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WO1999047685A1 - Perfectionnements apportes au metabolisme des acides gras ou s'y rapportant - Google Patents

Perfectionnements apportes au metabolisme des acides gras ou s'y rapportant Download PDF

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Publication number
WO1999047685A1
WO1999047685A1 PCT/GB1999/000846 GB9900846W WO9947685A1 WO 1999047685 A1 WO1999047685 A1 WO 1999047685A1 GB 9900846 W GB9900846 W GB 9900846W WO 9947685 A1 WO9947685 A1 WO 9947685A1
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Prior art keywords
fao
sequence
gene
host cell
substrate
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PCT/GB1999/000846
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English (en)
Inventor
Antoni Ryszard Slabas
Kieran Elborough
Sipo Vanhanen
Mark West
Qi Cheng
Nigel Lindner
John Casey
Dominique Sanglard
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Unichema Chemie B.V.
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Priority to CA002323762A priority Critical patent/CA2323762A1/fr
Priority to BR9909655-2A priority patent/BR9909655A/pt
Priority to KR1020007010305A priority patent/KR20010041979A/ko
Priority to JP2000536868A priority patent/JP2002506650A/ja
Priority to EP99910537A priority patent/EP1064386A1/fr
Priority to AU29469/99A priority patent/AU2946999A/en
Publication of WO1999047685A1 publication Critical patent/WO1999047685A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic

Definitions

  • This invention relates, inter alia, to certain nucleic acid sequences encoding polypeptides involved in fatty acid metabolism, constructs and host cells comprising the nucleic acid sequenes, host cells from which certain nucleic acid sequences have been deleted, and to methods of treating substrates and methods of preparing compounds.
  • Candida cloacae and a number of other industrial yeasts have the ability to use al anes or fatty acids as the sole carbon source (Watkinson & Morgan 1990 Biodegradation 1, 79-92; Kemp et al. 1994 Appl. Microbiol. Biotechnol. 40, 873-875) by a diterminal pathway (i.e. these organisms metabolise the substrate by oxidising both ends of the molecule, unlike most bacteria which -oxidise one end of the substrate molecule). This is achieved using metabolic pathways in three separate subcellular compartments: endoplasmic reticulum, peroxisomes and mitochondria (see Nauersberger et al, 1987 J. Basic Microbiol. 27, 565-582).
  • ⁇ , ⁇ - oxidation is catalyzed by three sequential enzymes: (a) a P450 linked hydrocarbon/fatty acid oxidase to yield a fatty alcohol, ⁇ , ⁇ -fatty diol or an ⁇ -hydroxy fatty acid; (b) a fatty alcohol/fatty diol oxidase, which in the presence of molecular oxygen yields the corresponding fatty aldehyde and H 2 O 2 , and (c) an aldehyde reductase.
  • the immediate product of step (c) is either a monocarboxylic acid or a dicarboxylic acid.
  • Subsequent metabolism of dicarboxylic acids occurs following activation to acyl CoA via 0-oxidation in the peroxisomes.
  • acetyl CoA is oxidised to CO 2 in the mitochondria.
  • Microorganisms possessing this determinal pathway are used commercially in the industrial production of dicarboxylic acids, which are important building blocks for the chemical industry and ingredients for personal care products. Another potential use for these organisms is to clean up environmental pollutants that are alkane-based. Specific strains of both C. cloacae and C. tropicalis which are disrupted in ⁇ -oxidation have been developed for the commercial production of dicarboxylic acids: these strains accumulate activated fatty acids at high levels in the medium (Casey et al. 1990, Picataggio et al. 1992, cited above).
  • the invention provides an isolated nucleic acid sequence encoding a polypeptide having fatty alcohol oxidase (FAO) activity.
  • Fatty alcohol oxidase activity is the catalysis of the oxidation of the terminal hydroxy group of a fatty alcohol, or an hydroxy fatty acid, in the presence of molecular oxygen, to form a fatty aldehyde or a fatty acid aldehyde, and H 2 O 2 .
  • FAO activity may conveniently be assayed by the method of Kemp et al, (1988 Appl. Microbiol. Biotechnol. 29, 370-374).
  • a "fatty alcohol oxidase” enzyme can be distinguished from an “alcohol oxidase” by virtue of the respective substrate specificities.
  • a fatty alcohol oxidase is an enzyme which, inter alia, exhibits greater specific activity for substrates having a chain length of 5 or more C atoms.
  • fatty alcohol oxidases may often oxidise secondary alcohols (e.g. dodecan-2-ol). Long chain (i.e. 5 or more, preferably 10 or more, C atoms) ⁇ -hydroxy fatty acids may be particularly preferred substrates of fatty alcohol oxidases.
  • Such a nucleic acid sequence has never before been obtained.
  • the present inventors successfully isolated two types of FAO coding sequence from the yeast C. cloacae. Using this information, the inventors were able successfully to isolate a similar further FAO coding sequence from the yeast C. tropicalis, by screening a cDNA library (prepared from cells grown under conditions in which expression of FAO is induced) with sequence- specific probes based on the C. cloacae FAO coding sequence.
  • a cDNA library prepared from cells grown under conditions in which expression of FAO is induced
  • the nucleic acid sequence of the invention is preferably a sequence obtainable from a yeast, especially from yeasts of the genus Candida. Examples include C. cloacae and C. tropicalis, the FAO coding sequences of which are disclosed herein.
  • the invention provides a nucleic acid sequence encoding a polypeptide having fatty alcohol oxidase activity and comprising substantially the amino acid sequence of one of the sequences shown in Figure 7, or a functional equivalent thereof.
  • Functionally equivalent amino acid sequences are those which possess FAO activity yet which are not identical with one of the sequences shown in Figure 7 - one or more amino acid substitutions may be present, without substantially affecting the FAO activity of the polypeptide, especially where the substitutions are conservative (e.g. leucine for isoleucine, threonine for serine etc).
  • a functionally equivalent amino acid sequence will possess at least 70% similarity (i.e. identical amino acid residues), preferably at least 75%, and more 4 preferably at least 80% similarity, with one of the sequences shown in Figure 7.
  • the cDNA library will be subjected to PCR amplification, using a pair of oligonucleotide primers, each primer hybridising to a sequence encoding a conserved amino acid motif.
  • stringent hybridisation conditions e.g. as described by Sambrook et al, Molecular Cloning. A Laboratory Manual, CSH i.e. washing with O. lx SSC, 0.5% SDS at 68°C
  • nucleic acid sequences very different to those 5 shown in Figures 6 and 8 may still encode polypeptides which are functional equivalents of those amino acid sequences shown in Figure 7 and accordingly are encompassed within the scope of the present invention.
  • the nucleic acid sequences of the invention will preferably comprise one or more regulatory elements in addition to the coding sequences. These may comprise, for example, 5' and/or 3' untranslated regions, promoters (for prokaryotic or eukaryotic expression systems), terminators, polyadenylation signals, enhancers and the like. Specific examples of regulatory elements are shown in Figures 6 and 8.
