329.
  • the selectable marker for deletion (HIS3) is amplified by PCR using oligonucleotides that contain homology to the
  • the PCR product is used to transform yeast to His+, which removes the target gene completely. Correct gene deletion is verified by PCR of fungal colonies, using a universal test oligo in HIS3, and an oligo in flanking chromosomal sequence. Only correctly deleted transformants yield a PCR product.
  • the fungal strain used for gene deletion should have a complete deletion of the selectable yeast marker that will be used to replace the gene of interest. Otherwise, most of the events are likely to be gene conversion and homologous recombination at the marker locus.
  • yeast S. cerevisiae
  • HIS3 is preferred as a selectable marker for the disruptions.
  • the selectable marker is cloned into a plasmid such as pBluescript® which will not replicate in yeast, thereby reducing background from yeast transformants containing template plasmid. . . . _ ;
  • yeast include those conferring resistance to antibiotics G418, hygromycin or bleomycin, or those (such as HIS3) which provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2 or TRP1 genes.
  • Fungi have many applications in biotechnology and traditional industries, including use in baking and brewing, which requires the production of significant yeast biomass, use as a source of single cell protein, in the production of fungal enzymes for use in the food industry and in the production of heterologous proteins by recombinant gene technology. Each of these applications is well known in the art.
  • Biomass production requires culture of fungal cells to high specific biomass . and subsequent isolation of the cells from the medium. Mutated fungi according to the present invention are particularly advantageously employed in such applications, since they can achieve a high specific biomass and utilise carbon sources more efficiently. For production of fungal enzymes, a high specific biomass is again desirable, since the concentration of the desired product may be increased, thus facilitating its purification. Techniques for culture of fungi to produce fungal enzymes, typically Aspergillus fungi, are well known in the art.
  • heterologous polypeptides of eukaryotic and prokaryotic origins
  • fungi for the recombinant production of heterologous polypeptides of eukaryotic and prokaryotic origins
  • a fungal host cell is transformed with a vector encoding the desired heterologous polypeptide, and cultured such that the heterologous polypeptide is produced. It may be produced intracellularly, or secreted.
  • vector refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are known to those skilled in the art. Many vectors are available, and • selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence a signal sequence.
  • Both expression and cloning vectors generally contain at least one nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and other fungi.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and fungal origins may. be employed in filamentous fungi, for example the amal replicon (Gems et al, (1991) Gene 98:61-67).
  • Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression.
  • a vector is cloned in E. coli and then the same vector is transfected into yeast or other fungal cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome.
  • an expression and cloning vector may contain a selection gene also referred to as selectable marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, into which it has been transferred.
  • This gene may encode a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from media.
  • any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.
  • Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, L ⁇ U2, LYS2, TRP1, or HIS3 gene.
  • a selectable marker has been used for mutation by homologous recombination
  • a different selectable marker is used for transformation of the host cells.
  • an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript® vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin.and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
  • the vector preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
  • Plasmids employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art.
  • Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
  • host cell includes any fungus, whether a unicellular organism, a cell derived from a multicellular organism and placed in tissue culture or a cell present as part of a multicellular organism, which is susceptible to transformation with a nucleic acid construct according to the invention.
  • host cells such as yeast and other fungal cells may be used for replicating DNA and producing polypeptides encoded by nucleotide sequences as used in the invention.
  • Suitable cells are generally filamentous fungi or yeasts.
  • cells from filamentous fungi preferably Aspergillus, such as A. niger and A. tubingensis.
  • Other preferred organisms include any one of Aspergillus oryzae, A. awamori, Trichoderma reesei, T. viride and T. longibrachiatum.
  • the transformed cell or organism may be used to prepare acceptable quantities of the desired gene product which is retrievable from the cell or organism.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression. Any suitable promoter may be used in connection with the present invention, with xylan-inducible promoters preferably of fungal origin being especially indicated.
  • the promoter of the present invention can additionally include features to ensure or to increase expression in a suitable host.
  • the features can be conserved regions such as a Pribnow Box or a TATA box.
  • the TATA box is typically found 30 bp upstream of the transcription initiation site, and is believed to be involved in the assembly of the transcriptional complex.
  • the promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the heterologous nucleotide sequence.
  • Fungal promoters typically contain both a TATA box and a UAS (Upstream Activating Site).
  • the UAS is a binding site for the activating regulator acting on the promoter in question.
  • the element of the present invention is believed to be a UAS.
  • Other sites, involved in various aspects of promoter regulation, may also be included.
  • heterologous nucleotide sequence with reference to the constructs according to the present invention means any sequence encoding a polypeptide of interest, other than the complete natural sequence normally associated with the promoter employed, or a sequence which is capable of expressing a nucleic acid, for example a regulatory RNA such as an antisense RNA or a ribozyme, or a tRNA or rRNA capable of regulating the metabolism of an organism.
  • nucleotide sequence includes homologous nucleotide sequences which are the sequences normally associated with the promoters employed. The invention includes the use of both homologous and heterologous nucleotide sequences.
  • heterologous nucleotide sequence can be any nucleotide sequence that is either foreign or natural to the organism (e.g. filamentous fungus, preferably of the genus Aspergillus, or a plant) in question.
  • the term "heterologous nucleotide sequence” also includes a homologous nucleotide sequence which has been mutated, such as by insertion, addition, deletion or alteration, such that it is no longer identical with the natural homologous nucleotide sequence.
  • heterologous nucleotide sequence examples include sequences coding for proteins and enzymes that modify metabolic and catabolic processes.
  • the heterologous nucleotide sequence may code for an agent for introducing or increasing pathogen resistance.