  • the invention provides replicable nucleic acid constructs comprising the nucleic acid sequence of the first aspect. Desirably the constructs will allow for expression of a fatty alcohol oxidase polypeptide in a suitable host cell, which may be prokaryotic or eukaryotic.
  • the construct may also advantageously comprise a selectable marker (e.g. a gene coding for resistance to an antibiotic such as hygromycin, neomycin, ampicillin, tetracycline and the like).
  • the invention provides a host cell into which has been introduced a nucleic acid sequence in accordance with the first aspect of the invention.
  • the host cell may be eukaryotic (e.g. mammalian cell, plant cell, yeast cell such as Candida or Pichia sp or a fungal cell) or prokaryotic (a bacterial cell such as E. coli).
  • Methods of introducing nucleic acid sequences into host cells are well-known. Such methods may generally be referred to as "transformation” and include classical Ca 2+ -mediated transformation of bacterial cells, transfection, transduction, protoplast fusion, “biolistic” methods, electroporation and the like.
  • the host cell may be one which does not naturally possess a FAO coding sequence, such that introduction of the nucleic acid sequence may allow the host cell to express a new protein (i.e. FAO).
  • the host cell may be one which already possesses a FAO coding sequence.
  • introduction of the sequence of the invention may allow the host cell to express a different FAO to that which is naturally produced by the host cell (e.g. an enzyme with a different pH optimum, or a different substrate specificity).
  • several copies of the sequence of the invention may be introduced into the host cell, so as to increase the copy number of the FAO coding sequence, thereby increasing the amount of FAO enzyme produced by the 6 host cell.
  • the invention also provides a method of transforming a host cell by introducing into the host cell a nucleic acid sequence directing the expression of a FAO polypeptide, preferably such that the transformed host cell produces FAO enzyme.
  • the invention provides a method of altering a substrate, the method comprising contacting the substrate with a host cell transformed with the nucleic acid sequence of the invention, whereby the substrate is metabolised by the host cell via a pathway involving FAO.
  • the products of the substrate may not be of interest (for example, where the host cell is added to an alkane-containing pollutant, such as an oil spill or slick, for the purpose of breaking down the alkane).
  • the metabolic products of the substrate may be of commercial value.
  • the method will conveniently comprise the additional step of recovering the product(s), from the host cell and/ or from the extra-cellular environment.
  • portions of at least 200 nucleotides (preferably 300-600 nucleotides or more) of sequences in accordance with the first aspect of the invention may be used to cause specific antisense inhibition of FAO expression in organisms which possess an endogenous FAO gene which exhibits homology with the sequence of the invention.
  • the invention thus provides a method of causing antisense inhibition of an FAO gene.
  • nucleic acid sequence comprising portions at opposed end regions of the FAO gene to delete FAO coding sequences from host cells.
  • Methods, disclosed in the prior art in relation to other genes are known whereby a nucleic acid sequence comprising opposed end regions of a gene (but from which the majority of the intervening portion has been removed), is introduced into a host cell, wherein the host cell comprises a functional gene whose end regions share a high level of nucleotide sequence identity with the introduced sequence.
  • a homologous recombination event occurs, in which the introduced (inactive) gene is integrated on the host cell chromosome, and the wild type functional gene is excised from the chromosome and subsequently degraded.
  • deletion mutants can be obtained in which a functional gene in a host cell is replaced with a partial fragment of the gene, which is 7 wholly inactive. Such deletion mutants can possess extremely useful properties.
  • the invention provides a method of deleting a functional FAO coding sequence from the chromosome of a host cell, the method comprising: preparing a non-functional fragment of an FAO gene (typically comprising opposed end regions thereof but substantially free of FAO coding sequence); introducing the nonfunctional FAO gene fragment into a host cell which comprises a functional FAO gene, the opposed end regions of which exhibit a high level of nucleotide sequence identity with the introduced non-functional FAO gene fragment, so as to cause replacement of the functional FAO gene by the introduced non-functional FAO gene fragment.
  • a non-functional fragment of an FAO gene typically comprising opposed end regions thereof but substantially free of FAO coding sequence
  • introducing the nonfunctional FAO gene fragment into a host cell which comprises a functional FAO gene, the opposed end regions of which exhibit a high level of nucleotide sequence identity with the introduced non-functional FAO gene fragment, so as to cause replacement of the functional FAO gene by the introduced non-functional FAO gene fragment.
  • the method may be repeated a number of times if desired, as several industrially significant organisms have polyploid genomes or may, for other reasons, comprise a number of copies of the FAO gene, all of which should preferably be deleted in order to obtain maximum phenotypic change in the deletion mutant.
  • the method may also comprise one or more screening steps (typically after each transformation) to select for further processing those cells which have desirable qualities (e.g. lowest levels of FAO activity, which could be assayed, for example, using the methods described in detail herein).
  • FAO coding sequence refers to that portion of the FAO gene which is translated into amino acid.
  • the FAO gene comprises the FAO coding sequence together with regulatory sequences such as the promoter, 5' and 3' untranslated regions, and the like.
  • the invention also provides a nucleic acid construct for deleting an FAO gene from a cell, the construct comprising a non- functional FAO gene fragment (i.e. a portion of an FAO gene, which portion is insufficient to code for a polypeptide having FAO activity), which fragment comprises one or more portions possessing sequence identity with the FAO gene to be deleted.
  • a non- functional FAO gene fragment i.e. a portion of an FAO gene, which portion is insufficient to code for a polypeptide having FAO activity
  • the non-functional FAO gene fragment typically comprises at least part of the 5' and 3' untranslated portions of the FAO gene, although it may also include a small amount (for example, up to about 200 nucleotides) of the 5' and/or 3' end portions of the FAO coding 8 sequence.
  • the majority (typically 80% or more) of the FAO coding sequence is advantageously omitted from the non-functional FAO gene fragment.
  • the omitted portion of the FAO gene in the non-functional FAO gene fragment is replaced by an approximately equivalent length of irrelevant DNA, such that the spacing between the 5' and 3' opposed end portions of the non-functional FAO gene fragment is substantially similar to the spacing between the corresponding 5' and 3' end portions of the functional FAO gene on the host cell chromosome to be deleted.
  • a high level of nucleotide identity is desirable between the opposed end regions of the functional FAO gene to be deleted, and the corresponding 5' and 3' opposed end regions of the introduced non-functional FAO gene fragment.
  • this nucleotide sequence identity is 90% or more, over a length of at least 50 nucleotides at each opposed end region.
  • the invention therefore also provides FAO " mutant cells and organisms, from which the FAO coding sequence has been specifically substantially deleted.
  • Such cells may be referred to as FAO deletion mutants.
  • the deletion mutant cell of the invention is a cell in which sufficient of the FAO gene has been specifically deleted so as substantially to abolish FAO activity (which typically will be less than 5 % , preferably less than 2 % , of the activity associated with an otherwise identical cell without the FAO " deletion).
  • Such deletion mutants, with specific, known mutations are greatly preferred to FAO " mutants which might conceivably be obtainable by random mutagenesis, which process is unpredictable and can cause undesirable mutations in other genes.
  • the deletion mutant of the invention is prepared using a deletion construct as defined above.
  • the deletion mutant will normally be an organism which, prior to deletion of the FAO coding sequence, is capable of utilising an alkane and/or a fatty acid as a substrate.
  • Preferred deletion mutant organisms are unicellular micro-organisms, such as yeasts, or 9 other organisms amenable to genetic manipulation (e.g. fungi), especially organisms which are commonly used in industry, such as Candida sp. (especially C. tropicalis), Pichia sp. and Torulopsis sp.
  • Such deletion mutants are expected to offer the possibility of synthesising certain organic compounds of commercial interest on a large scale and in a very cost-effective manner.
  • yeast mutants with other metabolic deficiencies
  • the FAO " deletion mutant may additionally be of a mutant phenotype with respect to one or more other defined characteristics, especially in relation to enzymes involved in metabolism of fatty alcohols, fatty acids, or dicarboxylic acids.
  • an existing /3-oxidation deletion mutant may be transformed with a fatty alcohol oxidase deletion or disruption construct in accordance with the invention to provide a /3-oxidation " , FAO " deletion mutant.
  • the FAO genes may be deleted first, followed by deletion of one or more /3-oxidation genes.
  • Yet another possibility would be to delete both FAO and /3-oxidation genes substantially simultaneously, either by using a single deletion construct comprising sequences to disrupt both sets of genes, or by using two or more deletion constructs. Methods of performing such work will be apparent to those skilled in the art with the benefit of the present specification. 10
  • deletion mutants accumulate large quantities of those compounds which would normally act as substrates for the missing enzyme. Often, these accumulated intermediate metabolites are produced in such large amounts that they also enter the extra-cellular environment, from where they are readily recovered.
  • the substrates for FAO are typically fatty alcohols (HO-CH 2 -R-CH 3 ), fatty diols (HO-CH 2 -R-CH 2 -OH), and ⁇ -hydroxy fatty acids (HO-CH 2 -R-COOH), (where R is a saturated or unsaturated hydrocarbon chain, which may be substituted or unsubstituted, branched or [preferably] straight chain, normally comprising 6-22 carbon atoms), so these compounds could be expected to be produced in large amounts by FAO deletion mutants growing on substrates rich in alkanes and/or fatty acids, or substrates which give rise to such compounds upon metabolism by the deletion mutant.
  • the invention provides a method of producing fatty alcohols and/or ⁇ , ⁇ -fatty diols, and/or ⁇ -hydroxy fatty acids, from a substrate comprising a hydrocarbon (particularly alkenes or alkanes) and/or a fatty acid and/or a fatty alcohol, the method comprising contacting the substrate with a plurality of FAO deletion mutant cells under conditions suitable for metabolism of the substrate by the deletion mutant cells, and recovering from the resulting mixture fatty alcohols, ⁇ , ⁇ -fatty diols and/or ⁇ -hydroxy fatty acids.
  • the preferred products are ⁇ , ⁇ -fatty diols and all ⁇ -hydroxy, saturated or unsaturated, substituted or unsubstituted C 8 -C 22 fatty acids, which compounds are commercially significant (fatty alcohols can generally be prepared by other means).
  • the product may be a fatty alcohol and/or an ⁇ , ⁇ -fatty diol; where the substrate is a fatty acid, the product may be an ⁇ -hydroxy fatty acid; and where the substrate is a fatty alcohol, the product may be an ⁇ , ⁇ -fatty diol.
  • the conditions generally suitable for metabolism of the substrate are known to those skilled in the art (e.g. typically 20-35 °C, preferably with forced aeration etc). The precise conditions will depend on the nature of the substrate and the desired product, the identity of the FAO deletion mutant etc. 11
  • Suitable fatty acid substrates particularly include C 8 -C 22 saturated or unsaturated fatty acids, such as erucic, oleic, linoleic, behenic, arachidic, stearic, palmitic, myristic and lauric acids and the like.
  • Suitable alkane substrates particularly include C 8 -C 22 saturated or unsaturated hydrocarbons.
  • Suitable fatty alcohol substrates comprise -C ⁇ saturated or unsaturated fatty alcohols.
  • a yeast FAO deletion mutant could be provided with an ester, which is readily converted into fatty acids by the deletion mutant.
  • Convenient sources of esters are cheap vegetable oils, such as sunflower oil, rapeseed oil, soya bean oil, palm oils, and the like.
  • Other possible substrates include hydroxy- or epoxy-substituted fatty acids.
  • the FAO deletion mutant cells may be grown in suspension in liquid batch culture in a fermenter.
  • the cells may be cultured immobilised as a solid matrix, with substrate passed substantially continuously across the matrix.
  • the cells are cultured until the majority of the alkane/fatty acid containing or producing substrate has been exhausted.
  • the fatty alcohols, ⁇ , ⁇ -fatty diols and/or ⁇ -hydroxy fatty acid products of interest are secreted into the extracellular medium, from which they can readily be 12 separated from the cells (e.g. by filtration, centrifugation etc).
  • the cells can be lysed or treated in other ways so as to release their intracellular contents, which may include the products of interest.
  • the products of interest may be extracted from broth culture using one or more known techniques (e.g. solvent extraction, or acid precipitation).
  • solvent extraction, or acid precipitation Useful information on these techniques is given, for example, by Hatton in Comprehensive Biotechnology Vol. 2 (1985, Eds. Cooney & Humphrey, published by Pergammon Press, Oxford, UK) and in US 4,339,536.
  • an acid precipitation method for the extraction of dioic acids at the end of fermentation the pH of the broth is increased to 11- 12 to dissolve the dioic acid product, and the cells removed by filtration or centrifugation. The dioic acid is then precipitated by acidification of the filtrate to pH4 or lower, and the product collected by filtration.
  • a generally similar approach should also be useful for extraction of ⁇ -hydroxy fatty acids.
  • the products of interest may be subjected to one or more conventional purification techniques (e.g. distillation, crystallization, precipitation or chromatography).
  • purification techniques e.g. distillation, crystallization, precipitation or chromatography.
  • Figure 1 is a graph of fatty alcohol oxidase activity against time, showing induction of expression of the enzyme in C. cloacae cells grown on oleic acid substrates;
  • Figure 2 is a photograph of SDS-PAGE analysis of C. cloacae polypeptides produced in cells grown on oleic acid substrates;
  • Figure 3A is an elution profile for elution of alcohol oxidase from a phenyl superose column used to purify the enzyme
  • Figure 3B is a photograph showing gel electrophoresis analysis of the purified enzyme
  • 13 Figure 4 is a graph showing the pH profile of the purified enzyme
  • Figure 5 shows sample Northern blot results of fatty alcohol oxidase mRNA
  • Figure 6 shows the DNA and deduced amnio acid sequence of a fatty alcohol oxidase gene
  • Figure 7 is a comparison of the amino acid sequence of three fatty alcohol oxidases
  • Figure 8 shows the DNA and deduced amino acid sequence of an alcohol oxidase gene
  • FIGS 9A,B and 10A,B are schematic representations of the preparation of various nucleic acid constructs in accordance with the invention.