  • the heterologous nucleotide sequence may be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues.
  • the heterologous nucleotide sequence may code for a non-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans.
  • nucleotide sequences according to the invention include pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, hexose oxidase, oxidoreductases, Upases, glucan lyase, rhamno-galacturonases, hemicellulases, endo- ⁇ - glucanases, arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof.
  • the heterologous nucleotide sequence may be a protein giving nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant).
  • the heterologous nucleotide sequence may code for an enzyme that can be used in food processing such as chymosin, thaumatin and ⁇ -galactosidase.
  • the heterologous nucleotide sequence can encode any one of a pest toxin, an antisense transcript such as that for patatin or ⁇ -amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense, a glucanase or genomic ⁇ -l,4-endoglucanase.
  • the heterologous nucleotide sequence may code for an intron of a particular nucleotide sequence, wherein the intron can be in sense or antisense orientation.
  • the promoter and the nucleotide sequence according to the invention are stably maintained within host cells or transgenic organisms.
  • the promoter and/or the nucleotide sequence may be maintained within the transgenic organism in a stable extrachromosomal construct. This is preferred for transgenic yeast or some filamentous fungi.
  • the promoter and/or the heterologous nucleotide sequence (such as the nucleotide sequence according to the present invention) may be stably incorporated within the transgenic organism's genome. This is preferred for some transgenic yeast, and most filamentous fungi.
  • a preferred host organism for the expression of the nucleic acid constructs of the present invention and/or for the preparation of the heterologous polypeptides according to the present invention is an organism of the genus Aspergillus, such as Aspergillus niger.
  • a transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant- DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D., Kinghorn J.R.
  • filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional Japanese koji and soy fermentations have used Aspergillus sp. Also, in this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry. There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracellular products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
  • expression constructs are prepared by inserting a heterologous nucleotide sequence (such as a nucleotide sequence coding for an amylase enzyme) into a construct designed for expression in filamentous fungi.
  • a heterologous nucleotide sequence such as a nucleotide sequence coding for an amylase enzyme
  • the constructs contain the promoter according to the present invention which is active in fungi.
  • the heterologous nucleotide sequence can be fused to a signal sequence which directs the protein encoded by the heterologous nucleotide sequence to be secreted.
  • a signal sequence of fungal origin is used.
  • a terminator active in fungi may also be employed.
  • heterologous nucleotide sequence is fused to a fungal gene encoding a stable protein.
  • This can stabilise the protein encoded by the heterologous nucleotide sequence which encodes a protein of interest.
  • a cleavage site recognised by a specific protease, can be introduced between the fungal protein and the protein encoded by the heterologous nucleotide sequence, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the heterologous nucleotide sequence.
  • a site which is recognised by a KEX-2 like peptidase found in at least some Aspergilli (Broekhuijsen et al 1993 J Biotechnol 31 135- 145).
  • a fusion leads to cleavage in vivo resulting in protection of the expressed product and not a larger fusion protein.
  • Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins.
  • the proteins can be deposited intracellularly if the nucleotide sequence according to the present invention (or another heterologous nucleotide sequence) is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention (or another heterologous nucleotide sequence) is equipped with a signal sequence the protein will accumulate extracellularly.
  • heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
  • filamentous fungi For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991, Op. Cit). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
  • auxotrophic markers such as argB, trpC, niaD and pyrG
  • antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance.
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • the species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression.
  • Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarranton, eds, pp 107-133, Blackie, Glasgow).
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting a nucleotide sequence comprising a DNA construct according to the present invention into a construct designed for expression in yeast.
  • a nucleotide sequence comprising a DNA construct according to the present invention
  • a construct designed for expression in yeast Several types of constructs used for heterologous expression have been developed, suitable component parts of which are discussed hereinbefore.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • S. cerevisiae YPH499 strain (MATa ade2-101 his3- ⁇ 200 Ieu2- ⁇ l ura3-52 trpl- ⁇ 63 lys2- 810) is used as a basis for the present experiments.
  • the yeast mitochondrial carrier genes YMCl (xvic2) and YMC2 (iicl) are deleted by homologous recombination with the auxotrophic marker HIS3 at the respective loci.
  • ymclymc2 deletion of YMC2 in ymcl yeast is carried out with the marker TRP1.
  • Yeast are grown in a synthetic medium (SM) containing 0.67% yeast nitrogen base, without amino acids (Difco), 0.1%KH 2 PO 4 , 0.12% (NH 4 ) 2 SO 4 , 200 mg/1 leucine, 40 mg/1 lysine, 40 mg/1 tryptophan, 40 mg/1 uracil and 10 mg/1 adenine supplemented with carbon sources as indicated below.
  • SM synthetic medium
  • the final pH is adjusted to 4.5 with KOH or 6.5 for growth on acetate.
  • Histidine 40 mg/1) is added to cultures of wild-type cells (his3).
  • Yeasts are precultured in complex medium (YP) supplemented with 2% glucose, harvested in the late exponential growth phase by centrifugation, washed twice with cold SM and diluted into the same medium as described above, supplemented with the stated carbon sources, for the experimental assessment.
  • YP complex medium
  • the biomass is determined by optical density measurement at 600nm when cell growth has reached a plateau. Wild-type cells, in each case, exhibit similar growth to ymc2 cells.
  • the ymclymc2 double mutant shows significantly higher growth on glucose (2x biomass), galactose (2.4x), and especially glycerol (almost 4x). Strikingly, the biomass increases over wild-type where the carbon source is changed from respiratory metabolism repression (glucose), to partial repression (galactose) to derepression (glycerol).
  • the ymclymc2 mutant also grows on other non-fermentative carbon sources better than wild-type or ymc2 mutants.
  • the ymcl single mutant also shows a significant increase in biomass over wild-type when grown on glucose (1.5x) and, to a lower extent, on ethanol and glycerol.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides a mutated yeast strain which grows to a higher specific biomass than the wild-type strain, wherein the mutation modulates the expression of at least one mitochondrial carrier protein.