  • Candida cloacae 3152 strain FERM P-736 (used throughout these experiments) originates from the Fermentation Research Institute, the Agency of Industrial Science and Technology, the Ministry of the Industrial Trade and Industry, Japan and is described in GB 1,300,455. It was maintained at 4°C on agar slopes containing 1.5g agar, 0.5g yeast extract, 0.5g peptone and lg sucrose per 100ml.
  • Starter cultures used to inoculate shake flasks and fermenters, were prepared by aseptically transferring a loop of agar slope culture into 50ml medium containing 0.25g yeast extract, 0.25g peptone and 0.5g sucrose and incubating 24hr in a baffled 250ml flask at 30°C and 90rpm shaking. All biochemical reagents, including the detergent CHAPS, were obtained from Sigma Chemical Co. Poole, Dorset, UK and were of the highest purity available. Reagents for electrophoresis were from BioRad (Hemel Hempstead, Herts, UK).
  • the minimal medium used at shake flask scale contained (per litre) 25g sucrose, 7.6g NH 4 C1, 1.5g Na 2 SO 4 , 300ml ImM pH 7.0 potassium phosphate buffer, 20mg ZnSO 4 .7H 2 0, 20mg MnSO 4 .4H 2 O, 20mg FeSO 4 .7H 2 O, 2g MgCl 2 .6H 2 O, lOO ⁇ g biotin, 20mg nicotinic acid, 20mg pyridoxine, 8mg thiamine and 6mg pantothenate.
  • the minimal medium cultures were grown for 48h at 30°C at 90 rpm on a shaker.
  • Cell-free extracts were made from the pellets by grinding each pellet in liquid nitrogen three times then rapidly resuspending the powder in 10 ml of 50mM potassium phosphate pH 7.5 containing 5% glycerol, 0.5% CHAPS and centrifuging it at 10,000g for 10 min for clarification. The alcohol oxidase activity in these extracts was then measured.
  • Fatty alcohol was assayed spectrophotometrically (Kemp et al., 1988 Appl. Microbiol. Biotechnol. 29, 2,10- 1 A).
  • the assay mixture contained 50 mM-Tris/HCl pH 8.5, 0.7 mg/ml ABTS (2,2'-azino-bis-[3-ethylbenzthiazoline-6-sulphonic acid]), 7U of horseradish peroxidase and 50 ⁇ M dodecanol previously dissolved in DMSO, in a final volume of 1.0ml unless otherwise specified. Reactions were initiated by addition of sample and the increase in absorbance at 405nm measured.
  • Figure 1 is a graph of alcohol oxidase activity (in units per gram cells wet weight) against time (hours) after addition of oleic acid; it rose 4-fold in six hours and reached a maximum of 7-fold at 24h.
  • cell extracts from 0, 6 and 125h post-induction on oleic acid were run on SDS-PAGE and stained for protein with Coomassie Blue ( Figure 2).
  • SDS-PAGE gels 15 consisted of a 5% stacking gel with a 10% running gel and were run on a mini BioRad Protean gel kit. The buffers used were as described by Laemmli (1970 Nature 227, 680- 685).
  • Figure 2 shows the results of SDS/PAGE analysis of protein profiles of C. cloacae cells prior to and after induction on oleic acid. Samples are 0, 6, 125 hours post induction. Arrows on the right show proteins which are elevated upon induction. The arrow on the left marked (a) indicates a protein of Mr 64 kDa which is reduced in quantity upon induction.
  • Native gel electrophoresis was performed to establish which of the bands identified by SDS-PAGE was associated with alcohol oxidase activity. This was carried out at 200V for 2.5h in a 10% resolving/5% stacking gel as described above for SDS-PAGE except that the SDS was replaced by 1 % and 0.5% sodium cholate in the gel and running buffer respectively.
  • the sample buffer consisted of lOmM-Tris/HCl pH 6.8, 2% glycerol, 1 % sodium cholate and sufficient bromophenol blue to make it visible.
  • the running buffer and apparatus were chilled on ice before and during the run. The sample was 50 ⁇ l of 0.2U of hydroxyapatite-purified C.
  • cloacae material prepared by the method of Dickinson & Wadworth (1992 Biochem. J. 282, 325-331).
  • buffer, ABTS and peroxidase at lOx the concentration used in the standard alcohol oxidase assay (described above) and dodecanol at 2.7 mM were applied to the surface of the gel, and it was incubated at room temperature for 5 mins. Alcohol oxidase activity was revealed as a region of green stain on the surface of the gel. The region containing the biological activity was cut out from the native gel and then subjected to SDS-PAGE.
  • the cells were harvested (3500g/10min), washed with 3L of 50mM HEPES/NaOH pH 7.5 and resuspended in 140ml of 50mM HEPES/NaOH pH 7.5, ImM EDTA, ImM DTT prior to cell disruption using a French pressure cell.
  • the wet weight of cells was typically 110-120g.
  • microsomes (within which the enzyme was presumed to be concentrated)
  • 132g (wet weight) of cells were passed through a French pressure cell 3 times at 20,000 psi, the disrupted cell extract centrifuged at 20,000g for 30min and the precipitate discarded.
  • the preparation was snap-frozen in liquid N 2 and stored at -80°C prior to further processing, for the sake of convenience.
  • the supernatant was thawed and lOOmM-Tris/HCl pH 7.5 added to give a final volume of 230ml.
  • the microsomal fraction was pelleted by ultracentrifugation at 140,000g for 1.5h at 4°C.
  • the pellet was washed by suspending in lOOmM-HEPES/NaOH, pH 8.0, containing 0.15M-KC1 (final volume 115ml) and re-pelleted by further ultracentrifugation at 140,000g for 1.5h at 4°C.
  • the washed microsomes were resuspended in 50mM-HEPES/NaOH, pH 8.0 (final vol. 62ml).
  • the resuspended pellet was made up to 500ml with 50mM- HEPES/NaOH, pH 8.0.
  • Sodium cholate was added to 1.0% and PMSF in isopropanol to ImM.
  • the pellet was resuspended in 50mM-HEPES/NaOH, pH 8.0, containing 1 % Na cholate to a final volume of 52 ml, dialysed against two changes of 2 litres 50mM-HEPES/NaOH pH 8.0 for 2 x lh and a further 500ml of 50mM-HEPES/NaOH, pH 8.0 containing 1% CHAPS for lh 17 and then centrifuged at 20,000g for 5min to give a clear supernatant.
  • the inventors initially adopted a similar procedure to that used for the purification of alcohol oxidase from C. tropicalis (Dickinson & Wadworth 1992 Biochem. J. 282, 325- 331). It was found essential to prepare the microsomes from alkane induced fermentation as the oleic acid induced cells accumulated fatty acids which interfere with the membrane pelleting during the ultracentrifugation step.
  • Use of this prior art procedure which involved preparation of microsomes, detergent solubilization, (NH 4 ) 2 SO 4 precipitation, QAE-cellulose chromatography and hydroxy apatite absorption, did not produce a homogeneous preparation as judged by SDS-PAGE when applied to C. cloacae ⁇ t was found that an additional gel filtration step on a Superose 12 column was required to obtain homogeneity.