Description

Fungal Strains
The present invention relates to improved fungal strains which possess the ability to grow to a greater specific biomass and thus possess enhanced properties for both fungal production and the expression of useful gene products. In particular, the invention relates to mutant yeast strains having one or more mutations in one or more yeast mitochondrial carrier genes.
Fungi, and especially yeasts, are well known for their commercial applications both as expression vehicles for polypeptide production and as products in their own right (for example in the form of baker's or brewing yeasts). Techniques for culturing yeasts for such applications are well known in the art.
Most if not all methods for culturing fungi and especially yeasts rely on carbohydrate feedstocks as energy sources for the growth of the organisms. These may be, for example, single carbohydrates or simple mixtures thereof, as commonly used in research applications, or complex natural sources such as molasses and wort, as commonly used in industrial applications. Facultative fermentative yeasts such as Saccharomyces cerevisiae can metabolise sugars either through aerobic respiration or alcoholic fermentation. alcoholic fermentation occurs to a certain degree even during aerobic cultivation. This tendency towards alcoholic fermentation limits the ability of S. cerevisiae to grow to a high specific biomass, since biomass yields are significantly reduced during fermentative growth. Fully respiratory metabolism is observed only at low specific growth rates in aerobic, sugar-limited cultures, which conditions can theoretically be met in fed-batch cultures as used in the industrial production of baker's yeast.
However, due to the high sugar concentration in the feed and imperfect mixing in large- scale bioreactors, local sugar concentrations often exceed the respiro-fermentative threshold resulting in local ethanol production and thus a decrease in the biomass yield even in fed-batch cultures.
The formation of fermentation by-products not only reduces biomass yield, but also reduces the formation of biomass related products, such as heterologous polypeptides expressed in yeasts. To facilitate downstream processing, high product concentrations are desirable. Large-scale heterologous polypeptide production therefore typically occurs at high biomass densities. At high biomass densities, even low specific rates of fermentation by-product formation will rapidly lead to the build-up of toxic metabolite levels, with detrimental effects on productivity. At present, only a small fraction of the carbon and nitrogen substrates fed to heterologous protein-producing cultures is converted into the product of interest. This means that cost of feedstocks remains a major factor in the economics of biomass-directed industrial applications for yeasts.
Techniques developed to increase fungal biomass in the prior art have concentrated on the optimisation of aerobic growth conditions. For example, fed-batch culture as referred to above may be designed to maintain nutrient concentration at a level which favours aerobic growth. The aim is to avoid fermentative sugar metabolism as far as it is possible to do so.
Mitochondrial carrier proteins are key components in the integration of mitochondria into the metabolism of the cell through their contribution to DNA synthesis, redox balance and by participating in various biosynthetic pathways. Yeast mitochondrial transport proteins have been reviewed by Walker and Runswick (Journal of Bioenergetics and Biomembranes (1993) 25:435) and more recently in Palmieri & van Ommen, (1999) in Frontiers in Cellular Bioenergetics (Papa, Guerrieri, and Tager, Eds.) pp. 489-519, Kluwer Academic/plenum Publishers, New York. 35 members of the mitochondrial carrier family are known, but the transport specificities are known for only a subset thereof.
Summary of the Invention
We have developed fungal strains which possess improved growth characteristics by mutating one or more yeast mitochondrial carrier genes. The improved fungi are able to metabolise carbohydrates more effectively than wild-type strains, possibly as a result of improved respiratory capacity. Surprisingly, biomass productivity is also increased. According to a first aspect of the invention, therefore, there is provided a mutated fungal strain which grows to a higher specific biomass than the wild-type strain, wherein the mutation modulates the expression of at least one mitochondrial carrier protein.
As used herein, a "mutated fungal strain" is one or more cells of a fungal strain which has been altered, with respect to the wild-type strain (see below). The mutation may be carried out by any one of a number of techniques, as set forth in more detail below, but the aim is to modulate the expression of at least one mitochondrial carrier protein.
The wild-type strain is a fungal strain upon which the mutated strain is based. This may be the parental strain, in other words the strain from which the mutated strain is directly generated by mutation, or a basic strain from which the mutated strain is derived after successive modifications. Preferably, it is the parental strain.
"Specific biomass" refers to the biomass produced per unit volume of the fungal culture at the termination of the growth phase, when cell density reaches a plateau. As used herein, where reference is made to "cells", it should be understood to include hyphal structures in filamentous fungi.
The present invention involves the modulation of the expression of mitochondrial carrier proteins in fungi. Modulation, in this respect, refers to any change in the activity of the protein, whether achieved by alteration of that activity, or by potentiation or repression of the wild-type activity, or by an increase or decrease in the levels of expression of the wild-type protein. It includes complete or partial loss of protein activity through gene deletion and/or mutation.
The mutated fungal strains according to the invention are advantageously more efficient in the metabolism of at least one carbohydrate source than the wild-type strain. The advantage may be specific to a single carbohydrate, or common to more than one or even substantially all carbohydrates. It is postulated that the increase in specific biomass production observed in fungi according to the invention is linked to an increase in respiratory capacity, which is associated with an increased efficiency of carbohydrate utilisation. Preferably, the fungal strains according to the invention are yeasts, amongst which S. cerevisiae is preferred.
Preferably, the expression of two or more yeast carrier proteins is modulated. It has been found that simultaneous deletion of two mitochondrial carrier proteins has highly beneficial effects on fungal strains. Preferred mitochondrial carrier proteins in S. cerevisiae are YMC1 (xvic2; chromosome 15, carrier 2) and YMC2 (iicl; chromosome 2, carrier 1). Advantageously, the expression of one or both of these proteins is do nregulated. In a particularly preferred embodiment, the invention relates to a ymclymc2 yeast strain, in which both YMC1 and YMC2 gene loci have been disrupted or deleted so as to prevent YMC1 and YMC2 protein production.
In a further aspect, the invention relates to a method for preparing a mutated fungal strain which grows to a higher specific biomass than the wild-type strain, comprising the steps of:
(a) modifying one or more fungi such that the expression of one or more mitochondrial carrier proteins is modulated;
(b) assaying the fungi for improved biomass production; and (c) selecting those fungi which display improved biomass production and culturing those fungi to produce the mutant fungal strain.