  • FIG. 3b shows the results of this SDS-PAGE analysis of fractions 3-6 from the phenyl superose column. Lanes 1, 2, 3 and 4 are fractions 3, 4, 5 and 6 respectively. Molecular weight markers are indicated on the left hand side. The arrow on the right hand side corresponds to a Mr of 73 kDa.
  • Table 1 shows the overall purification procedure, giving an enzyme preparation purified 230-fold with a 10.7% recovery of biological activity.
  • the new procedure was faster than that disclosed in the prior art and gave rise to essentially homogeneous enzyme.
  • the enzyme activity was visualized directly in the gel as a green band when the gel was incubated with enzyme assay reagents. This band was excised and subject to SDS/PAGE, and a major band at 73 kDa was seen, confirming assignment of this band as alcohol oxidase.
  • the purified enzyme was assayed for biological activity at several pHs using dodecanol as the substrate. Assays were performed at least in duplicate and usually the variability was less than 5% .
  • the enzyme preparation was homogenous, as judged by SDS-PAGE, and was diluted one in ten in O.lmg/ml BSA, 0.5% CHAPS, 20% glycerol and lOOmM- Tris pH8.5. Each assay contained lO ⁇ l of enzyme.
  • the low pH buffer was Bis/Tris/Propane 50mM and the high pH buffer glycine 50mM.
  • the substrate was lO ⁇ l of 5mM dodecanol in DMSO (final concentration 50 ⁇ M).
  • Figure 4 is a graph of enzyme units against pH.
  • the experiment involved the use of two different batches of enzyme preparation (shown by circle or square symbols respectively).
  • the enzyme exhibits a broad pH optimum between 8.0 and 9.5 but shows much lower activity below pH 7.5. This is similar to the properties of the enzyme from Candida tropicalis, which has maximal activity at pH 9.0 and has no activity at pH 5.5. 20
  • the purified enzyme was also the subject of investigations into reaction kinetics.
  • three separate experiments were performed using 10 or 20 ⁇ l of 1 in 10 diluted enzyme as described above for the pH profile experiment, using 50mM Tris/HCl pH7.5 as buffer and dodecanol stock at 5 and 0.5mM in DMSO. This was to prevent any misinterpretation due to dilution effects. It was found that the apparent K,,, for dodecanol is between 4.0 and 5.0 ⁇ M, and the V ⁇ 110s 1 . Determination of KTM and N ⁇ for decanol did not give reliable results: the K,,, determined from three consecutive experiments on the same day increased from 35 ⁇ M in the first determination to 65 ⁇ M in the third. The reason for this variation is unknown.
  • the protein preparation was used for the generation and sequencing of alcohol oxidase peptides. This was performed using the Promega procedure with Chromaphor green to detect the protein in the first gel and Endoproteinase Glu C for "in gel” digestion (Elborough et ⁇ Z., 1994 Plant Mol. Biol. 24, 21-34).
  • the partially pure protein (1300pmol based on a specific activity of 80 U/mg of pure material) from the (NH 4 ) 2 SO 4 dialyzed pellet was separated using SDS-PAGE, stained with dye Chromophor Green, and the major band running at 73 kDa excised from the gel. Only one band in the region of 73 kDa was evident at this stage.
  • the specific activity of this protein was 8.5 U/mg, approximately one tenth of the purified protein. However, most of the impurities were of low molecular 21 weight, and there was only one dominant band on the gel which was at approximately 73 kDa.
  • the excised band was loaded onto a second gel where in-gel digestion with glu-C and separation of the resulting peptides was carried out.
  • Undigested excised 73 kDa band run at the same time as this gel gave a single high molecular weight band, verifying its purity.
  • the resultant peptides were transferred to Problott for sequencing. Three bands were separately applied to the sequencer, but only one gave any sequence data.
  • Example 1 describes the purification of the fatty alcohol oxidase (FAO) protein and determination of its internal amino acid sequence. Based on that work, the inventors attempted to clone and characterize FAO coding sequences from C. cloacae.
  • FEO fatty alcohol oxidase
  • genomic and cDNA libraries were prepared.
  • C. cloacae DNA was isolated from cells grown on YPD (10 g/1 yeast extract, 20 g/1 peptone, 20g/l glucose) according to Philippsen et al. (1991 Methods in Enzymology 194, 169-182). Partially Sau 3A-digested DNA was size fractionated to 14-23 kb via a 10-40% sucrose gradient and ligated into ⁇ Bluestar BamHI arms (Novagen). Gigapack II Gold packaged particles were transfected into E. coli ER1647 (Novagen).
  • Poly (A)+ mRNA from the frozen cell samples was used for the construction of a random-primed non-directional cDNA library in Ec ⁇ RI-digested alkaline phosphatase treated ⁇ zapll vector (Stratagene). Approximately 5.0 ⁇ g of mRNA was used in the reverse transcriptase reaction according to instructions of the TimeSaver cDNA synthesis kit (Pharmacia). EcoKL/Notl adapters were added to the ends and cDNA ligated with EcoRI digested ⁇ zapll. Gigapack II Gold packaging extract (Stratagene) was used to form phage particles followed by transfection of E. coli XL 1 -Blue cells (Stratagene).
  • FAO fatty alcohol oxidase
  • the fragments were cloned directly into pGEM-T vectors (Promega) using standard techniques and the length of all clones was analyzed by PCR using internal FAO and T7 (5' GTA ATA CGA CTC ACT ATA GGG CG 3' Seq. ID No. 13) or SP6 (5' GCT ATT TAG GTG ACA CTA TAG 3' Seq. ID No. 14) primers essentially as described previously, except that annealing was at 58 °C for 30 seconds.
  • Plasmid AX 17 sequence was digested with PvwII and Hindlll to remove poly linker sites and the resulting 1 kb fragment was used as a probe to screen the 24 h oleic acid induced C. cloacae cDNA library.
  • Figure 5 shows the induction of FAO mRNA upon 24 h growth on oleic acid probed with PvwII-Hmdlll fragment from pAX17.
  • Lane 1 is mRNA from cells grown on 2% glucose
  • lane 2 is mRNA from cells grown on 1 % oleic acid.
  • Molecular size markers are indicated at the side.
  • the signal of 2.4 kb, the size of which corresponds to a protein of 25 approximately 73 kDa known to represent FAO protein was induced 5-7 times on oleic acid compared to glucose grown C. cloacae. A low level of constitutive expression on glucose was also observed, although this is not apparent from the Figure.
  • pgFAO 14 contained an 18kb fragment insert which was further characterised. Sequencing of 4.3 kb of the genomic clone showed that the whole class 1 type gene was represented.
  • the deduced amino acid sequence of FAOl contains a carboxy-terminal peroxisome targeting sequence SKL (Gould et al, 1989 J. Cell. Biol. 108, 1657-1664). In FAOl the corresponding carboxy-terminal sequence is TKL, which fits to the more general consensus (uncharged (neutral) - basic - hydrophobic residue). 2 6
  • Both E O1 and FA02 contain the consensus sequence of Cys-X-X-Cys-His for the cytochrome c family heme-binding site (Mathews 1985 Prog. Biophys. Mol. Biol. 45, 1- 56). Identity between FAOl and FAOl was 80.0% at nucleotide level, and deduced amino acid similarity was 89.4% . The augment of deduced FA ⁇ 1 and FAO2 amino acid sequences is shown in Figure 7 (which also includes the amino acid sequence of another alcohol oxidase, FAOT, cloned from C. tropicalis described in detail in Example 3 below). An exhaustive search of EMBL/Genbank databases did not reveal any related sequences, confirming the novelty of the two FAO genes. The deduced amino acid sequence of FAO2 and FAOT are shown as Seq. ID Nos. 20 and 21 respectively in the attached sequence listing.
  • FAOl also contains a typical eukaryotic polyadenylation signal AATAAA (Proudfoot & Brownlee 1976 Nature 263, 211-214) as well as a consensus sequence for transcription termination TAG...TA T/A GT...TTT in S. cerevisiae (Zaret & Sherman 1982 Cell 28, 563-573) (see Figure 6).
  • AATAAA eukaryotic polyadenylation signal
  • Codon usage of the two genes is not particularly biased as 89% and 90% of all codons available are used by FAOl and FAOl respectively, though preferred codons are slightly different. The most prominent difference relates to codons specifying: tyrosine, where FAOl uses TAC (72.0%), whilst FAOl favours TAT (52.2%); and aspartic acid, where FAOl uses GAC (60.5%) whereas FAOl uses GAT (64.3%).
  • tyrosine where FAOl uses TAC (72.0%), whilst FAOl favours TAT (52.2%)
  • aspartic acid where FAOl uses GAC (60.5%) whereas FAOl uses GAT (64.3%).
  • Candida species use the universal leucine codon CUG as a serine codon (Ohama et al. 1993 Nucl. Acids 27
  • FAOl and FAOl were isolatable from a cDNA library, excluding the possibility that one of them might be a pseudogene. Isolation of the FAOl and FAOl genes allows the execution of knock-out experiments to determine their exact functions and enables construction of new industrially significant strains in which the ⁇ oxidation pathway is blocked at the alcohol oxidase stage.
  • C. tropicalis NCYC 470 the strain used in this example, originates from the National Collection of Yeast Cultures, Brewing Industry Research Foundation, Great Britain.
  • C. tropicalis cells were grown for 24 hours in minimal medium [1.5g/l Na 2 SO 4 , 20mg/l ZnSO 4 .H 2 0, 20mg/lFeSO4.7H 2 0, 100 ⁇ g/l biotin, 20mg/l pyridoxine, 6mg/l pantothenate, 7.6g/l NH 4 C1, 40.4g/l KH 2 PO 4 , 20mg/l MnSO 4 .4H 2 O, 2g/l MgCl 2 .H 2 O, 30mg/l nicotinic acid, 8mg/l thiamine] containing 1 % oleic acid as a carbon source, harvested and ground in mortar and pestle under liquid N 2 .
  • minimal medium [1.5g/l Na 2 SO 4 , 20mg/l ZnSO 4 .H 2 0, 20mg/lFeSO4.7H 2 0, 100 ⁇ g/l biotin, 20mg/l pyridoxine, 6mg/l panto
  • EcoW/Notl adapters were added to the ends and cDNA was 28 ligated with EcoRI digested ⁇ zapll.
  • Gigapack II Gold packaging extract (Stratagene) was used to form phage particles followed by transfection of E. coli XL 1 -Blue cells (Stratagene).
  • cloacae library with the insert from pAX17) as a probe under the following low stringency hybridization conditions: prehybridization 2h at 55°C in 6xSSC-lxDenhardt's, 0.5% SDS, 0.05% sodium pyrophosphate with 0.05mg/ml herring sperm DNA; hybridization overnight at 55' C in essentially the same mix except that instead of herring sperm DNA, 1 mM ⁇ DTA was used. Filters were washed twice with 2xSSC-0.1 % SDS at 55 °C (30 minutes per wash).
  • C. tropicalis DNA was isolated from cells grown overnight in YPD according to Philippsen et al. (1991, cited above). Partially Sau3A-digested DNA, size fractionated through a 10-40% sucrose gradient to 10-12 kb, was ligated into Zap ⁇ xpress Bamffl arms (Stratagene). PhageMaker pakaging extract (Novagen) was used to form phage particles, which were then transfected into XL 1 -Blue MRF' cells (Stratagene).
  • the resulting product of approximately 4.5 kb was diluted 1 :5 in buffer and 1 ⁇ l amplified with 2.5 U BIOTAQTM DNA Polymerase (Bioline), 250 ⁇ M dNTPs, and PCR buffer (Bioline) containing 1.5 mM MgCl 2 , using 30 pmol of FT5 (5' GAT GGT AAA GGA CAT GGC 3' Seq. ID No. 23) and T7 primers in following conditions: 1 cycle 30 denaturation at 94°C 5 minutes; 30 cycles denaturation at 94°C 1 minute, annealing at 56 °C 1 minute, elongation at 72 °C 2 minutes; 1 cycle prolonged elongation at 72 °C for 5 minutes.
  • the resulting 1 kb product was reamplified with gFT7 (5' CCA AGG GAT GAA CGA TCC 3' Seq. ID No. 24) and T7 primers in PCR under essentially the same conditions.
  • This PCR product of approximately 750 bp was purified using Wizard PCR Preps DNA Purification System for Rapid Purification of DNA Fragments (Promega), ligated into pT7Blue (R) vector (Novagen) and transformed into Epicurian coli * XL2-Blue MRF' ultracompetent cells (Stratagene). 733 bp of the resulting plasmid pFAOT3 was sequenced using sequence specific primers. The total FAOT sequence of 4233 bp (Seq. ID No. 25), which contains an open reading frame of 2112 nt (704 amino acid residues) is shown in Figure 8.
  • primers FI or FI', F2, F3, F4 or F4', F5 or F5 ⁇ F6, F7, F8 or F8' are underlined (note that primers F2, F4, F6, F8 are from the opposite DNA strand to that shown in the figure).
  • the deduced amino acid sequence is also shown beneath the DNA sequence.
  • a plasmid for gene disruption using the FAOT 5' and 3' untranslated regions was constructed in two steps: (1) amplication of the 5' -UTR sequence of FAOT and ligation with C. tropicalis transformation vector pDS148 (a generous gift from Dr Kevin Sanglard, CHUV, Lausanne, Switzerland), yielding plasmid plSV; and (2) amplificiation of the 3' -UTR sequence of FAOT and ligation with plSV, yielding plasmid pSVUl which was used as template for PCR. Further subcloning resulted in the final FAOT disruption plasmid pSVU2. The process is illustrated schematically in Figures 9 A and 9B.