Modification of the fungi advantageously comprises gene deletion or mutation. This is preferably carried out by homologous recombination, using a selectable marker such as HIS3, which can be inserted into the yeast genome to replace or disrupt endogenous genes by known techniques.
Yeast strains according to the invention are useful for biomass production, and for the production of heterologous polypeptides. Therefore, the invention provides a method for producing a polypeptide by recombinant expression in fungal cells, comprising the steps of transforming a mutated fungal strain according to the invention with a nucleotide sequence encoding the polypeptide under the control of suitable expression sequences, culturing the fungal cells and expressing the polypeptide therein, and isolating the polypeptide from the fungal cells.
Moreover, the invention provides a method for producing fungal biomass, comprising the steps of culturing mutated fungal strain according to previous aspects of the invention, and optionally isolating the fungal cells from the culture medium.
Isolation, as referred to herein, refers to complete or partial isolation of the cells from the medium, or from at least one component of the medium. In general, isolation of cells or biomass may be achieved by any suitable technique, including sedimentation or centrifugation, drying, and the like. Polypeptide isolation is likewise carried out by conventional techniques, including affinity techniques, sedimentation and metal ion chelation, as are known in the art.
In a further embodiment, the invention provides the use of a mutated yeast strain according to previous aspects for the production of heterologous polypeptides or biomass, as desired.
Detailed Description of the Invention
Fungal Strains
Mitochondrial carrier proteins may be found in any cells which comprise mitochondria, and in the context of the present invention in all fungal cells, including yeasts. Although the invention is not intended to be limited, otherwise than as stated, to any particular fungal organism, fungi which are in common use in commercial production of heterologous polypeptides or fungal biomass, such as for animal feed or other industrial uses, are particularly preferred for modification in accordance with the invention. The invention may be applied to fungi of Aspergillus spp., such as A. niger, A. oryzae or A. awάmori, commonly used for both endogenous enzyme production and production of heterologous polypeptides. Other suitable fungi include Mucor and Endothia spp. (used in the production of fungal rennins), Pennicillium citrinum (nucleases), Pennicilium chrysogenum (glucose oxidase), Kluyveromyces (lactase) and Trychoderma (cellulase). Further suitable fungi are mentioned below in connection with recombinant polypeptide production. It will be understood that they need not necessarily be used for such purposes, but may be equally siotable for biomass or endogenous polypeptide production.
Biomass production, in the form of single-cell protein (SCP) is commonly based on yeasts such as Candida, Khiyveromyces, Endomycopsis or Pichia.
Saccharomyces spp. is highly important in commercial applications and is a preferred organism for use in the present invention. S. cerevisiae is commonly used, but other varieties, such as S. carlsbergensis, S. ellipsoideus and Schizosaccharomyces axe also used.
Saccharomyces, and especially S. cerevisiae, are particularly preferred in the context of the present invention.
Mitochondrial Carrier Proteins
35 mitochondrial carriers are known, and are present in mitochondria from all eukaryotic species, including fungi and mammals.
At least the following mitochondrial carriers known to be highly similar to yeast mitochondrial carriers (ymc) are known and can be identified in GenBank (4 April 2000):
1: 031778
HYPOTHETICAL 58.2 KD PROTEIN IN KLB-COTE INTERGENIC REGION gi 16226482 | s | 031778 | YMCB_BACSϋ [6226482]
2: NP_015383 putative mitochondrial carrier protein; Ymclp [Saccharomyces cerevisiae] gi|6325315|ref |NP_015383.1 | Y C1 | [6325315]
3: NP_009662 " mitochondrial carrier protein; Ymc2p [Saccharomyces cerevisiae] gi|6319580|ref | NP_009662.1 | YMC2 | [6319580]
4: P75885
HYPOTHETICAL 10.2 KD PROTEIN IN APPA-CSPH INTERGENIC REGION gi I 3025225 | sp | P75885 | YMCD_ECOLI [3025225]
P75882 HYPOTHETICAL 78.7 KD LIPOPROTEIN IN APPA-CSPH INTERGENIC REGION
PRECURSOR gi|2497709|sp|P75882|YMCA_ECOLI[2497709] 6: P75884
HYPOTHETICAL 24.3 KD LIPOPROTEIN IN APPA-CSPH INTERGENIC REGION
PRECURSOR gi|2497708|sp|P75884|Y CC_ECOLI[2497708] 7: CAA17781
(AL022070) hypothetical protein [Schizosaccharomyces pombe] gi I 2950467 I emb I CAA17781.il [2950467]
8: CAA18388 (AL022299) mitochondrial carrier protein [Schizosaccharomyces pombe] gi|3006147|emb|CAA18388.1] [3006147]
9: CAA93570
(Z69727) putative mitochondrial carrier protein [Schizosaccharomyces pombe] gi 11204220 I emb I CAA93570.il [1204220]
10: P75883
HYPOTHETICAL 27.3 KD PROTEIN IN APPA-CSPH INTERGENIC REGION PRECURSOR gi|2495585|sp|P75883|YMCB_ECOLI[2495585]
11: P38087
MITOCHONDRIAL CARRIER PROTEIN YMC2 PRECURSOR gil 5867841 sp] P38087 ) YMC2_YEAST [586784].
12: P38262
HYPOTHETICAL 59.1 KD PROTEIN IN VPS15-YMC2 INTERGENIC REGION gi I 586529 | sp | P38262 | YBV3_YEAST [586529] 13: P38261
HYPOTHETICAL 85.5 KD PROTEIN IN VPS15-YMC2 INTERGENIC REGION gi I 586528 | sp | P382611 YBV2_YEAST [586528]
14: P38260 HYPOTHETICAL 32.6 KD PROTEIN IN VPS15-YMC2 INTERGENIC REGION gi|586527|sp|P38260|YBVl_YEAST[586527]
15: P38259
HYPOTHETICAL 12.9 KD PROTEIN IN VPS15-YMC2 INTERGENIC REGION gi I 586526 I sp | P38259 I YBV0_YEAST [586526]
16: P38258
HYPOTHETICAL 14.8 KD PROTEIN IN VPS15-YMC2 INTERGENIC REGION gi|586525|sp|P38258|YBU9_YEAST[586525]
17: P38257
HYPOTHETICAL 52.8 KD PROTEIN IN VPS15-YMC2 INTERGENIC REGION gi I 586524 | sp | P38257 | YBU8_YEAST [586524] 18: P32331
MITOCHONDRIAL CARRIER PROTEIN YMC1 PRECURSOR • gi| 418574 I sp| P323311 YMC1_YEAS [418574]
19: P32689 HYPOTHETICAL 78.5 KD LIPOPROTEIN IN PGI-XYLE INTERGENIC REGION PRECURSOR gi I 418539 | sp|P32689 |YJBH_ECOLI[ 418539]
20: P32688
HYPOTHETICAL 26.3 KD PROTEIN IN PGI-XYLE INTERGENIC REGION PRECURSOR gi I 418538 | sp | P32688 | YJBG_ECOLI [418538]
21: P32687
HYPOTHETICAL 25.0 KD LIPOPROTEIN IN PGI-XYLE INTERGENIC REGION PRECURSOR gi I 418537 | sp | P32687 | YJBF_ECOLI [418537]
10
22: AAD22974
(AF126444) extended-spectrum beta-lactamase variant [Salmonella enteritidis serovar Saintpaul] 15 gi I 4559300 I gb| AAD22974.il [4559300]
23: CAA22786
(AL035212) putative AraC family transcriptional regulator [Streptomyces coelicolor A3 (2)] 20 gi|4160315|emb|CAA22786.1| [4160315]
24: AAC74072
(AE000200) orf, hypothetical protein [Escherichia coli] gi 11787222 I gb I AAC74072.il [1787222] -25
25: AAC7407?
(AE000200) putative regulator [Escherichia coli] gil 17872211 gb I AAC74071.il [1787221]
30 26: AAC74070
(AE000200) orf, hypothetical protein [Escherichia coli] gi I 1787220 I gb I AAC74070.il [1787220]
27: AAC74069 35 (AE000200) orf, hypothetical protein [Escherichia coli] gi| 1787219 I gb I AAC74069.il [1787219]
28: AAC73681
(AE000163) orf, hypothetical protein [Escherichia coli] 40 gi|1786794|gblAAC73681.1| [1786794]
29: CAB13579
(Z99112) ymcC [Bacillus subtilis] gil 2634078 I emb I CAB13579.il [2634078] 45
30,: CAB13575
(Z99112) similar to hypothetical proteins [Bacillus subtilis] gil 2634074 I emb I CAB13575.il [2634074]
50 31: CAB13574
(Z99112) similar to hypothetical proteins [Bacillus subtilis] gil 2634073 I emb I CAB13574.il [2634073]
32: CAA85059 55 (Z35973) ORF YBR104w [Saccharomyces cerevisiae] gil 536390 I emb 1CAA85059.1| [536390]
33: CAA89176
(Z49219) Ymclp [Saccharomyces cerevisiae] 60 gi| 805039 I emb I CAA89176.il [805039] 34: S48269 mitochondrial carrier protein YMC2 precursor - yeast (Saccharomyces cerevisiae) gi|1078116|pir| | S48269 [1078116]
35: S54080 carrier protein YMCl, mitochondrial - yeast (Saccharomyces cerevisiae) gi|1084942|pir| | S54080 [1084942]
36: CAA47602
(X67122) mitochondrial carrier protein [Saccharomyces cerevisiae] gi|48241emb|CAA47602.1| [4824] 37: CAA95003
( Z71255 ) Ymclp [ Saccharomyces cerevisiae ] gi | 1314127 I emb I CAA95003 . i l [ 1314127 ]
A far greater number is known (449) which show high similarity to mitochondrial carriers other than YMC proteins.
The fungal equivalents of known mitochondrial carriers, and any other proteins which are discovered to possess evolutionary homology with known mitochondrial carriers, may be targets for modification in accordance with the present invention.
Particularly preferred are yeast mitochondrial carriers YMC 1 and YMC2, and fungal homologues thereof. In an advantageous aspect of the present invention, such carrier proteins are downregulated in the mutated fungal strains, and are preferably entirely deleted by genetic manipulation. The polypeptide sequences of YMCl and YMC 2 are set forth herein as SEQ. ID. No. 1 and SEQ. ID. No. 2, respectively. The nucleic acid sequence of YMCl is SEQ. ID. No. 3.
Modulation of Mitochondrial Carrier Activity
The modulation of the activity of fungal mitochondrial carriers may be achieved in any one of a number of ways. As set forth above,, "modulation" refers to the alteration, upregulation or downregulation of the activity of a wild-type mitochondrial carrier, and can encompass deletion of the activity through genetic manipulation. A number of strategies for modulation of mitochondrial carriers is set forth below; the examples are non-limiting and should be seen as illustrative of the types of techniques which may be used in the present invention.
In a first illustration, antisense technology may be used to regulate the expression of a mitochondrial carrier. In general, antisense approaches downregulate or eliminate the expression of the target protein (for example, see Selinfreund, R. H., et al, (1990) J. Cell Biol. I l l, 2021-2028; Bourque 1995 Plant Science 105 pp 125-149).
Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to the expression control sequences or mRNA of the target mitochondrial carrier are introduced into cells (e.g., by transformation with a nucleic acid vector or by transfer of naked DNA). The antisense nucleic acid binds to the target sequence in the fungal cell and prevents transcription or translation of the target sequence. In many embodiments, the antisense molecule recruits cellular nucleases which cleave the antisense: sense complex. Phosphothioate and methylphosphate antisense oligonucleotides are more stable and are specifically contemplated for use in the invention. The antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end.
In an advantageous embodiment, fungal cells are transformed with vectors which express the required antisense oligonucleotides, such that expression of the target mitochondrial carrier protein is inhibited permanently in the fungal cell.
In a second illustration, mutations may be made to the regulatory or structural regions of a gene encoding the target mitochondrial carrier in order to alter the levels of expression of the carrier protein or its activity. Although such mutations may in theory be designed according to rational design procedures to achieve desired effects, such as the introduction or deletion of promoter elements, it is preferred that such mutations be effected in an at least partially randomised manner, and selected using the enhanced screening possibilities offered by fungal cells.
For example, fungal cells may be subjected to entirely random mutagenesis, for example using mutagenic chemical agents or radiation, and selected according to their ability to achieve high biomass after a period of growth. Such assays may be automated, by determining biomass according to, for example, optical density (such as OD60o) and conducting the growth experiments in multiwell plates, or other test systems suitable for high-throughput automated analysis.
Alternatively, the mutagenesis may be directed, using site-directed mutagenesis procedures as known in the art, and or mutagenesis kits available from a variety of manufacturers. Such procedures can be conducted in vitro or in vivo, and may be randomised to a greater or lesser degree.
Such mutation events may result in the potentiation or inhibition of the activity of a mitochondrial carrier, or in a qualitative change in its activity. Advantageous mutations are generally selected by screening of numerous mutations, some of which may be neutral or disadvantageous in the context of the present invention.
In a third illustration, modulation of mitochondrial carrier activity may be achieved by disruption or deletion of the gene encoding the carrier protein by homologous recombination.
Homologous recombination is highly developed for fungal organisms, and techniques are well known in the art. Briefly, a nucleic acid encoding a selectable marker, such as a HIS 3 marker, is fused to flanking sequences which are complementary to sequences flanking the gene which it is desired to delete. Such flanking sequences may be obtained by PCR amplification from fungal genomic nucleic acid, or by synthesis.
The nucleic acid encoding the selectable marker is subsequently used to replace or disrupt the target gene by homologous recombination, for example using a PCR-based approach.
• PCR-based approaches are preferred, and an exemplary approach may be based largely on the method described in Baudin et al., 1993, NAR 21: 3329. The selectable marker for deletion (HIS3) is amplified by PCR using oligonucleotides that contain homology to the
HIS3 gene and also 45 bp of homology immediately 5' and 3' to the target mitochondrial carrier protein gene being deleted. The PCR product is used to transform yeast to His+, which removes the target gene completely. Correct gene deletion is verified by PCR of fungal colonies, using a universal test oligo in HIS3, and an oligo in flanking chromosomal sequence. Only correctly deleted transformants yield a PCR product.
The fungal strain used for gene deletion should have a complete deletion of the selectable yeast marker that will be used to replace the gene of interest. Otherwise, most of the events are likely to be gene conversion and homologous recombination at the marker locus. In yeast (S. cerevisiae) strains carrying the his3D-200 deletion, which removes all sequences homologous to the HIS3 gene, are suitable. HIS3 is preferred as a selectable marker for the disruptions.
The selectable marker is cloned into a plasmid such as pBluescript® which will not replicate in yeast, thereby reducing background from yeast transformants containing template plasmid. . . . _ ;
Alternative selectable markers for yeast include those conferring resistance to antibiotics G418, hygromycin or bleomycin, or those (such as HIS3) which provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2 or TRP1 genes.
Use of Mutant Fungi according to the Invention
Fungi have many applications in biotechnology and traditional industries, including use in baking and brewing, which requires the production of significant yeast biomass, use as a source of single cell protein, in the production of fungal enzymes for use in the food industry and in the production of heterologous proteins by recombinant gene technology. Each of these applications is well known in the art.