  • plasmid pDS148 mentioned above was constructed previously by replacing the URA3 Hindlll fragment of S. cerevisiae present in pNKY51 (see Alani et al, 1897 Genetics 116, 541-545) with an equivalent URA3 fragment from C. tropicalis. 3 1
  • FIG. 10A and 10B Another construct for gene disruption was prepared, again in a two-step process, using FAOT 5' and 3' coding sequences (CDS): (1) amplification of the 5' CDS sequence of FAOT and ligation with C. tropicalis vector pDS148, yielding plasmid p2SV; and (2) amplification of the 3' CDS seuqence of FAOT and ligation with p2SV, yielding plasmid pSVCl which was also used as template for PCR. Further subcloning resulted the final FAOT disruption plasmid pSVC2. Preparation of pSVC2 is illustrated schematically in Figures 10A and 10B.
  • a 400 bp 5'-UTR FAOT Bglll fragment was amplified from 10 ng of gFAOT4, described above, using 50 pmol of primers FI (5' GAA AAG ATC TGT TAT TAG AAG AGT TAC 3' Seq. ID No. 26) and F2 (5' AAA CAA TTA GAT CTC CGA AAC ACA GGC 3' Seq. ID No. 27) (see Fig.
  • the resulting fragment was purified using the Wizard PCR Preps DNA Purification System for Rapid Purification of DNA Fragments (Promega). Subsequently the PCR product was ligated into pT7Blue (R) vector and transformed in E. coli XL2-Blue MRF ultracompetent cells to ascertain that Bglll restriction enzyme digestion released the required fragment. After Bglll digestion, the fragment was ligated to 5g///-digested, Shrimp Alkaline Phosphatase (Boehringer Mannheim) -treated pDS 148 and transformed into XL2-Blue MRF ultracompetent cells.
  • plSV The orientation of the 400 bp Bglll insert in the resulting plasmid, termed plSV, was checked by PCR of 1:5 dilution of a transformed bacterial colony resuspended in TE with 2.5 U BiotaqTM DNA Polymerase (Bioline), 250 ⁇ M dNTPs, PCR buffer containing 1.5 mM MgCl 2 using 30 pmol of primers FI and URA1 (5' CTG GTT GTT CTT CTG GTG 32
  • a 225 bp 3-UTR FAOT BamHI fragment was amplified from 10 ng of gFAOT4, described above, using 50 pmol of primers F3 (5' CCG GAT CCA GCT TGT TGA TTG AAT CC 3' Seq. ID No. 29) and F4 (5' GAA GGA TCC ACA ATT GAT TGC ACA GC 3' Seq. ID No. 30) (see Fig. 8), with 2U Vent DNA polymerase (New England Biolabs), 250 ⁇ M dNTPs in PCT buffer in the same conditions as in the step (1) above.
  • the resulting fragment was purified using the Promega Wizard System. Subsequently the PCR product was ligated into pT7Blue (R) vector and transformed into E. coli XL2-Blue MRF ultracompetent cells to ascertain that BamHI restriction enzyme digestion released the required fragment. After BamHI digestion, the fragment was ligated to BamHI- digested, Shrimp Alkaline Phosphatase (Boehringer Mannheim)-treated plSV and transformed into XL2-Blue MRF ultracompetent cells.
  • pSVUl The orientation of the 225 bp BamHI insert in the resulting plasmid, termed pSVUl, was checked by PCR of 1:5 dilution of a transformed bacterial colony resuspended in TE with 2.5 U BiotaqTM DNA Polymerase (Bioline), 250 ⁇ M dNTPs, PCR buffer containing 1.5 mM MgCl 2 using 30 pmol of primers F4 and URA2 (5' GGT TGG AAC GCC TAC TTG 3' Seq. ID No. 31, from the C.
  • PCR was performed using the Boehringer Expand Long Template PCR System in the following conditions: 1 cycle denaturation at 94 °C 5 minutes; 35 cycles denaturation at 94 °C 1 minute, annealing at 64.5°C 1 minute, elongation at 72°C 5 minutes; 1 cycle prolonged elongation at 72°C for 10 minutes.
  • the resulting fragment was purified using the Promega Wizard system.
  • EcoRI digestion the PCR product was ligated into EcoR/-digested andshrimp Alkaline Phosphatase-treated pBuescript SK+ vector to give plasmid pSVU2.
  • pSVU2 was transformed into E. coli XL 1 -Blue competent cells to ensure that EcoRI restriction enzyme digestion released the required fragment.
  • a 437 bp 5-UTR FAOT Bglll fragment was amplified from 10 ng of gFAOT4, using 50 pmol of primers F5 (5' GAA AGA GAT CTA CGT GAA AAG GCA TA 3' Seq. ID No. 34) and F6 (5' AAC AAG ATC TTG CGC CAT GTC ATA TG 3' Seq. ID No. 35) (see Fig.
  • the resulting fragment was purified using the Promega Wizard System. Subsequently the 34
  • PCR product was ligated into pT7Blue (R) vector and transformed into E. coli XL2-Blue MRF ultracompetent cells to ensure that Bglll restriction enzyme digestion released the required fragment. After Bglll digestion, the fragment was ligated to 5g 7/-digested, Shrimp Alkaline Phosphatase (Boehringer Mannheim)-treated pDS 148 and transformed into XL2-Blue MRF ultracompetent cells.
  • the orientation of the 437 bp Bglll insert in the resulting plasmid p2SV was checked by PCR of 1.5 dilution of a transformed bacterial colony resuspended in T ⁇ with 2.5U BiotaqTM DNA Polymerase (Bioline), 250 ⁇ M dNTPs, PCR buffer containing 1.5 mM MgCl 2 using 30 pmol of primers F5 and URA1 in the following conditions: 1 cycle denaturation at 94°C 1 minute, annealing at 54°C 1 minute, elongation at 72°C 2 minutes; 1 cycle prolonged elongation at 72 °C for 5 minutes. Only the correct orientation results in the expected product of 1.4 kb. This was also confirmed by sequencing plasmid p2SV using the F5 oligonucleotide as primer, which showed that the 5 '-CDS FAOT sequence is followed by the hisG sequence from the plasmid pDS148.
  • a 525 bp 3'-CDS FAOT BamHI fragment was amplified from 10 ng of gFAOT4, using 50 pmol of primers F7 (5' TCC ATG GAT CCA AGC TTC GTT GTT AC 3) and F8 (5 CAT TGG ATC CGC ACC ACT TGC AGT T 3') (Seq. ID Nos. 36 and 37 respectively), with 2U Vent DNA polymerase (New England Biolabs), 250 ⁇ M dNTPs in PCR buffer in the same conditions as in the step (1) above.
  • the resulting fragment was purified using the Promega Wizard System. Subsequently the PCR product was ligated into pT7Blue (R) vector and transformed into E. coli XL2-Blue MRF ultracompetent cells to ascertain that BamHI restriction enzyme digestion released the required fragment. After BamHI digestion, the fragment was ligated to BamHI- digested, Shrimp Alkaline Phosphatase (Boehringer Mannheim)-treated p2SV and transformed into XL2-Blue MRF ultracompetent cells.
  • the orientation of the 525 bp BamHI insert in the resulting plasmid was checked by PCR of 1:5 35 dilution of a transformed bacterial colony resuspended in TE with 2.5 U BiotaqTM DNA Polymerase (Bioline), 250 ⁇ M dNTPs, PCR buffer containing 1.5 mM MgCl 2 using 30 pmol of primers F8 and URA2 (5' GGT TGG AAC GCC TAC TTG 3', Seq. ID No. 38 from the C.