Biomass production, whether for baking/brewing or SCP applications, requires culture of fungal cells to high specific biomass . and subsequent isolation of the cells from the medium. Mutated fungi according to the present invention are particularly advantageously employed in such applications, since they can achieve a high specific biomass and utilise carbon sources more efficiently. For production of fungal enzymes, a high specific biomass is again desirable, since the concentration of the desired product may be increased, thus facilitating its purification. Techniques for culture of fungi to produce fungal enzymes, typically Aspergillus fungi, are well known in the art.
The use of fungi for the recombinant production of heterologous polypeptides of eukaryotic and prokaryotic origins is likewise well known. Typically a fungal host cell is transformed with a vector encoding the desired heterologous polypeptide, and cultured such that the heterologous polypeptide is produced. It may be produced intracellularly, or secreted.
As used herein, "vector" refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are known to those skilled in the art. Many vectors are available, and • selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence a signal sequence.
Both expression and cloning vectors generally contain at least one nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and other fungi. The origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, the 2μ plasmid origin is suitable for yeast, and fungal origins may. be employed in filamentous fungi, for example the amal replicon (Gems et al, (1991) Gene 98:61-67). Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or other fungal cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome.
Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, into which it has been transferred. This gene may encode a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from media.
As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LΕU2, LYS2, TRP1, or HIS3 gene. Advantageously, where a selectable marker has been used for mutation by homologous recombination, a different selectable marker is used for transformation of the host cells.
Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript® vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin.and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin. Moreover, the vector preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
The term "host cell", as used herein, includes any fungus, whether a unicellular organism, a cell derived from a multicellular organism and placed in tissue culture or a cell present as part of a multicellular organism, which is susceptible to transformation with a nucleic acid construct according to the invention. Such host cells, such as yeast and other fungal cells may be used for replicating DNA and producing polypeptides encoded by nucleotide sequences as used in the invention. Suitable cells are generally filamentous fungi or yeasts.
Particularly preferred are cells from filamentous fungi, preferably Aspergillus, such as A. niger and A. tubingensis.
■ Other preferred organisms include any one of Aspergillus oryzae, A. awamori, Trichoderma reesei, T. viride and T. longibrachiatum.
The transformed cell or organism may be used to prepare acceptable quantities of the desired gene product which is retrievable from the cell or organism. The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression. Any suitable promoter may be used in connection with the present invention, with xylan-inducible promoters preferably of fungal origin being especially indicated.
Fungal promoters are known in the literature (for example, see Gurr, et al, (1987) The structure and organisation of nuclear genes of filamentous fungi. In Kinghorn, J.R. (ed), Gene Structure in Eukaryotic Microbes, IRL Press, Oxford, pp. 93-139).
In addition to the nucleotide sequences described above, the promoter of the present invention can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The TATA box is typically found 30 bp upstream of the transcription initiation site, and is believed to be involved in the assembly of the transcriptional complex.
The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the heterologous nucleotide sequence. Fungal promoters, for instance, typically contain both a TATA box and a UAS (Upstream Activating Site). The UAS is a binding site for the activating regulator acting on the promoter in question. The element of the present invention is believed to be a UAS. Other sites, involved in various aspects of promoter regulation, may also be included.
The term "heterologous nucleotide sequence" with reference to the constructs according to the present invention means any sequence encoding a polypeptide of interest, other than the complete natural sequence normally associated with the promoter employed, or a sequence which is capable of expressing a nucleic acid, for example a regulatory RNA such as an antisense RNA or a ribozyme, or a tRNA or rRNA capable of regulating the metabolism of an organism. Conversely, the term "nucleotide sequence" includes homologous nucleotide sequences which are the sequences normally associated with the promoters employed. The invention includes the use of both homologous and heterologous nucleotide sequences. A heterologous nucleotide sequence can be any nucleotide sequence that is either foreign or natural to the organism (e.g. filamentous fungus, preferably of the genus Aspergillus, or a plant) in question. The term "heterologous nucleotide sequence" also includes a homologous nucleotide sequence which has been mutated, such as by insertion, addition, deletion or alteration, such that it is no longer identical with the natural homologous nucleotide sequence.
Typical examples of a heterologous nucleotide sequence include sequences coding for proteins and enzymes that modify metabolic and catabolic processes. The heterologous nucleotide sequence may code for an agent for introducing or increasing pathogen resistance. The heterologous nucleotide sequence may be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. The heterologous nucleotide sequence may code for a non-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans. Examples of nucleotide sequences according to the invention include pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, hexose oxidase, oxidoreductases, Upases, glucan lyase, rhamno-galacturonases, hemicellulases, endo-β- glucanases, arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof. The heterologous nucleotide sequence may be a protein giving nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant).
The heterologous nucleotide sequence may code for an enzyme that can be used in food processing such as chymosin, thaumatin and α-galactosidase. The heterologous nucleotide sequence can encode any one of a pest toxin, an antisense transcript such as that for patatin or α-amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense, a glucanase or genomic β-l,4-endoglucanase.
The heterologous nucleotide sequence may code for an intron of a particular nucleotide sequence, wherein the intron can be in sense or antisense orientation. Preferably the promoter and the nucleotide sequence according to the invention are stably maintained within host cells or transgenic organisms. By way of example, the promoter and/or the nucleotide sequence may be maintained within the transgenic organism in a stable extrachromosomal construct. This is preferred for transgenic yeast or some filamentous fungi. Alternatively, the promoter and/or the heterologous nucleotide sequence (such as the nucleotide sequence according to the present invention) may be stably incorporated within the transgenic organism's genome. This is preferred for some transgenic yeast, and most filamentous fungi.