  • PCR was performed using the Boehringer Expand Long Template PCR system in the following conditions: 1 cycle denaturation at 94°C 5 minutes; 35 cycles denaturation at 94°C 1 minute, annealing at 60°C 1 minute, elongation at 72°C 5 minutes; 1 cycle prolonged elongation at 72°C for 10 minutes.
  • the resulting fragment was purified using the Promega Wizard System.
  • the PCR product was ligated into pGEM-T vector to give pSVC2 which was transformed into E. coli XLl-Blue competent cells to ensure that EcoRI restriction enzyme digestion will release the required fragment.
  • the EcoRI fragment generated from pSNU2 has the 5' and 3' UTR available for homologous recombination with the FAO gene (as it is specific for it) and the URA3 gene 3 6 inside the UTRs to use as a positive auxotrophic marker.
  • the transformation cassette also has between the 5' UTR and 3' UTR two identical regions (hisG) which can recombine, so removing the selectable marker URA3 but having completely disrupted the FAO gene in the host cell.
  • the EcoRI fragment generated from ⁇ SNC2 has the 5' and 3' CDS available for homologous recombination with the FAO gene or with a second FAO-like gene (which normally would have nearly 80% homology as is the case in C. cloacae), where second or further alleles of the FAO gene exist (e.g. in diploid or pofyploid organisms).
  • the genotypes of Ura+ transformants were analysed by PCR to verify the deletion of one of the FAO alleles.
  • the following primers were used in this PCR assay: one specific for the FAO gene (5' GAA GGA TCC ACA ATT GAT TGC ACA GC 3', Seq. ID No. 41) and one specific for the hisG DNA fragment present in the disruption cassette (5' CGC GCG ATA CAG ACC GGT TC 3' Seq. ID No. 42).
  • the PCR reaction comprised yeast DNA mixed with primers and conventional PCR buffers with 30 cycles of: annealing at 54°C for 1 min; elongation at 72°C for 2 min; followed by denaturation at 94°C for 30 seconds.
  • Ura+ transformants obtained from the pSVC2 linear DNA were observed in Ura+ transformants obtained from the pSVC2 linear DNA and with a length of 1.5 kb in Ura+ transformants obtained from the pSVU2 linear DNA.
  • the positive transformants were 37 named DSY1670 (from pSVC2) and DSY1671 (from pSVU2).
  • Regeneration of the ura3 genetic marker was obtained by incubating 10 6 cells from DSY1670 and DSY1671 respectively in a medium containing 5-fluoroorotic acid (5-FOA), which counter-selects for the absence of the URA3 gene contained in the disruption cassette (yeast cells with a functional URA3 gene convert 5-FOA into a toxic compound which kills the cells).
  • Single Ura- derivatives were obtained from DSY1670 and DSY1671 and one transformant from each strain (DSY1688 and DSY1692 respectively) were retained for further work.
  • Ura- strains were re-transformed with linear EcoRI fragments as described above. Ura+ transformants were obtained and their genotypes analysed by PCR. Two primers were used which allowed for distinguishing of the FAO wild type allele and the disrupted FAO alleles. These primers had the following sequences 5' TTG CAT TTG AAA GCC ATC CAT TAC CCT 3'(Seq. ID No. 43) and 5' CAA ACA ATC CGA AAC ACC AAC AAC AGT 3' (Seq. ID No. 44). The PCR products obtained from the amplification of the FAO wild type allele and the FAO disrupted allele were expected to be 1.9 kb and 2.1 kb, respectively.
  • DSY1700-1, DSY1700-2 and DSY1700-3 Positive transformants, in which the FAO alleles were disrupted, were obtained from the transformation of DSY1688 with linear DNA fragment from pSVC2. In these strains (DSY1700-1, DSY1700-2 and DSY1700-3), no wild type FAO allele could be amplified, only the disrupted FAO allele.
  • the genotype of DSY1700 derivatives is: (Afao::hisG/Afao::hisG- URA3-hisG) .
  • the representative strain DSY 1700-2 has been made the subject of a deposit under the Budapest Treaty at the National Collection of Yeast Cultures (NCYC, AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, United Kingdom). The date of deposit is March 1999, and the accession number is

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Abstract

La présente invention concerne une séquence d'acide nucléique isolée codant un polypeptide présentant une activité alcool-oxydase d'acide gras ou FAO (Fatty Alcohol Oxidase). L'invention concerne également des constructions de suppression d'acide nucléique comprenant un fragment non fonctionnel d'un gène FAO, des cellules mutantes de suppression de FAO, et leur utilisation.
PCT/GB1999/000846 1998-03-18 1999-03-18 Perfectionnements apportes au metabolisme des acides gras ou s'y rapportant WO1999047685A1 (fr)

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CA002323762A CA2323762A1 (fr) 1998-03-18 1999-03-18 Perfectionnements apportes au metabolisme des acides gras ou s'y rapportant
BR9909655-2A BR9909655A (pt) 1998-03-18 1999-03-18 Aperfeiçoamentos em ou com relação ao metabolismo de ácido graxo
KR1020007010305A KR20010041979A (ko) 1998-03-18 1999-03-18 지방산 대사에 관한 개선
JP2000536868A JP2002506650A (ja) 1998-03-18 1999-03-18 脂肪酸代謝のまたは脂肪酸代謝に関する改良
EP99910537A EP1064386A1 (fr) 1998-03-18 1999-03-18 Perfectionnements apportes au metabolisme des acides gras ou s'y rapportant
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Cited By (6)

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WO2003006509A3 (fr) * 2001-07-10 2003-11-20 Univ Napoli Federico Ii Mini-anticorps humain qui est cytotoxique pour des cellules tumorales exprimant le recepteur erbb2
US7585952B2 (en) 2001-07-10 2009-09-08 Biotechnol S.A. Human mini-antibody cytotoxic for tumor cells which express the ErbB2 receptor
US8227585B2 (en) 2001-07-10 2012-07-24 Biotecnol S.A. Human mini-antibody cytotoxic for tumor cells which express the ERBB2 receptor
EP1576095A2 (fr) * 2002-04-19 2005-09-21 Cognis IP Management GmbH Genes et proteines d'oxydase d'alcool gras de i candida troplicalis /i et methodes associees
EP1576095A4 (fr) * 2002-04-19 2006-10-11 Cognis Ip Man Gmbh Genes et proteines d'oxydase d'alcool gras de i candida troplicalis /i et methodes associees
US7160708B2 (en) 2002-04-19 2007-01-09 Cognis Corporation Fatty alcohol oxidase genes and proteins from Candida tropicalis and methods relating thereto

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AU2946999A (en) 1999-10-11
BR9909655A (pt) 2000-11-21
GB9805660D0 (en) 1998-05-13
EP1064386A1 (fr) 2001-01-03
JP2002506650A (ja) 2002-03-05
KR20010041979A (ko) 2001-05-25
CA2323762A1 (fr) 1999-09-23

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