A preferred host organism for the expression of the nucleic acid constructs of the present invention and/or for the preparation of the heterologous polypeptides according to the present invention is an organism of the genus Aspergillus, such as Aspergillus niger. In this regard, a transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant- DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D., Kinghorn J.R. (Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong, S.A., Berka R.M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.D., Kinghorn J.R. (Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). The following commentary provides a summary of those teachings for producing transgenic Aspergillus according to the present invention. "
For almost a century, filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional Japanese koji and soy fermentations have used Aspergillus sp. Also, in this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry. There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracellular products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
In order to prepare the transgenic Aspergillus, expression constructs are prepared by inserting a heterologous nucleotide sequence (such as a nucleotide sequence coding for an amylase enzyme) into a construct designed for expression in filamentous fungi.
Several types of constructs used for heterologous expression have been developed. The constructs contain the promoter according to the present invention which is active in fungi. The heterologous nucleotide sequence can be fused to a signal sequence which directs the protein encoded by the heterologous nucleotide sequence to be secreted. Usually a signal sequence of fungal origin is used. A terminator active in fungi may also be employed.
Another type of expression system has been developed in fungi where the heterologous nucleotide sequence is fused to a fungal gene encoding a stable protein. This can stabilise the protein encoded by the heterologous nucleotide sequence which encodes a protein of interest. In such a system a cleavage site, recognised by a specific protease, can be introduced between the fungal protein and the protein encoded by the heterologous nucleotide sequence, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the heterologous nucleotide sequence. By way of example, one can introduce a site which is recognised by a KEX-2 like peptidase found in at least some Aspergilli (Broekhuijsen et al 1993 J Biotechnol 31 135- 145). Such a fusion leads to cleavage in vivo resulting in protection of the expressed product and not a larger fusion protein.
Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited intracellularly if the nucleotide sequence according to the present invention (or another heterologous nucleotide sequence) is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention (or another heterologous nucleotide sequence) is equipped with a signal sequence the protein will accumulate extracellularly.
With regard to product stability and host strain modifications, some heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991, Op. Cit). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarranton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting a nucleotide sequence comprising a DNA construct according to the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed, suitable component parts of which are discussed hereinbefore.
For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
Examples
Preparation of mutated yeast strains and analysis of biomass production
S. cerevisiae YPH499 strain (MATa ade2-101 his3-Δ200 Ieu2-Δl ura3-52 trpl-Δ63 lys2- 810) is used as a basis for the present experiments. The yeast mitochondrial carrier genes YMCl (xvic2) and YMC2 (iicl) are deleted by homologous recombination with the auxotrophic marker HIS3 at the respective loci. In order to construct a double mutant, ymclymc2, deletion of YMC2 in ymcl yeast is carried out with the marker TRP1.
Deletants are verified by PCR and western blot analysis. Yeast are grown in a synthetic medium (SM) containing 0.67% yeast nitrogen base, without amino acids (Difco), 0.1%KH2PO4, 0.12% (NH4)2SO4, 200 mg/1 leucine, 40 mg/1 lysine, 40 mg/1 tryptophan, 40 mg/1 uracil and 10 mg/1 adenine supplemented with carbon sources as indicated below. The final pH is adjusted to 4.5 with KOH or 6.5 for growth on acetate. Histidine (40 mg/1) is added to cultures of wild-type cells (his3). Yeasts are precultured in complex medium (YP) supplemented with 2% glucose, harvested in the late exponential growth phase by centrifugation, washed twice with cold SM and diluted into the same medium as described above, supplemented with the stated carbon sources, for the experimental assessment.
The biomass production for different yeasts in a variety of carbon sources is shown below (Table 1).
Figure imgf000023_0001
In each case, the biomass is determined by optical density measurement at 600nm when cell growth has reached a plateau. Wild-type cells, in each case, exhibit similar growth to ymc2 cells.
In contrast, the ymclymc2 double mutant shows significantly higher growth on glucose (2x biomass), galactose (2.4x), and especially glycerol (almost 4x). Strikingly, the biomass increases over wild-type where the carbon source is changed from respiratory metabolism repression (glucose), to partial repression (galactose) to derepression (glycerol). The ymclymc2 mutant also grows on other non-fermentative carbon sources better than wild-type or ymc2 mutants.
The ymcl single mutant also shows a significant increase in biomass over wild-type when grown on glucose (1.5x) and, to a lower extent, on ethanol and glycerol.
Sequence Listing
SEQ. ID. No. 1:
YMCl
MSEEFPSPQLIDDLEEHPQHDNARVVKDLLAGTAGGIAQVLVGQPFDTTKVRLQTSSTPTTAMEVVRKLLA EGPRGFYKGTLTPLIGVGACVSLQFGVNQAMKRFFHHRNADMSSTLSLPQYYACGVTGGIVNSFLASPIEHV RIRLQTQTGSGTNAEFKGPLECIKKLRHNKALLRGLTPTILREGHGCGTYFLVYEALIANQMNKRRGLERKD IPAWKLCIFGALSGTALWLMVYPLDVIKSVMQTDNLQKPKFGNSISSVAKTLYANGGIGAFFKGFGPTMLRA APANGATFATFELAMRLLG
SEQ. ID. No.2
YMC2
MSEEFPTPQL LDELEDQQKV TTPNEKRELS SNRVLKDIFA GTIGGIAQVL VGQPFDTTKV RLQTATTRTT TLEVLRNLVK NEGVFAFYKG ALTPLLGVGI CVSVQFGVNE AMKRFFQNYN ASKNPNMSSQ DVDLSRSNTL PLSQYYVCGL TGG VNSFLA SPIEQIRIRL QTQTSNGGDR EFKGPWDCIK KLKAQGGLMR GLFPTMIRAG HGLGTYFLVY EALVAREIGT GLTRNEIPPW KLCLFGAFSG TML LTVYPL DVVKSIIQND DLRKPKYKNS ISYVAKTIYA KEGIRAFFKG FGPTMVRSAP VNGATFLTFE LVMRFLGEE
SEQ. ID. No.3
YMCl nucleotide sequence
atcaatcaat gtcaataagc taataacaac tatcatctat actccaacgc agctctcttg tttaggtttc tcgtcaatca aattcgcatc acgaagaagt atgtctgaag aatttccatc tcctcaacta atcgatgatt tggaagaaca tccacagcat gataatgctc gagtcgtgaa
- . agatttgctt gcaggtacag cgggtggtat tgcgcaagtg ctagtgggcc agccctttga tacgacaaaa gttaggttac aaacatcgag caccccaaca acagccatgg aagtcgtcag aaagctgctt gccaatgaag ggcctcgcgg gttttacaaa ggaactctga cgccattaat tggtgttggt gcatgtgttt cattacaatt tggtgttaat caagctatga agagattttt tcatcatcgc aatgctgata tgtcatcgac tttgtcattg ccacagtatt acgcatgtgg tgtcacaggc ggtatagtaa actcattctt ggcgtcccca attgagcatg tcaggattcg cttgcaaaca cagactggct caggcaccaa cgcagaattc aagggtcctt tggaatgcat caaaaaatta agacataaca aggccttgct acgtggttta acacctacaa tattgagaga aggtcatgga tgtggcacat atttcttagt gtatgaagcg ttgattgcta accaaatgaa caaaagacgt ggactagaga gaaaggacat tcctgcatgg aaactttgta tttttggagc attgtctggc actgccttat ggttgatggt atatccatta gatgtcatca agtctgtcat gcaaacggat aatttacaaa agcctaaatt tggtaattct atttccagtg tagccaagac tttatatgcc aatggaggga taggcgcttt tttcaaaggg tttggtccta ccatgctaag agctgctccc gccaatggtg ccacttttgc tacttttgaa ttagcgatga ggttattggg ttgataattc ctatagaatt tatgagccat cgtttaagaa aatagtacaa tataatgtat aacttgaaac tctatcttct tatattcaat agatgctact atgcgtacat atatatggtg agtgtgtgtg tttatatatg cgtagtaatc actcggcaat gtggaattgt taccgtgata gccttcatgc ttactcttcc aaccttctta gcaagtattc cacctcaact tccttagtga taggtataac aatttcctta taaattttta ccaaatattc tggagtaatc cttctttcac ctgactcgtt ggtagggtcc acaccataga cttcagcctt cttagttaat ctttctaga

Claims

Claims
1. A mutated fungal strain which grows to a higher specific biomass than the wild- type strain, wherein the mutation modulates the expression of at least one mitochondrial carrier protein.
2. A mutated fungal strain according to claim 1, wherein the expression of two or more yeast carrier proteins is modulated.
3. A mutated fungal strain according to claim 1 or claim 2, wherein the mutation is selected from the group consisting of a gene deletion, a mutation which renders the gene product inactive, a mutation which modifies the activity of the gene product, and a mutation which modifies the level of production of the gene product.
4. A mutated fungal strain according to any preceding claim which utilises at least one carbohydrate more efficiently than the wild-type strain.
5. A mutated fungal strain according to any preceding claim, which is a yeast strain.
6. A mutated fungal strain according to claim 5, which is mutated in the YMCl gene.
7. A mutated fungal strain according to claim 6, which is mutated in the YMCl and YMC2 genes.
8. A method for preparing a mutated fungal strain which grows to a higher specific biomass than the wild-type strain, comprising the steps of:
(a) modifying one or more fungi such that the expression of one or more mitochondrial carrier proteins is modulated;
(b) assaying the fungi for improved biomass production; and
(c) selecting those fungi which display improved biomass production and culturing those fungi to produce the mutant fungal strain.
. A method according to claim 8, wherein the fungi are modified by gene disruption or deletion by targeted homologous recombination.
10. A method for producing a polypeptide by recombinant expression in fungal cells, comprising the steps of transforming a mutated fungal strain according to any one of claims 1 to 7 with a nucleotide sequence encoding the polypeptide under the control of suitable expression sequences, culturing the fungal cells and expressing the polypeptide therein, and isolating the polypeptide from the fungal cells.
11. A method for producing fungal biomass, comprising the steps of culturing mutated fungal strain according to any one of claims 1 to 7, and optionally isolating the fungal cells from the culture medium.
12. Use of a mutated fungal strain according to any one of claims 1 to 7 in the production of a heterologous polypeptide.
13. Use of a mutated fungal strain according to any one of claims 1 to 7 in the production of fungal biomass.
PCT/GB2001/003457 2000-08-01 2001-08-01 Yeast strains WO2002010342A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU7574101A AU7574101A (en) 2000-08-01 2001-08-01 Fungal strains

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0018882.1 2000-08-01
GBGB0018882.1A GB0018882D0 (en) 2000-08-01 2000-08-01 Fungal strains

Publications (2)

Publication Number Publication Date
WO2002010342A2 true WO2002010342A2 (en) 2002-02-07
WO2002010342A3 WO2002010342A3 (en) 2002-07-04

Family

ID=9896773

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/003457 WO2002010342A2 (en) 2000-08-01 2001-08-01 Yeast strains

Country Status (3)

Country Link
AU (1) AU7574101A (en)
GB (1) GB0018882D0 (en)
WO (1) WO2002010342A2 (en)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CRABEEL M. ET AL.: "The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis" THE JOURNAL OF BIOLOGICLA CHEMISTRY, vol. 271, no. 40, 4 October 1996 (1996-10-04), pages 25011-25018, XP002192717 *
FERNÁNDEZ M. ET AL.: "ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae" MOLECULAR & GENERAL GENETICS, vol. 242, 1994, pages 727-735, XP002193350 *
GRAF R. ET AL.: "YMC1, a yeast gene encoding a new putative mitochondrial carrier protein" YEAST, vol. 9, no. 3, March 1993 (1993-03), pages 301-305, XP001064327 *
HAGEN T. ET AL.: "Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system" FEBS LETTERS, vol. 454, 1999, pages 201-206, XP002192718 *
PALMIERI L. ET AL.: "Yeast mitochondrial carriers: bacterial expression, biochemical identification and metabolic significance" JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, vol. 32, no. 1, February 2000 (2000-02), pages 67-77, XP001064365 *

Also Published As

Publication number Publication date
GB0018882D0 (en) 2000-09-20
WO2002010342A3 (en) 2002-07-04
AU7574101A (en) 2002-02-13

Similar Documents

Publication Publication Date Title
Li et al. Developing Aspergillus niger as a cell factory for food enzyme production
CN101128581B (en) Mutant aox 1 promoters
Rubio‐Texeira et al. Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae
US8404472B2 (en) System for producing aromatic molecules by bioconversion
JP2018504936A (en) Fungal strains and methods of use
US20110104769A1 (en) Improved yeast strains for organic acid production
CN109988714B (en) Trichoderma reesei and application thereof
Böer et al. Large-scale production of tannase using the yeast Arxula adeninivorans
Giga-Hama et al. Foreign gene expression in fission yeast: Schizosaccharomyces pombe
KR101187081B1 (en) Method of obtaining lipid-producing fungus
US20170029853A1 (en) Acid-tolerant yeast cell, method of producing organic acid using the same, and method of producing the yeast cell
Oliveira et al. Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger β-galactosidase production
CN101473035B (en) Method for reinforcing secretion efficiency of recombination exogenous protein in sprout fungi expression system
Martín Fungal transformation: from protoplasts to targeted recombination systems
EP3054004B1 (en) Aspergillus mutant strain and transformant thereof
EP2062967A1 (en) Genetically engineered aspergillus
Camattari et al. Induction by hypoxia of heterologous-protein production with the Kl PDC1 promoter in yeasts
JP2015039349A (en) Glucose inhibition gene disruption strain and material production method using the same
Lin et al. Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome
WO2003016525A9 (en) Process for producing alcohol from starch
Böer et al. Arxula adeninivorans
JPH08501695A (en) Increased production of secreted proteins by recombinant eukaryotic cells
Ibragimova et al. A strategy for construction of industrial strains of distiller's yeast
WO2002010342A2 (en) Yeast strains
KR101264294B1 (en) Yeast Variants Having Resistance to Low Temperatures

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP