WO2000065061A2 - Nucleic acid sequences from candida yeasts which code cytochrome b5 polypeptides - Google Patents

Nucleic acid sequences from candida yeasts which code cytochrome b5 polypeptides Download PDF

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WO2000065061A2
WO2000065061A2 PCT/DE2000/001246 DE0001246W WO0065061A2 WO 2000065061 A2 WO2000065061 A2 WO 2000065061A2 DE 0001246 W DE0001246 W DE 0001246W WO 0065061 A2 WO0065061 A2 WO 0065061A2
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cytochrome
nucleic acid
acid sequences
polypeptides
maltosa
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WO2000065061A3 (en
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Wolf-Hagen Schunck
Alexei Chernogolov
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Max-Delbrück-Centrum für Molekulare Medizin
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    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/80—Cytochromes

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  • the invention relates to nucleic acid sequences from Candida yeasts, preferably from Candida maltosa, which encode cytochrome b5 polypeptides and the corresponding cytochrome b5 polypeptides and their use for increasing the activity of cytochrome P450 systems, in particular for stimulating the activity of alkane and fatty acid hydroxylating agents Cytochrome P450 systems in the production of long-chain dicarboxylic acids (> C10)
  • Cytochrome b5 are hemoproteins that are involved in various electron transfer processes in eukaryotic cells [reviews by: Oshino, N., Pharmac. Ther. A. 2, (1978) 477-515 and Vergeres and Waskell Biochemie 77 (1995) 604-620]. Electrons are transferred to cytochrome b5 by NADH- or NADPH-dependent reductases. The reduced cytochrome b5 can then transfer the electron taken up to various other proteins. These electron acceptors include cytochrome P450, fatty acid desaturases, methemoglobin and methionine synthase.
  • cytochrome b5 forms can be distinguished: a) a membrane-bound form, which is located in the endoplasmic reticulum (microsomal form) and interacts there with fatty acid desaturases and cytochrome P450 b) another membrane-bound form, but which is specific in is located in the outer mitochondrial membrane and c) a soluble form of cytochrome b5, as found in erythrocytes (reduction of methaemoglobin) and, according to more recent studies, also in liver cells (reductive activation of methionine synthase [Chen and Banerjee, J. Biol. Chem. 2__ (1998) 26248-26255] occurs.
  • cytochrome b5 consist of a cytoplasmic domain (length approx. 90 to 100 amino acid residues), a membrane-spanning segment (approx. 20 predominantly hydrophobic amino acid residues) and a C-terminal part (approx. 10 hydrophilic amino acid residues), which according to recent studies is luminally oriented [Vergeres et al., J. Biol. Chem. 270 (1995) 3414-3422]. Contains the cytoplasmic domain the prosthetic heme group and is directly involved in the electron transfer function of the cytochrome b5. The three-dimensional structure of the cytoplasmic domain of bovine microsomal cytochrome b5 is known [Argos and Mathews J. Biol.
  • Cytochrome b5 belong to the class of "tail-anchored" proteins.
  • the membrane-spanning segment and the C-terminal luminal part contain all determinants for its intracellular localization. Differences in the presence of individual charged amino acids in the luminal part determine whether the respective cytochrome b5 form is located in the endoplasmic reticulum or in the outer mitochondrial membrane [Kuroda et al., J. Biol. Chem. 273 (1998) 31097-31102].
  • cytochrome b5 proteins of the endoplasmic reticulum it is also known that the length of the membrane-spanning segment is decisive for their permanent localization (retention) in this cell compartment [Pedrazzini et al. Proc. Natl. Acad. Be. USA 93 (1996) 4207-4212].
  • the soluble form in erythrocytes is a C-terminally shortened cytochrome b5, which accordingly has no membrane anchor [VanDerMark and Steggles, Biochem. Biophys. Res. Commun. 240 (1997) 80-83].
  • Cytochrome b5 play a complex role in the regulation of the enzymatic activity of microsomal cytochrome P450 systems [Perret and Pompon, Biochemistry 3 Z (1998) 11412-11424 and literature cited therein].
  • cytochrome b5 can inhibit or considerably stimulate the P450-catalyzed substrate reactions.
  • the stimulating effect is generally based on an improvement in the coupling of Electron transfer and substrate hydroxylation in the reaction cycle of the cytochrome P450 systems.
  • Cytochrome b5 can act as a redox partner (transfer of the second electron in the reaction cycle) as well as cause conformational changes on the cytochrome P450, which lead to an increase in substrate affinity.
  • the binding sites of cytochrome b5 and NADPH-P450 reductase on cytochrome P450 are probably not identical but may be partially overlapping [Bridges et al. J. Biol. Chem. 273 (1998) 17036-17049].
  • Some authors describe that apocytochrome b5 can already stimulate the activity of individual cytochrome P450 systems [Yamazaki et al. J. Biol. Chem. 271 (1996) 27438-27444]. The actual effects of cytochrome b5 on a specific cytochrome P450 system have so far not been predictable.
  • cytochrome P450 systems to regio- and partially. Stereospecific hydroxylation of various chemical compounds is also of considerable biotechnological interest.
  • One area of application is, for example, the production of long-chain dicarboxylic acids using alkane and fatty acid-hydroxylating cytochrome P450 systems.
  • Processes patented for this purpose already include overexpression of the direct components of these cytochrome P450 systems (P450 and NADPH-P450 reductase) in alkane-utilizing yeasts (Candida tropicalis, WO-A 91/14781) or in Saccharomyces cerevisiae (DE 195 07 546 AI) .
  • P450 and NADPH-P450 reductase direct components of these cytochrome P450 systems
  • yeasts Candida tropicalis, WO-A 91/14781
  • Saccharomyces cerevisiae DE 195 07 546 AI
  • the object of the invention was therefore to find nucleic acid sequences which enable the production of cytochrome b5 polypeptides, which stimulate the activity of alkane and fatty acid hydroxylating P450 enzymes and thus increase the rate and efficiency of the production of dicarboxylic acids.
  • the task was solved by nucleic acid sequences from Candida yeasts, preferably from Candida maltosa, which encode cytochrome b5 polypeptides, and their fragments, variants and mutations.
  • C. maltosa cytochrome b5 was purified, its complete cDNA was cloned and the formation of a functional cytochrome b5 was detected by heterologous expression of the cloned cDNA.
  • cytochrome b5 from C. maltosa with its sequence according to the invention could be achieved, since it can be used to stimulate the activity of cytochrome P450 systems from the same organism or from related alkane-utilizing yeasts.
  • the invention thus relates to nucleic acid sequences from alkane-utilizing Candida yeasts which encode a cytochrome b5 polypeptide and their fragments, variants and mutations, in particular the corresponding cDNA.
  • nucleic acid sequences with SEQ ID No. 1 and SEQ ID No. 2 or their variants.
  • the invention relates to cytochrome b5 polypeptides which are encoded by the nucleic acid sequences, preferably a cytochrome b5 polypeptide of SEQ ID No. 3 and variants thereof which have deletions or modifications and have cytochrome b5 activities.
  • the invention relates to plasmids and vectors which contain a nucleic acid sequence according to the invention, as well as host cells which have a vector and antibodies which bind specifically to the polypeptides.
  • the nucleic acid sequences or polypeptides are used to stimulate the activity of cytochrome P50 enzymes in the biotechnological production of fatty acids and dicarboxylic acids by oxidation of long-chain n-alkanes (> CIO).
  • cytochrome P50 enzymes in the biotechnological production of fatty acids and dicarboxylic acids by oxidation of long-chain n-alkanes (> CIO).
  • more than 4-fold stimulation is achieved in the oxidation of dodecane using P450 Cml from Candida maltosa, and more than 7-fold stimulation in the oxidation of lauric acid using P450 Cm2 from Candida maltosa.
  • the microsomal cytochrome b5 of the alkane-utilizing yeast C. maltosa was purified, tryptically cleaved and partially characterized in terms of its amino acid sequence.
  • the sequence of one of the tryptic peptides formed the basis for the derivation of degenerate oligonucleotides for the amplification of partial sequences of the cytochrome b5 cDNA using the polymerase chain reaction (PCR).
  • the complete coding region was then amplified and cloned on the basis of the sequences obtained.
  • the protein derived from the DNA sequences consists of 126 amino acids, contains the partial sequences as determined for the cytochrome b5 purified from C.
  • the yeast strain C. maltosa ATCC 28140 was cultivated in a 5 1 bioreactor on hexadecane as a carbon source.
  • the media composition and general growth conditions were as in Huth et al., J. Basic Microbiol. __ (1990) 481-488.
  • the yeast cells were harvested shortly before entering the stationary growth phase.
  • the mechanical cell disruption and the extraction of the microsomal Membrane fractions were carried out as previously described [Riege et al. Biochem. Biophys. Res. Commun. 98 (1981) 527-534].
  • the microsomal cytochrome b5 content was approx. 0.5 nmol / mg protein.
  • cytochrome b5 The purification of the cytochrome b5 was carried out in accordance with methods as were published for cytochrome b5 from other organisms [Guzov et al. J. Biol. Chem. 271 (1996) 26637-26645; Strittmatter et al., Meth. Enzymol. 52 (1978) 97-101].
  • cytochrome b5 preparation with a purity of approx. 60% was obtained (the ratio of the absorptions at 412 and 280 nm was approx. 1.4).
  • the purified cytochrome b5 is characterized by the following spectral properties: absorption maxima at 412 (type band), 525 and 527 nm in the oxidized state; Shift of the Soret maximum to 424 nm after reduction with dithionite.
  • the preparation obtained was separated in a 15% SDS polyacrylamide gel.
  • the main bands obtained with an apparent molecular weight of 17.5 and 20 kDa were then blotted onto a polyvinylidene difluoride membrane (Pro Blott, Applied Biosystems, USA) and cleaved in situ with trypsin [method according to: Fernandez et al. Anal. Biochem. 218 (1994) 112-117].
  • the tryptic peptides were eluted and separated by reverse phase HPLC on a microcolumn (RPC C2 / C18 SC; 100 x 2.1 mm) (Smart System, Pharmacia, Sweden). Selected fractions were then applied to polybrene-containing glass fiber filters and sequenced (gas phase sequencer, procise, applied biosystems).
  • T27 (derived from the 20 kDa band): LYIGNLK
  • T53 (derived from the 20 kDa band): VYDITSYIDEHPGGE
  • T54 (derived from the 17.5 kDa band): VYDITSYIDE
  • the peptides T53 and T54 match in the sequence of 10 amino acids. This result indicates that the 17.5 kDa band is probably a proteolytic fragment of the 20 kDa band. 1.3. PCR amplification of the cytochrome b5 cDNA
  • the template for the PCR amplification of cytochrome b5 cDNA fragments was initially the plasmid pool from a previously produced C. maltosa EH 15 cDNA library [Schunck et al., Biochem Biophys. Res. Commun. 181: 843-850 (1989)].
  • the cDNAs contained in this library are inserted into the Hind II site of the plasmid pUC119.
  • Cytochrome b5-specific primers were derived from the sequence of the peptide T53 and used in combination with plasmid-derived primers for the PCR. The following primers were successfully used for the following experiments:
  • T53-1-4 5'- GAT / C ATT / C ACT / C AGT / C TAT / C ATT / C GAT / C GAA C -3 'T53-2-2: 5'- ATT / C GAT GAA CAT / C CCA / T GGT / A GG -3 'pUCl 19-2CR: 5'- ACG GCC AGT GAA TTC GAG -3'
  • the PCR Ready-To-Go PCR Kit, Amersham Pharmacia Biotech
  • the PCR carried out over 35 cycles consisting of the steps: 95 ° C, 45 sec / 53 ° C, 45 sec / 72 ° C, 45 sec (+ 1 sec per cycle).
  • the DNA fragment obtained was subjected to a second PCR with the primer combination T53-2-2 (3 'offset from T53-1-4) / pUC119-2CR (25 cycles, same temperature regime). The fragment amplified in this way was sequenced.
  • T53-4CR 5'- GTTTTA TTT CTA AGG GTT TTG GAG -3 'pUCl19-1: 5'- ACA GCTATG ACC ATG ATT ACG -3'
  • T53-4CR was derived from the sequence of the first amplified fragment and attaches to the 3 'end of the suspected cytochrome b5 cDNA.
  • the PCR was carried out over 30 cycles consisting of the steps: 95 ° C., 45 sec / 55 ° C., 45 sec / 72 ° C., 45 sec.
  • the product obtained with a length of approx. 400 bp was sequenced. The sequence showed an open reading frame for a polypeptide with 126 amino acids.
  • T53-7Sal 5'- GAA GTC GAC GTC ATG TCT GAT ACT ACA -3 'T53-8BamCR: 5'- CGC GGA TCC ATT ATG TTT TAT TTC TAA G -3'
  • primers start at the 5 'or 3' end of the sequence of the 400 bp fragment described above.
  • the underlined sequence areas are added restriction sites for Sal I or Bam HI for subsequent cloning into the yeast expression vector YEp51 (see point 1.5).
  • the poly (A) RNA from cells of the C. maltosa strain ATCC 28140 served as template after growth on hexadecane.
  • the RNA was isolated after mechanical cell disruption using the Oligotex Direct mRNA Mini Kit (QIAGEN).
  • the Ready To Go RT-PCR kit from Amersham Pharmacia Biotech was used for the transcription into single-stranded cDNA and the subsequent amplification of the cytochrome b5 cDNA.
  • 100 ng poly (A) RNA and an oligo (dT) primer were used for reverse transcription (30 min, 42 ° C.). After inactivation for 5 min at 95 ° C., the cytochrome b5-specific primers (T53-7Sal and T53-8Bam) were added at 65 ° C.
  • the subsequent PCR amplification was carried out over 35 cycles consisting of the steps: 95 ° C, 45 sec / 53 ° C, 45 sec / 72 ° C, 45 sec (+ 1 sec per cycle).
  • the TOPO T-A cloning kit from INVITROGEN was used for the subsequent cloning of the RT-PCR product obtained. According to the manufacturer's instructions, the RT-PCR product was ligated into the plasmid pCR 2.1 TM and then transformed into E. coli TOP 10.
  • the cloned cDNAs (SEQ ID No. 1 and 2) code for a polypeptide (SEQ ID No. 3) with a length of 126 amino acids (FIG. 1).
  • the peptide sequences (see point 2) determined for the purified cytochrome b5 from C. maltosa correspond exactly with the regions 31 to 45 (T53 peptide) and 76 to 82 (T27 peptide) in the derived amino acid sequence (Fig. 1).
  • the determined amino acid sequence of C. maltosa cytochrome b5 shows a moderate but significant homology to that of cytochrome b5 from other organisms.
  • the sequence identities are, for example, 33% when compared with the human protein and surprisingly not more than 37% when compared with the only known cytochrome b5 from another type of yeast (S. cerevisiae).
  • the corresponding alignments are shown in Fig. 2.
  • the cytochrome b5 cDNA was obtained from one of the sequenced clones SEQ ID No. 2 isolated by restriction cleavage with Sal I and Bam HI and into the yeast expression vector YEp51 [Broach et al., In: Experimental Manipulation of Gene Expression, M. Inoye, ed. Academic Press, NY, 1983, pp. 83-117].
  • the plasmid thus constructed contained the cytochrome b5 cDNA under the control of the galactose-inducible GALIO promoter. and was then used to transform the S. cerevisiae strain GRF18 ( ⁇ , his 3-11, his 3-15, leu 2-3, leu 2-112, can 1 ).
  • the cells were harvested 24 hours after the galactose had been added and mechanically disrupted with glass balls (in 100 mM Tris / HCl buffer pH 7.7 with 5 mM EDTA and a protease inhibitor cocktail, Boehringer-Mannheim).
  • the subsequent differential centrifugation comprised the following steps: 15 min, 3000 g; 15 min, 10,000 g; 90 min, 100,000 g.
  • the 100,000 g sediment (microsomes) was resuspended in the Tris buffer indicated above.
  • the cytochrome b5 content was determined spectrally in the individual cell fractions: For this purpose, the difference spectra between the dithionite-reduced and the sample oxidized with H 2 O 2 (final concentration: 0.003%) were recorded. The extinction coefficient of 185 mJVT'xcm "1, which is customary for cytochrome b5 proteins, was used for the calculation of the difference in the absorptions at 424 and 409 nm [Estabrook and Werringloer, Meth. Enzymol. 52 (1978) 212-221].
  • Fig. 3 show that the cDNA expressed under the control of the GALIO promoter actually codes for a microsomal cytochrome b5.
  • An expression level of approx. 300 to 400 nmol cytochrome b5 per 1 culture can be estimated from the difference spectra of the homogenates.
  • the microsomes isolated from the strain GRF18 / YEp51-b5 had a cytochrome b5 content of approx. 0.5 nmol / mg protein.
  • the content of the host's own cytochrome b5 was significantly lower under the chosen test conditions and was at the detection limit, as shown by the difference spectra with the corresponding cell fractions of the control strain GRF18 / YEp51 (Fig. 3).
  • the heterologously expressed microsomal cytochrome b5 (see 1.5.) was solubilized with the aid of detergents and purified by chromatography on DEAE-Sepharose, hydroxyapatite and DEAE-Toyoperl.
  • the purified protein showed an apparent molecular weight of about 20 kDa.
  • the following two sequences were determined when determining the N-terminal amino acid sequence:
  • cytochrome b5 has a strong stimulating effect on the activity of the investigated alkane and fatty acid oxidizing P450 forms.
  • an S. cerevisiae strain was constructed by integrative transformation, which carries in its genome a cassette for the expression of the C. maltosa cytochrome b5.
  • the integrative expression cassette consisted of the following elements: flanking partial sequences of the TRPl gene for integration into the yeast genome, a URA3 marker gene as well as the GAL 10 promoter, the cytochrome b5 cDNA and the ADH 1 terminator .
  • the partial sequences of the TRPl gene were amplified by PCR from genomic DNA. The following were used as primer pairs:
  • TRP-Al 5'-TCTAGCCATGGTGACTATTGAGCACG-3 '/ TRP-A3CR: 5 * - ACAGGTACCGATATCAATGCCGTAATC-3' and
  • TRP-B4 5'-TATTGCGGCCGCTTAGATTAAATGG-37
  • TRP-B2CR 5'-CTAGGAATTCGGCACACAGTGG-3 '
  • the amplified fragments correspond to regions 49-390 and 670-1425 within the sequence TRP1 gene (underlying sequence: V01341, Gene Bank). These fragments were inserted into the already described plasmid "pBM21-Ex2" ((Zimmer, T., Kaminski, K., Scheller, U., Vogel, F., and Schunck, W.-H. DNA and Cell Biology 14 (1995 ) 619-628) using the restriction sites PmaCl I EcoRV (TRP-A) or Notl EcoRI (TRP-B) (resulting plasmid: "Ex2-TRAB").
  • the URA3 marker gene was isolated from the plasmid pSEY304 (Bankaitis, VA, Johnson, LM, and Emr, SD Proc.Natl.Acad.Sci. USA 83 (1986) 9075-9079) by restriction with Pvu I / Nru I and in cloned the Eco RV site of the plasmid Ex2-TRAB (resulting plasmid: Ex2-TRAB / URA).
  • the cytochrome b5 cDNA was obtained from the plasmid pCR 2.1 TM (see 1.3.) By restriction isolated with Sal I and Bam HI and cloned into the plasmid Ex2-TRAB / URA opened with the same restrictases (resulting plasmid: pEx2 / Cmb5, see FIG. 4).
  • the cytochrome b5 cDNA used corresponds to SEQ ID No: 2.
  • the integrative expression cassette thus constructed was then isolated from the plasmid pEx2 / Cmb5 by restriction with Pma CI and Nhe I and into the S. cerevisiae strain YS 18 (isogenic to GRF18 but ura3; Sengstag and Hinnen, Gene 62 (1988) 223-228 ) transformed.
  • the strains YSCmb5Cl and YSCmb5C10 constructed in this way and selected for the further experiments each carry an expression cassette which is stably integrated into the yeast genome and which enables galactose-inducible expression of the C. maltosa cytochrome b5.
  • the YSCmb5C10 strain is characterized by an approximately 2.5-fold higher cytochrome b5 expression compared to YSCmb5Cl.
  • yeast strains were then generated which enable the simultaneous expression of cytochrome P450, NADPH-P450 reductase and cytochrome b5.
  • the YSCmb5 strains were transformed with the autonomously replicating plasmids YEp51Cml-R and YEp51Cm2-R. These plasmids have been described previously: Zimmer, T., Kaminski, K., Scheller, U., Vogel, F., and Schunck, W.-H. DNA and Cell Biology 14 (1995) 619-628 and DE 195 07 546 AI).
  • YSCmb5Cl / CmlR and YSCmb5vC10 / CmlR - for coexpression of cytochrome b5, P450 Cml and NADPH-P450 reductase;
  • YSCmb5Cl / Cm2R and YSCmb5C10 / Cm2R - for coexpression of cytochrome b5, P450 Cm2 and NADPH-P450 reductase;
  • reaction mixtures (1 ml) contained in 100 mM KK phosphate buffer pH 7.25, 10 mM KC1, 5 mM MgCl 2 , approx. 0.2 mg microsomal protein, 100 nmol NADPH, a NADPH regenerating system consisting of 3.1 ⁇ mol glucose-6-phosphate and 1 U glucose-6-phosphate dehydrogenase, as well as the substrate either [1- 14 C] dodecane (1000 nmol, lxlO 6 dpm) or [1- 14 C] lauric acid (250 nmol, 5xl0 5 dpm).
  • the reactions were started by adding NADPH; in some experiments NADH (500 nmol) was added at the same time.
  • the rate of NADPH-dependent, P450 Cm2 catalyzed oxidation of lauric acid was increased approximately 4-fold by the presence of cytochrome b5 (see Table 1).
  • cytochrome b5 see Table 1.
  • the synergistic effect of NADPH and NADH only occurred in the presence of cytochrome b5 (see Table 1).
  • cytochrome b5 caused a more than 4-fold increase in the P450 Cml-catalyzed oxidation of dodecane (Table 2).
  • stimulation of the P450 activity can also be achieved by adding purified cytochrome b5 to microsomes that only contained P450 and NADPH-P450 reductase (Table 2).
  • 1-dodecanol and 12-hydroxylauric acid were the main products of the P450-dependent dodecane and lauric acid oxidations, respectively.
  • Fig. 1 Nucleic acid sequence and derived amino acid sequence for the cytochrome b5 of the alkane-utilizing yeast Candida maltosa.
  • Fig. 2 Comparison of the amino acid sequence of the cytochrome b5 from Candida maltosa with the known cytochrome b5 proteins.
  • A - AHgnment with the sequence of the cytochrome b5 from Saccharomyces cerevisiae (Swiss Prot Accession number: P40312);
  • B - AHgnment with the sequence of the human cytochrome b5 (microsomal form, Swiss Prot Accession number: P00167).
  • Fig. 3 Heterologous expression of the Candida maltosa cytochrome b5 cDNA in Saccharomyces cerevisiae. The difference spectra (reduced versus oxidized sample) for the detection of cytochrome b5 in different cell fractions are shown: A: homogenate; B: 10,000 g of supernatant; C: 100,000 g of supernatant; D: 100,000 g sediment (microsomes). # 1 cell fractions of strain GRF18 / YEp51-b5 (expression of the C. maltosa cytochrome b5); # 2- cell fractions of the control strain GRF18 / YEp51; # 0- baseline to # 1.
  • Fig.4 Map of the plasmid pEx2 / Cmb5. This plasmid contains the constructed expression cassette for the heterologous expression of the cytrochrome b5 from Candida maltosa in Saccharomyces cerevisiae.

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Abstract

The invention relates to nucleic acid sequences from Candida yeasts, preferably from Candida maltosa, which code cytochrome b5 polypeptides, as well as to the corresponding cytochrome b5 polypeptides and their use for increasing the activity of cytochrome P450 systems, especially for stimulating the activity of alkane-hydroxylating and fatty acid-hydroxylating cytochrome P450 systems during the production of long-chained dicarboxylic acids (>/=C10).

Description

Nukleinsäure-Sequenzen aus Candida Hefen, die Cytochrom b5-Polypeptide kodierenNucleic acid sequences from Candida yeasts that encode cytochrome b5 polypeptides
Die Erfindung betrifft Nukleinsäure-Sequenzen aus Candida Hefen vorzugsweise aus Candida maltosa, die Cytochrom b5-Polypeptide kodieren sowie die entsprechenden Cytochrom b5-Polypeptide und ihre Verwendung zur Erhöhung der Aktivität von Cytochrom P450 Systemen, insbesondere zur Stimulierung der Aktivität Alkan- und Fettsäure- hydroxylierender Cytochrom P450 Systeme bei der Produktion langkettiger Dicarbonsäuren (> C10)The invention relates to nucleic acid sequences from Candida yeasts, preferably from Candida maltosa, which encode cytochrome b5 polypeptides and the corresponding cytochrome b5 polypeptides and their use for increasing the activity of cytochrome P450 systems, in particular for stimulating the activity of alkane and fatty acid hydroxylating agents Cytochrome P450 systems in the production of long-chain dicarboxylic acids (> C10)
Cytochrome b5 sind Hämoproteine, die an verschiedenen Elektronentransfer-Prozessen in eukaryontischen Zellen beteiligt sind [Übersichten bei: Oshino, N., Pharmac. Ther. A. 2, (1978) 477-515 und Vergeres and Waskell Biochemie 77 (1995) 604-620]. Die Übertragung von Elektronen auf Cytochrom b5 erfolgt durch NADH- bzw. auch NADPH-abhängige Reduktasen. Das reduzierte Cytochrom b5 kann dann das aufgenommene Elektron auf verschiedene andere Proteine übertragen. Zu diesen Elektronenakzeptoren gehören Cytochrome P450, Fettsäure-Desaturasen, Methämoglobin und Methionin-Synthase.Cytochrome b5 are hemoproteins that are involved in various electron transfer processes in eukaryotic cells [reviews by: Oshino, N., Pharmac. Ther. A. 2, (1978) 477-515 and Vergeres and Waskell Biochemie 77 (1995) 604-620]. Electrons are transferred to cytochrome b5 by NADH- or NADPH-dependent reductases. The reduced cytochrome b5 can then transfer the electron taken up to various other proteins. These electron acceptors include cytochrome P450, fatty acid desaturases, methemoglobin and methionine synthase.
Nach ihrer intrazellulären Lokalisierung können drei verschiedene Cytochrom b5 Formen unterschieden werden: a) eine membrangebundene Form, die im Endoplasmatischen Retikulum lokalisiert ist (mikrosomale Form) und dort mit Fettsäure-Desaturasen und Cytochromen P450 wechselwirkt b) eine weitere membrangebundene Form, die aber spezifisch in der äußeren Mitochondrienmembran lokalisiert ist und c) eine lösliche Form des Cytochrom b5, wie sie in Erythrocyten (Reduktion von Methämoglobin) und nach neueren Untersuchungen auch in Leberzellen (reduktive Aktivierung der Methionin-Synthase [Chen and Banerjee, J. Biol. Chem. 2__ (1998) 26248- 26255] vorkommt.After their intracellular localization, three different cytochrome b5 forms can be distinguished: a) a membrane-bound form, which is located in the endoplasmic reticulum (microsomal form) and interacts there with fatty acid desaturases and cytochrome P450 b) another membrane-bound form, but which is specific in is located in the outer mitochondrial membrane and c) a soluble form of cytochrome b5, as found in erythrocytes (reduction of methaemoglobin) and, according to more recent studies, also in liver cells (reductive activation of methionine synthase [Chen and Banerjee, J. Biol. Chem. 2__ (1998) 26248-26255] occurs.
Die membranständigen Formen des Cytochrom b5 bestehen aus einer cytoplasmatischen Domäne (Länge ca. 90 bis 100 Aminosäurereste), einem membranspannenden Segment (ca. 20 überwiegend hydrophobe Aminosäurereste) und einem C-terminalen Teil (ca. 10 hydrophile Aminosäurereste), der nach neueren Untersuchungen luminal orientiert ist [Vergeres et al., J. Biol. Chem. 270 (1995) 3414-3422]. Die cytoplasmatische Domäne enthält die prosthetische Hämgruppe und ist direkt an der Elektronentransfer-Funktion des Cytochrom b5 beteiligt. Die dreidimensionale Struktur der cytoplasmatischen Domäne des mikrosomalen Cytochrom b5 vom Rind ist bekannt [Argos and Mathews J. Biol. Chem. 250 (1975) 747-751]. Danach fungieren zwei Histidinreste als axiale Liganden des Hämeisens. Cytochrome b5 gehören zur Klasse der "tail-anchored" Proteine. Das membranspannende Segment und der sich C-terminal anschließende luminale Teil enthalten alle Determinanten für seine intrazelluläre Lokalisierung. Unterschiede im Vorliegen einzelner geladener Aminosäuren im luminalen Teil bestimmen darüber, ob die jeweilige Cytochrom b5 Form im Endoplasmatischen Retikulum oder in der äußeren Mitochondrienmembran lokalisiert wird [Kuroda et al., J. Biol. Chem. 273 (1998) 31097-31102]. Bei den Cytochrom b5 Proteinen des Endoplasmatischen Retikulums ist darüberhinaus bekannt, daß die Länge des membranspannenden Segments entscheidend für ihre permanente Lokalisierung (Retention) in diesem Zellkompartiment ist [Pedrazzini et al. Proc. Natl. Acad. Sei. USA 93 (1996) 4207- 4212]. Bei der löslichen Form in Erythrozyten handelt es sich um ein C-terminal verkürztes Cytochrom b5, welches dementsprechend über keinen Membrananker verfügt [VanDerMark and Steggles, Biochem. Biophys. Res. Commun. 240 (1997) 80-83].The membrane-related forms of cytochrome b5 consist of a cytoplasmic domain (length approx. 90 to 100 amino acid residues), a membrane-spanning segment (approx. 20 predominantly hydrophobic amino acid residues) and a C-terminal part (approx. 10 hydrophilic amino acid residues), which according to recent studies is luminally oriented [Vergeres et al., J. Biol. Chem. 270 (1995) 3414-3422]. Contains the cytoplasmic domain the prosthetic heme group and is directly involved in the electron transfer function of the cytochrome b5. The three-dimensional structure of the cytoplasmic domain of bovine microsomal cytochrome b5 is known [Argos and Mathews J. Biol. Chem. 250 (1975) 747-751]. Thereafter, two histidine residues act as axial ligands of the hemis. Cytochrome b5 belong to the class of "tail-anchored" proteins. The membrane-spanning segment and the C-terminal luminal part contain all determinants for its intracellular localization. Differences in the presence of individual charged amino acids in the luminal part determine whether the respective cytochrome b5 form is located in the endoplasmic reticulum or in the outer mitochondrial membrane [Kuroda et al., J. Biol. Chem. 273 (1998) 31097-31102]. In the case of the cytochrome b5 proteins of the endoplasmic reticulum, it is also known that the length of the membrane-spanning segment is decisive for their permanent localization (retention) in this cell compartment [Pedrazzini et al. Proc. Natl. Acad. Be. USA 93 (1996) 4207-4212]. The soluble form in erythrocytes is a C-terminally shortened cytochrome b5, which accordingly has no membrane anchor [VanDerMark and Steggles, Biochem. Biophys. Res. Commun. 240 (1997) 80-83].
Die ersten vollständigen Aminosäuresequenzen mikrosomaler Cytochrom b5 Proteine wurden vor etwa 10 Jahren aufgeklärt (vom Menschen und vom Huhn, [Ozols, J. Biochim. Biophys. Acta 997 (1989) 121-130]. Inzwischen sind die cDNAs bzw. Gene für Cytochrome b5 aus mindestens 20 unterschiedlichen Organismen kloniert worden. Die entsprechenden DNA- und abgeleiteten Aminosäuresequenzen sind in verschiedenen Datenbanken über Internet zugänglich. Eine spezielle Zusammenstellung als Zugang zu den publizierten Cytochrom b5 Sequenzen und den entsprechenden Literaturstellen findet sich unter: http://www.icgeb.trieste.it/p450/cytb5.html. Die Sequenz eines menschlichen Cytochrom b5 ist in WO-A-98/36071 beschrieben. Die Sequenz eines Cytochrom b5 aus alkanverwertenden Hefen war bisher unbekannt.The first complete amino acid sequences of microsomal cytochrome b5 proteins were elucidated about 10 years ago (by humans and by chickens, [Ozols, J. Biochim. Biophys. Acta 997 (1989) 121-130]. In the meantime, the cDNAs or genes for cytochrome b5 cloned from at least 20 different organisms The corresponding DNA and derived amino acid sequences are available in various databases on the Internet A special compilation as access to the published cytochrome b5 sequences and the corresponding literature can be found at: http: //www.icgeb. trieste.it/p450/cytb5.html The sequence of a human cytochrome b5 is described in WO-A-98/36071 The sequence of a cytochrome b5 from alkane-utilizing yeast was previously unknown.
Cytochrome b5 spielen eine komplexe Rolle bei der Regulation der enzymatischen Aktivität von mikrosomalen Cytochrom P450 Systemen [Perret and Pompon, Biochemistry 3 Z (1998) 11412-11424 und darin zitierte Literatur]. Je nach untersuchter Cytochrom P450 Form, dem gewählten Substrat und den Reaktionsbedingungen, kann Cytochrom b5 eine Hemmung bzw. beträchtliche Stimulierung der P450-katalysierten Substratumsetzungen bewirken. Die stimulierende Wirkung beruht im allgemeinen auf einer Verbesserung der Kopplung von Elektronentransfer und Substrathydroxylierung im Reaktionszyklus der Cytochrom P450 Systeme. Dabei kann Cytochrom b5 sowohl als Redox-Partner fungieren (Übertragung des zweiten Elektrons im Reaktionszyklus) als auch Konformationsveränderungen am Cytochrom P450 bewirken, die zu einer Erhöhung der Substrataffinität führen. Die Bindungsstellen von Cytochrom b5 und NADPH-P450 Reduktase am Cytochrom P450 sind wahrscheinlich nicht identisch aber möglicherweise partiell überlappend [Bridges et al. J. Biol. Chem. 273 (1998) 17036-17049]. Einige Autoren beschreiben, daß bereits Apo-Cytochrom b5 die Aktivität von einzelnen Cytochrom P450 Systemen stimulieren kann [Yamazaki et al. J. Biol. Chem. 271 (1996) 27438-27444]. Die tatsächlichen Effekte von Cytochrom b5 auf ein konkretes Cytochrom P450 System sind bisher nicht voraussagbar.Cytochrome b5 play a complex role in the regulation of the enzymatic activity of microsomal cytochrome P450 systems [Perret and Pompon, Biochemistry 3 Z (1998) 11412-11424 and literature cited therein]. Depending on the investigated cytochrome P450 form, the substrate selected and the reaction conditions, cytochrome b5 can inhibit or considerably stimulate the P450-catalyzed substrate reactions. The stimulating effect is generally based on an improvement in the coupling of Electron transfer and substrate hydroxylation in the reaction cycle of the cytochrome P450 systems. Cytochrome b5 can act as a redox partner (transfer of the second electron in the reaction cycle) as well as cause conformational changes on the cytochrome P450, which lead to an increase in substrate affinity. The binding sites of cytochrome b5 and NADPH-P450 reductase on cytochrome P450 are probably not identical but may be partially overlapping [Bridges et al. J. Biol. Chem. 273 (1998) 17036-17049]. Some authors describe that apocytochrome b5 can already stimulate the activity of individual cytochrome P450 systems [Yamazaki et al. J. Biol. Chem. 271 (1996) 27438-27444]. The actual effects of cytochrome b5 on a specific cytochrome P450 system have so far not been predictable.
Die Anwendung des menschlichen Cytochrom b5 zur Stimulierung der Aktivität menschlicher Cytochrom P450 Systeme nach heterologer Expression in S. cerevisiae ist aus einer Reihe von Publikationen [Übersicht bei Pompon et al., Toxicol. Lett. 82/83 (1995) 815- 822] und aus WO-A 97/10344 und US 5,635,369 bekannt. Die Frage, ob zur Stimulierung der Aktivität ausgewählter Cytochrom P450 Systeme auch Cytochrome b5 aus anderen Organismen eingesetzt werden können, ist bisher nicht systematisch untersucht. Aufgrund der zum Teil geringen Sequenzhomologien erscheint dies jedoch eher als unwahrscheinlich.The use of human cytochrome b5 to stimulate the activity of human cytochrome P450 systems after heterologous expression in S. cerevisiae has been described in a number of publications [review by Pompon et al., Toxicol. Lett. 82/83 (1995) 815-822] and from WO-A 97/10344 and US 5,635,369. The question of whether cytochrome b5 from other organisms can also be used to stimulate the activity of selected cytochrome P450 systems has not yet been systematically investigated. However, due to the sometimes low sequence homologies, this seems rather unlikely.
Die Fähigkeit von Cytochrom P450 Systemen zur regio- und z.T. auch stereospezifischen Hydroxylierung verschiedenster chemischer Verbindungen ist von erheblichem biotechnologischen Interesse. Ein Anwendungsgebiet ist beispielsweise die Produktion von langkettigen Dicarbonsäuren mit Hilfe Alkan- und Fettsäure-hydroxylierender Cytochrom P450 Systeme. Hierzu patentierte Verfahren beinhalten bisher zwar bereits eine Überexpression der unmittelbaren Komponenten dieser Cytochrom P450 Systeme (P450 und NADPH-P450 Reduktase) in alkanverwertenden Hefen (Candida tropicalis, WO-A 91/14781) bzw. in Saccharomyces cerevisiae (DE 195 07 546 AI). Zum Einsatz von Cytochrom b5 in derartigen Verfahren liegen aber weder publizierte Ergebnisse noch Patente vor.The ability of cytochrome P450 systems to regio- and partially. Stereospecific hydroxylation of various chemical compounds is also of considerable biotechnological interest. One area of application is, for example, the production of long-chain dicarboxylic acids using alkane and fatty acid-hydroxylating cytochrome P450 systems. Processes patented for this purpose already include overexpression of the direct components of these cytochrome P450 systems (P450 and NADPH-P450 reductase) in alkane-utilizing yeasts (Candida tropicalis, WO-A 91/14781) or in Saccharomyces cerevisiae (DE 195 07 546 AI) . However, there are no published results or patents on the use of cytochrome b5 in such processes.
Der Erfindung lag deshalb die Aufgabe zugrunde, Nukleinsäuresequenzen zu finden, die die Produktion von Cytochrom b5-Polypeptiden ermöglichen, welche die Aktivität von Alkan- und Fettsäure-hydroxylierenden P450-Enzymen stimulieren und damit die Rate und Effizienz der Produktion von Dicarbonsäuren erhöhen. Die Aufgabe konnte durch Nukleinsäure-Sequenzen aus Candida Hefen, vorzugsweise aus Candida maltosa, die Cytochrom b5-Polypeptide kodieren, sowie deren Fragmente, Varianten und Mutationen gelöst werden.The object of the invention was therefore to find nucleic acid sequences which enable the production of cytochrome b5 polypeptides, which stimulate the activity of alkane and fatty acid hydroxylating P450 enzymes and thus increase the rate and efficiency of the production of dicarboxylic acids. The task was solved by nucleic acid sequences from Candida yeasts, preferably from Candida maltosa, which encode cytochrome b5 polypeptides, and their fragments, variants and mutations.
Erfindungsgemäß wurde C. maltosa Cytochrom b5 gereinigt, seine vollständige cDNA wurde kloniert und die Bildung eines funktionsfähigen Cytochrom b5 durch heterologe Expression der Monierten cDNA nachgewiesen.According to the invention, C. maltosa cytochrome b5 was purified, its complete cDNA was cloned and the formation of a functional cytochrome b5 was detected by heterologous expression of the cloned cDNA.
Überraschend konnte ein spezifischer Vorteil des Cytochrom b5 aus C. maltosa mit seiner erfindungsgemäßen Sequenz erreicht werden, da es zur Stimulierung der Aktivität von Cytochrom P450 Systemen aus dem gleichen Organismus bzw. aus verwandten alkanverwertenden Hefen eingesetzt werden kann.Surprisingly, a specific advantage of cytochrome b5 from C. maltosa with its sequence according to the invention could be achieved, since it can be used to stimulate the activity of cytochrome P450 systems from the same organism or from related alkane-utilizing yeasts.
Gegenstand der Erfindung sind somit Nukleinsäure-Sequenzen aus alkanverwertenden Candida Hefen, die ein Cytochrom b5-Polypeptid kodieren sowie ihre Fragmente, Varianten und Mutationen, insbesondere die entsprechende cDNA.The invention thus relates to nucleic acid sequences from alkane-utilizing Candida yeasts which encode a cytochrome b5 polypeptide and their fragments, variants and mutations, in particular the corresponding cDNA.
Vorzugsweise handelt es sich um die Nukleinsäure-Sequenzen mit der SEQ ID No. 1 und SEQ ID No. 2 oder deren Varianten.These are preferably the nucleic acid sequences with SEQ ID No. 1 and SEQ ID No. 2 or their variants.
Desweiteren betrifft die Erfindung Cytochrom b5-Polypeptide, die von den Nukleinsäure- Sequenzen kodiert werden, vorzugsweise ein Cytochrom b5-Polypeptid der SEQ ID No. 3 sowie Varianten davon, die Deletionen oder Modifikationen aufweisen und Cytochrom b5- Aktivitäten aufweisen.Furthermore, the invention relates to cytochrome b5 polypeptides which are encoded by the nucleic acid sequences, preferably a cytochrome b5 polypeptide of SEQ ID No. 3 and variants thereof which have deletions or modifications and have cytochrome b5 activities.
Außerdem betrifft die Erfindung Plasmide und Vektoren, die eine erfindungsgemäße Nukleinsäure-Sequenz enthalten sowie Wirtszellen, die einen Vektor aufweisen und Antikörper, die spezifisch an die Polypeptide binden.In addition, the invention relates to plasmids and vectors which contain a nucleic acid sequence according to the invention, as well as host cells which have a vector and antibodies which bind specifically to the polypeptides.
Erfindungsgemäß werden die Nukleinsäuresequenzen oder Polypeptide zur Stimulierung der Aktivität von Cytochrom P50-Enzymen bei der biotechnologischen Herstellung von Fettsäuren und Dicarbonsäuren durch Oxidation von langkettigen n-Alkanen (> CIO) verwendet. Mit der Erfindung wird u. a. bei der Oxidation von Dodekan unter Verwendung von P450 Cml aus Candida maltosa eine mehr als 4fache Stimulierung erreicht, bei der Oxidation von Laurinsäure unter Verwendung von P450 Cm2 aus Candida maltosa eine mehr als 7fache Stimulierung.According to the invention, the nucleic acid sequences or polypeptides are used to stimulate the activity of cytochrome P50 enzymes in the biotechnological production of fatty acids and dicarboxylic acids by oxidation of long-chain n-alkanes (> CIO). With the invention, among other things, more than 4-fold stimulation is achieved in the oxidation of dodecane using P450 Cml from Candida maltosa, and more than 7-fold stimulation in the oxidation of lauric acid using P450 Cm2 from Candida maltosa.
Anschließend wird die Erfindung an Beispielen näher erläutert, auf die sie jedoch nicht beschränkt sein soll.The invention is subsequently explained in more detail using examples, to which, however, it should not be restricted.
Ausführungsbeispieleembodiments
1. Klonierung und heterologe Expression der vollständigen codierenden Region für das Cytochrom b5 der alkanverwertenden Hefe Candida maltosa1. Cloning and heterologous expression of the complete coding region for the cytochrome b5 of the alkane-utilizing yeast Candida maltosa
Wie nachfolgend im Einzelnen beschrieben wurde das mikrosomale Cytochrom b5 der alkanverwertenden Hefe C. maltosa gereinigt, tryptisch gespalten und hinsichtlich seiner Aminosäuresequenz partiell charakterisiert. Die Sequenz eines der tryptischen Peptide bildete die Grundlage für die Ableitung von degenerierten Oligonucleotiden zur Amplifikation von Teilsequenzen der Cytochrom b5 cDNA mit Hilfe der Polymerase-Kettenreaktion (PCR). Ausgehend von den erhaltenen Sequenzen erfolgte dann die Amplifikation und Klonierung der vollständigen codierenden Region. Das aus den DNA-Sequenzen abgeleitete Protein besteht aus 126 Aminosäuren, beinhaltet die Teilsequenzen wie sie für das aus C. maltosa gereinigte Cytochrom b5 ermittelt wurden und weist eine Homologie von etwa 30 % zu Cytochromen b5 aus andereren Organismen auf. Als weiterer Beweis für die Identität der Monierten cDNA wurde gezeigt, daß die heterologe Expression in S. cerevisiae zur Bildung eines spektral intakten, mikrosomalen Cytochrom b5 führt.As described in detail below, the microsomal cytochrome b5 of the alkane-utilizing yeast C. maltosa was purified, tryptically cleaved and partially characterized in terms of its amino acid sequence. The sequence of one of the tryptic peptides formed the basis for the derivation of degenerate oligonucleotides for the amplification of partial sequences of the cytochrome b5 cDNA using the polymerase chain reaction (PCR). The complete coding region was then amplified and cloned on the basis of the sequences obtained. The protein derived from the DNA sequences consists of 126 amino acids, contains the partial sequences as determined for the cytochrome b5 purified from C. maltosa and has a homology of approximately 30% to cytochrome b5 from other organisms. As further proof of the identity of the cloned cDNA, it was shown that the heterologous expression in S. cerevisiae leads to the formation of a spectrally intact, microsomal cytochrome b5.
1. 1. Reinigung des Cytochrom b5 Proteins1. 1. Purification of the cytochrome b5 protein
Der Hefestamm C. maltosa ATCC 28140 wurde in einem 5 1 Bioreaktor auf Hexadekan als Kohlenstoffquelle kultiviert. Die Medienzusammensetzung und die allgemeinen Wachstumsbedingungen waren wie bei Huth et al., J. Basic Microbiol. __ (1990) 481-488, beschrieben. Die Hefezellen wurden kurz vor Eintritt in die stationäre Wachstumsphase geerntet. Der mechanische Zellaufschluß und die Gewinnung der mikrosomalen Membranfraktion erfolgten wie früher beschrieben [Riege et al. Biochem. Biophys. Res. Commun. 98 (1981) 527-534]. Der mikrosomale Cytochrom b5 Gehalt lag bei ca. 0.5 nmol/mg Protein. Die Reinigung des Cytochrom b5 erfolgte in Anlehnung an Methoden wie sie für Cytochrome b5 aus anderen Organismen publiziert wurden [Guzov et al. J. Biol. Chem. 271 (1996) 26637-26645; Strittmatter et al., Meth. Enzymol. 52 (1978) 97-101].The yeast strain C. maltosa ATCC 28140 was cultivated in a 5 1 bioreactor on hexadecane as a carbon source. The media composition and general growth conditions were as in Huth et al., J. Basic Microbiol. __ (1990) 481-488. The yeast cells were harvested shortly before entering the stationary growth phase. The mechanical cell disruption and the extraction of the microsomal Membrane fractions were carried out as previously described [Riege et al. Biochem. Biophys. Res. Commun. 98 (1981) 527-534]. The microsomal cytochrome b5 content was approx. 0.5 nmol / mg protein. The purification of the cytochrome b5 was carried out in accordance with methods as were published for cytochrome b5 from other organisms [Guzov et al. J. Biol. Chem. 271 (1996) 26637-26645; Strittmatter et al., Meth. Enzymol. 52 (1978) 97-101].
Im Ergebnis wurde ein Cytochrom b5 Präparat mit einer Reinheit von ca. 60 % erhalten (das Verhältnis der Absorptionen bei 412 und 280 nm lag bei ca. 1.4). Das gereinigte Cytochrom b5 zeichnet sich durch folgende spektrale Eigenschaften aus: Absorptionsmaxima bei 412 (Sortebande), 525 und 527 nm im oxidierten Zustand; Verschiebung des Soretmaximums auf 424 nm nach Reduktion mit Dithionit.As a result, a cytochrome b5 preparation with a purity of approx. 60% was obtained (the ratio of the absorptions at 412 and 280 nm was approx. 1.4). The purified cytochrome b5 is characterized by the following spectral properties: absorption maxima at 412 (type band), 525 and 527 nm in the oxidized state; Shift of the Soret maximum to 424 nm after reduction with dithionite.
1.2. Bestimmung von partiellen Aminosäuresequenzen für das gereinigte Cytochrom b51.2. Determination of partial amino acid sequences for the purified cytochrome b5
Das unter 1.1. erhaltene Präparat wurde in einem 15 %-igen SDS-Polyacrylamidgel aufgetrennt. Die erhaltenen Hauptbanden mit einem scheinbaren Molekulargewicht von 17.5 und 20 kDa wurden dann auf eine Polyvinylidendifluorid-Membran geblottet (Pro Blott, Applied Biosystems, USA) und in situ mit Trypsin gespalten [Methode nach: Fernandez et al. Anal. Biochem. 218 (1994) 112 -117]. Die tryptischen Peptide wurden eluiert und über eine Reverse-phase-HPLC an einer Mikrosäule (RPC C2/C18 SC; 100 x 2.1 mm) aufgetrennt (Smart System, Pharmacia, Sweden). Ausgewählte Fraktionen wurden dann auf Polybren- haltige Glasfaser-Filter aufgetragen und sequenziert (Gasphasen- Sequenator, Procise, Applied Biosystems).That under 1.1. The preparation obtained was separated in a 15% SDS polyacrylamide gel. The main bands obtained with an apparent molecular weight of 17.5 and 20 kDa were then blotted onto a polyvinylidene difluoride membrane (Pro Blott, Applied Biosystems, USA) and cleaved in situ with trypsin [method according to: Fernandez et al. Anal. Biochem. 218 (1994) 112-117]. The tryptic peptides were eluted and separated by reverse phase HPLC on a microcolumn (RPC C2 / C18 SC; 100 x 2.1 mm) (Smart System, Pharmacia, Sweden). Selected fractions were then applied to polybrene-containing glass fiber filters and sequenced (gas phase sequencer, procise, applied biosystems).
Im Ergebnis wurden folgende Peptidsequenzen erhalten:As a result, the following peptide sequences were obtained:
T27 (abgeleitet von der 20 kDa-Bande): LYIGNLKT27 (derived from the 20 kDa band): LYIGNLK
T53 (abgeleitet von der 20 kDa-Bande): VYDITSYIDEHPGGET53 (derived from the 20 kDa band): VYDITSYIDEHPGGE
T54 (abgeleitet von der 17.5 kDa-Bande): VYDITSYIDET54 (derived from the 17.5 kDa band): VYDITSYIDE
Die Peptide T53 und T54 stimmen in der Sequenz von 10 Aminosäuren überein. Dieses Ergebnis zeigt an, daß es sich bei der 17.5 kDa-Bande wahrscheinlich um ein proteolytisches Fragment der 20 kDa-Bande handelt. 1.3. PCR-Ampliflkation der Cytochrom b5 cDNAThe peptides T53 and T54 match in the sequence of 10 amino acids. This result indicates that the 17.5 kDa band is probably a proteolytic fragment of the 20 kDa band. 1.3. PCR amplification of the cytochrome b5 cDNA
Als Template für die PCR-Amplifikation von Cytochrom b5 cDNA-Fragmenten diente zunächst der Plasmidpool aus einer bereits früher hergestellten C. maltosa EH 15 cDNA-Bank [Schunck et al., Biochem Biophys. Res. Commun. 181 (1989) 843-850]. Die in dieser Bank enthaltenen cDNAs sind in den Hind II-Ort des Plasmids pUC119 inseriert. Cytochrom b5-spezifische Primer wurden aus der Sequenz des Peptids T53 abgeleitet und in Kombination mit Plasmid-abgeleiteten Primern für die PCR eingesetzt. Folgende Primer wurden für die nachstehenden Experimente erfolgreich verwendet:The template for the PCR amplification of cytochrome b5 cDNA fragments was initially the plasmid pool from a previously produced C. maltosa EH 15 cDNA library [Schunck et al., Biochem Biophys. Res. Commun. 181: 843-850 (1989)]. The cDNAs contained in this library are inserted into the Hind II site of the plasmid pUC119. Cytochrome b5-specific primers were derived from the sequence of the peptide T53 and used in combination with plasmid-derived primers for the PCR. The following primers were successfully used for the following experiments:
T53-1-4: 5'- GAT/C ATT/C ACT/C AGT/C TAT/C ATT/C GAT/C GAA C -3' T53-2-2: 5'- ATT/C GAT GAA CAT/C CCA/T GGT/A GG -3' pUCl 19-2CR: 5'- ACG GCC AGT GAA TTC GAG -3'T53-1-4: 5'- GAT / C ATT / C ACT / C AGT / C TAT / C ATT / C GAT / C GAA C -3 'T53-2-2: 5'- ATT / C GAT GAA CAT / C CCA / T GGT / A GG -3 'pUCl 19-2CR: 5'- ACG GCC AGT GAA TTC GAG -3'
Die Kombination von T53-1-4 mit pUCl 19-2CR führte zur Amplifikation eines etwa 380 bp großen DNA-Fragments. Die dazu durchgeführte PCR (Ready-To-Go PCR Kit, Amersham Pharmacia Biotech) verlief über 35 Zyklen bestehend aus den Schritten: 95 °C, 45 sec / 53 °C, 45 sec / 72 °C, 45 sec (+ 1 sec pro Zyklus). Zur Erhöhung der Spezifität wurde das erhaltene DNA-Fragment einer zweiten PCR mit der Primerkombination T53-2-2 (3' versetzt gegenüber T53-1-4) / pUC119-2CR unterworfen (25 Zyklen, gleiches Temperaturregime). Das so amplifizierte Fragment wurde sequenziert.The combination of T53-1-4 with pUCl 19-2CR led to the amplification of an approximately 380 bp DNA fragment. The PCR (Ready-To-Go PCR Kit, Amersham Pharmacia Biotech) carried out over 35 cycles consisting of the steps: 95 ° C, 45 sec / 53 ° C, 45 sec / 72 ° C, 45 sec (+ 1 sec per cycle). To increase the specificity, the DNA fragment obtained was subjected to a second PCR with the primer combination T53-2-2 (3 'offset from T53-1-4) / pUC119-2CR (25 cycles, same temperature regime). The fragment amplified in this way was sequenced.
Für die anschließende Amplifikation des fehlenden 5'-Bereichs der Cytochrom b5 cDNA wurde folgendes Primerpaar eingesetzt:The following primer pair was used for the subsequent amplification of the missing 5 'region of the cytochrome b5 cDNA:
T53-4CR: 5'- GTTTTA TTT CTA AGG GTT TTG GAG -3' pUCl19-1: 5'- ACA GCTATG ACC ATG ATT ACG -3'T53-4CR: 5'- GTTTTA TTT CTA AGG GTT TTG GAG -3 'pUCl19-1: 5'- ACA GCTATG ACC ATG ATT ACG -3'
T53-4CR wurde aus der Sequenz des zuerst amplifϊzierten Fragments abgeleitet und setzt am 3'-Ende der vermuteten Cytochrom b5 cDNA an. Die PCR verlief über 30 Zyklen bestehend aus den Schritten: 95 °C, 45 sec / 55 °C, 45 sec / 72 °C, 45 sec. Das erhaltene Produkt mit einer Länge von ca. 400 bp wurde sequenziert. Die Sequenz zeigte eine offenen Leserahmen für ein Polypeptid mit 126 Aminosäuren.T53-4CR was derived from the sequence of the first amplified fragment and attaches to the 3 'end of the suspected cytochrome b5 cDNA. The PCR was carried out over 30 cycles consisting of the steps: 95 ° C., 45 sec / 55 ° C., 45 sec / 72 ° C., 45 sec. The product obtained with a length of approx. 400 bp was sequenced. The sequence showed an open reading frame for a polypeptide with 126 amino acids.
Im letzten Schritt erfolgte die Gewinnung der vollständigen Cytochrom b5 cDNA mit Hilfe des folgenden Primerpaars:In the last step, the complete cytochrome b5 cDNA was obtained using the following primer pair:
T53-7Sal: 5'- GAA GTC GAC GTC ATG TCT GAT ACT ACA -3' T53-8BamCR: 5'- CGC GGA TCC ATT ATG TTT TAT TTC TAA G -3'T53-7Sal: 5'- GAA GTC GAC GTC ATG TCT GAT ACT ACA -3 'T53-8BamCR: 5'- CGC GGA TCC ATT ATG TTT TAT TTC TAA G -3'
Diese Primer setzen am 5'- bzw. 3'-Ende der Sequenz des oben beschriebenen 400 bp Fragments an. Die unterstrichenen Sequenzbereiche sind hinzugefügte Restriktionsorte für Sal I bzw. Bam HI zur anschließenden Umklonierung in den Hefe-Expressionsvektor YEp51 (siehe Punkt 1.5).These primers start at the 5 'or 3' end of the sequence of the 400 bp fragment described above. The underlined sequence areas are added restriction sites for Sal I or Bam HI for subsequent cloning into the yeast expression vector YEp51 (see point 1.5).
Als Template diente die Poly(A)RNA aus Zellen des C. maltosa Stammes ATCC 28140 nach Wachstum auf Hexadekan. Die Isolierung der RNA erfolgte nach mechanischem Zellaufschluß mit Hilfe des Oligotex Direct mRNA Mini Kits (QIAGEN). Für die Umschreibung in einzelsträngige cDNA und die anschließende Amplifikation der Cytochrom b5 cDNA wurde der Ready To Go RT-PCR Kit von Amersham Pharmacia Biotech verwendet. Zur reversen Transkription (30 min, 42 °C) wurden 100 ng Poly(A)RNA und ein Oligo(dT) Primer eingesetzt. Nach einer Inaktivierung für 5 min bei 95 °C erfolgte bei 65 °C die Zugabe der Cytochrom b5-spezifischen Primer (T53-7Sal und T53-8Bam). Die anschließende PCR-Amplifikation verlief über 35 Zyklen bestehend aus den Schritten: 95 °C, 45 sec / 53 °C, 45 sec / 72°C, 45 sec (+ 1 sec pro Zyklus).The poly (A) RNA from cells of the C. maltosa strain ATCC 28140 served as template after growth on hexadecane. The RNA was isolated after mechanical cell disruption using the Oligotex Direct mRNA Mini Kit (QIAGEN). The Ready To Go RT-PCR kit from Amersham Pharmacia Biotech was used for the transcription into single-stranded cDNA and the subsequent amplification of the cytochrome b5 cDNA. 100 ng poly (A) RNA and an oligo (dT) primer were used for reverse transcription (30 min, 42 ° C.). After inactivation for 5 min at 95 ° C., the cytochrome b5-specific primers (T53-7Sal and T53-8Bam) were added at 65 ° C. The subsequent PCR amplification was carried out over 35 cycles consisting of the steps: 95 ° C, 45 sec / 53 ° C, 45 sec / 72 ° C, 45 sec (+ 1 sec per cycle).
Für die nachfolgende Klonierung des erhaltenen RT-PCR Produkts wurde der TOPO T-A Cloning Kit der Firma INVITROGEN verwendet. Entsprechend den Instruktionen des Herstellers erfolgte eine Ligation des RT-PCR Produkts in das Plasmid pCR 2.1™ und eine anschließende Transformation in E. coli TOP 10.The TOPO T-A cloning kit from INVITROGEN was used for the subsequent cloning of the RT-PCR product obtained. According to the manufacturer's instructions, the RT-PCR product was ligated into the plasmid pCR 2.1 ™ and then transformed into E. coli TOP 10.
1.4. Charakterisierung der Cytochrom b5 cDNA Sequenz1.4. Characterization of the cytochrome b5 cDNA sequence
Zur Sequenzierung wurden insgesamt 21 Klone zufällig ausgewählt, die Plasmide mit einem RT-PCR Insert trugen. Alle sequenzierten Inserts enthielten einen offenen Leserahmen von 378 bp. Signifikante Unterschiede in den analysierten Sequenzen traten an zwei Positionen (147 und 156 gerechnet vom Start-ATG) auf: T(147)-C(156) bei 8 Klonen im Gegensatz zu C(147)-T(156) bei den übrigen. Dieses Ergebnis deutet auf das Vorliegen von zwei allelen Varianten des Cytochrom b5 Gens in C. maltosa hin. Die unterschiedlichen Basen an den Positionen 147 und 156 haben jedoch keinen Einfluß auf die Aminosäuresequenz des kodierten Proteins.A total of 21 clones were randomly selected for sequencing and carried plasmids with an RT-PCR insert. All sequenced inserts contained an open reading frame from 378 bp. Significant differences in the analyzed sequences occurred at two positions (147 and 156 calculated from the start ATG): T (147) -C (156) in 8 clones in contrast to C (147) -T (156) in the others. This result indicates the presence of two allelic variants of the cytochrome b5 gene in C. maltosa. However, the different bases at positions 147 and 156 have no influence on the amino acid sequence of the encoded protein.
Die Monierten cDNAs (SEQ ID No. 1 und 2) kodieren für ein Polypeptid (SEQ ID No. 3) mit einer Länge von 126 Aminosäuren (Abb. 1). Die für das gereinigte Cytochrom b5 aus C. maltosa ermittelten Peptidsequenzen (vgl. Punkt 2) stimmen exakt mit den Regionen 31 bis 45 (T53 Peptid) bzw. 76 bis 82 (T27 Peptid) in der abgeleiteten Aminosäuresequenz überein (Abb. 1).The cloned cDNAs (SEQ ID No. 1 and 2) code for a polypeptide (SEQ ID No. 3) with a length of 126 amino acids (FIG. 1). The peptide sequences (see point 2) determined for the purified cytochrome b5 from C. maltosa correspond exactly with the regions 31 to 45 (T53 peptide) and 76 to 82 (T27 peptide) in the derived amino acid sequence (Fig. 1).
Die ermittelte Aminosäuresequenz des C. maltosa Cytochrom b5 zeigt eine mäßige aber signifikante Homologie zu der von Cytochromen b5 aus anderen Organismen. Die Sequenzidentitäten betragen beispielsweise 33 % beim Vergleich mit dem menschlichen Protein und überraschenderweise auch nicht mehr als 37 % beim Vergleich mit dem bisher einzigen bekannten Cytochrom b5 aus einer anderen Hefeart (S. cerevisiae). Die entsprechenden Alignments sind in Abb. 2 dargestellt.The determined amino acid sequence of C. maltosa cytochrome b5 shows a moderate but significant homology to that of cytochrome b5 from other organisms. The sequence identities are, for example, 33% when compared with the human protein and surprisingly not more than 37% when compared with the only known cytochrome b5 from another type of yeast (S. cerevisiae). The corresponding alignments are shown in Fig. 2.
1.5. Heterologe Expression des Candida maltosa Cytochrom b5 in Saccharomyces cerevisiae1.5. Heterologous expression of the Candida maltosa cytochrome b5 in Saccharomyces cerevisiae
Die Cytochrom b5 cDNA wurde aus einem der sequenzierten Klone SEQ ID No. 2 durch Restriktionsspaltung mit Sal I und Bam HI isoliert und in den Hefeexpressionsvektor YEp51 [Broach et al., In: Experimental Manipulation of Gene Expression, M. Inoye, ed. Academic Press, N.Y, 1983, pp. 83-117] umkloniert. Das so konstruierte Plasmid enthielt die Cytochrom b5 cDNA unter Kontrolle des Galaktose-induzierbaren GALIO-Promotors. und wurde dann zur Transformation des S. cerevisiae Stammes GRF18 (α, his 3-11, his 3-15, leu 2-3, leu 2-112, can1) eingesetzt. Die Kultivierung der Transformanden (GRF18/YEp51-b5) in einem Raffmose-haltigen Medium und die Induktion der Expression der Cytochrom b5 cDNA durch Zugabe von Galaktose erfolgte analog zu früheren Arbeiten zur Expression von Cytochromen P450 in diesem Wirts/Vektor- System [Menzel et al, Arch. Biochem. Biophys. 330 (1996) 97-109]. Als Kontrolle dienten parallel durchgeführte Kulturen des gleichen Hefestammes nach Transformation mit dem Ausgangsplasmid (GRF18/YEp51).The cytochrome b5 cDNA was obtained from one of the sequenced clones SEQ ID No. 2 isolated by restriction cleavage with Sal I and Bam HI and into the yeast expression vector YEp51 [Broach et al., In: Experimental Manipulation of Gene Expression, M. Inoye, ed. Academic Press, NY, 1983, pp. 83-117]. The plasmid thus constructed contained the cytochrome b5 cDNA under the control of the galactose-inducible GALIO promoter. and was then used to transform the S. cerevisiae strain GRF18 (α, his 3-11, his 3-15, leu 2-3, leu 2-112, can 1 ). The cultivation of the transformants (GRF18 / YEp51-b5) in a medium containing raffmose and the induction of the expression of the cytochrome b5 cDNA by adding galactose was carried out analogously to previous work on the expression of cytochrome P450 in this host / vector system [Menzel et al, Arch. Biochem. Biophys. 330 (1996) 97-109]. Cultures of the same carried out in parallel served as controls Yeast strain after transformation with the starting plasmid (GRF18 / YEp51).
Die Zellen wurden 24 h nach Zugabe der Galaktose geerntet und mechanisch mit Glaskugeln aufgeschlossen (in 100 mM Tris/HCl-Puffer pH 7.7 mit 5 mM EDTA und einem Protease- Inhibitor-Cocktail, Boehringer-Mannheim). Die anschließende differentielle Zentrifugation beinhaltete folgende Schritte: 15 min, 3000 g; 15 min, 10 000 g; 90 min, 100 000 g. Das 100 000 g Sediment (Mikrosomen) wurde in dem oben angegebenen Tris-Puffer resuspendiert.The cells were harvested 24 hours after the galactose had been added and mechanically disrupted with glass balls (in 100 mM Tris / HCl buffer pH 7.7 with 5 mM EDTA and a protease inhibitor cocktail, Boehringer-Mannheim). The subsequent differential centrifugation comprised the following steps: 15 min, 3000 g; 15 min, 10,000 g; 90 min, 100,000 g. The 100,000 g sediment (microsomes) was resuspended in the Tris buffer indicated above.
Der Cytochrom b5 Gehalt wurde in den einzelnen Zellfraktionen spektral bestimmt: Dazu wurden die Differenzspektren zwischen der Dithionit-reduzierten und der mit H2O2 (Endkonzentration: 0.003 %)-oxidierten Probe aufgenommen. Zur Berechnung diente der für Cytochrom b5 Proteine übliche Extinktionskoeffizient von 185 mJVT'xcm"1 für die Differenz der Absorptionen bei 424 und 409 nm [Estabrook and Werringloer, Meth. Enzymol. 52 (1978) 212-221].The cytochrome b5 content was determined spectrally in the individual cell fractions: For this purpose, the difference spectra between the dithionite-reduced and the sample oxidized with H 2 O 2 (final concentration: 0.003%) were recorded. The extinction coefficient of 185 mJVT'xcm "1, which is customary for cytochrome b5 proteins, was used for the calculation of the difference in the absorptions at 424 and 409 nm [Estabrook and Werringloer, Meth. Enzymol. 52 (1978) 212-221].
Die erhaltenen Ergebnisse (Abb. 3) belegen, daß die unter Kontrolle des GALIO-Promotors exprimierte cDNA tatsächlich für ein mikrosomales Cytochrom b5 kodiert. Aus den Differenzspektren der Homogenate kann eine Expressionshöhe von ca. 300 bis 400 nmol Cytochrom b5 pro 1 Kultur abgeschätzt werden. Die aus dem Stamm GRF18/YEp51-b5 isolierten Mikrosomen hatten einen Cytochrom b5 Gehalt von ca. 0.5 nmol/mg Protein. Der Gehalt an wirtseigenem Cytochrom b5 war unter den gewählten Versuchsbedingungen wesentlich geringer und lag an der Nachweisgrenze wie die Differenzspektren mit den entsprechenden Zellfraktionen des Kontrollstammes GRF18/YEp51 zeigen (Abb. 3).The results obtained (Fig. 3) show that the cDNA expressed under the control of the GALIO promoter actually codes for a microsomal cytochrome b5. An expression level of approx. 300 to 400 nmol cytochrome b5 per 1 culture can be estimated from the difference spectra of the homogenates. The microsomes isolated from the strain GRF18 / YEp51-b5 had a cytochrome b5 content of approx. 0.5 nmol / mg protein. The content of the host's own cytochrome b5 was significantly lower under the chosen test conditions and was at the detection limit, as shown by the difference spectra with the corresponding cell fractions of the control strain GRF18 / YEp51 (Fig. 3).
1.6. Reinigung des heterolog exprimierten Candida maltosa Cytochrome b51.6. Purification of the heterologously expressed Candida maltosa cytochrome b5
Das heterolog exprimierte mikrosomale Cytochrom b5 (vgl 1.5.) wurde mit Hilfe von Detergentien solubilisiert und durch Chromatographie an DEAE-Sepharose, Hydroxyapatit und DEAE-Toyoperl gereinigt. Das gereinigte Protein zeigte ein scheinbares Molekulargewicht von etwa 20 kDa. Bei der Bestimung der N-terminalen Aminosäuresequenz wurden folgende zwei Sequenzen ermittelt:The heterologously expressed microsomal cytochrome b5 (see 1.5.) Was solubilized with the aid of detergents and purified by chromatography on DEAE-Sepharose, hydroxyapatite and DEAE-Toyoperl. The purified protein showed an apparent molecular weight of about 20 kDa. The following two sequences were determined when determining the N-terminal amino acid sequence:
Met-Ser-Asp-Thr-Thr-Thr-Thr-Val-Tyr-Asp-Tyr-Glu-Glu-Ile-Ser-... Ser-Asp-Thr-Thr-Thr-Thr-Val-Tyr-Asp-Tyr-Glu-Glu-Ile-Ser-Lys-... Diese unterscheiden sich lediglich durch die An- bzw. Abwesenheit des Start-Methionins, und stimmen exakt mit der aus der cDNA-Sequenz abgeleiteten Aminosäuresequenz des C. maltosa Cytochrom b5 überein. Dieses Ergebnis bestätigt eindeutig die Identität des heterolog exprimierten Proteins.Met-Ser-Asp-Thr-Thr-Thr-Thr-Val-Tyr-Asp-Tyr-Glu-Glu-Ile-Ser -... Ser-Asp-Thr-Thr-Thr-Thr-Val-Tyr-Asp -Tyr-Glu-Glu-Ile-Ser-Lys -... These differ only in the presence or absence of the start methionine, and exactly match the amino acid sequence of the C. maltosa cytochrome b5 derived from the cDNA sequence. This result clearly confirms the identity of the heterologously expressed protein.
2. Stimulierung der Aktivität von Alkan- und Fettsäure-oxidierenden Cytochrom P450 Enzymen durch das C. maltosa Cytochrom b52. Stimulation of the activity of alkane and fatty acid oxidizing cytochrome P450 enzymes by the C. maltosa cytochrome b5
Wie nachfolgend im Einzelnen beschrieben wurde der Einfluß des C. maltosa Cytochrom b5 auf die Aktivität von zwei C. maltosa Cytochrom P450 Formen untersucht. Diese werden als P450 Cml und P450 Cm2 bezeichnet (Schunck et al., Eur. J. Cell Biol. 55 (1991) 336-345). Sie bilden bei Rekonstitution mit der ebenfalls aus C. maltosa Monierten NADPH-P450 ReduMase enzymatisch aktive Monooxy genäse- Systeme, welche die Oxidation verschiedener n-Alkane und Fettsäuren katalysieren (Scheller, U., Zimmer, T., Kärgel, E. and Schunck, W.- H. Arch. Biochem. Biophys. _2_ (1996) 245-254). Sowohl P450 Cml als auch P450 Cm2 können nicht nur die primäre terminale Hydroxylierung von Alkanen und Fettsäuren katalysieren, sondern auch alle weiteren Oxidationsschritte bis zur Bildung der entsprechenden Dicarbonsäuren (Scheller, U., Zimmer, T., Becher, D., Schauer, F., Schunck, W.-H., J. Biol. Chem. 211 (1998) 32528-32534).As described in detail below, the influence of the C. maltosa cytochrome b5 on the activity of two C. maltosa cytochrome P450 forms was investigated. These are referred to as P450 Cml and P450 Cm2 (Schunck et al., Eur. J. Cell Biol. 55 (1991) 336-345). When reconstituted with NADPH-P450 ReduMase, which is also cloned from C. maltosa, they form enzymatically active monooxy genesis systems which catalyze the oxidation of various n-alkanes and fatty acids (Scheller, U., Zimmer, T., Kärgel, E. and Schunck , W.-H. Arch. Biochem. Biophys. _2_ (1996) 245-254). Both P450 Cml and P450 Cm2 can not only catalyze the primary terminal hydroxylation of alkanes and fatty acids, but also all further oxidation steps up to the formation of the corresponding dicarboxylic acids (Scheller, U., Zimmer, T., Becher, D., Schauer, F ., Schunck, W.-H., J. Biol. Chem. 211 (1998) 32528-32534).
Um Enzymsysteme bestehend aus P450 Cml bzw. P450 Cm2 und NADPH-P450 Reduktase zu rekonstituieren und den Einfluß von Cytochrom b5 zu testen, wurden diese drei Komponenten in S. cerevisiae coexprimiert. Das dazu entwickelte System ist unter 2.1. erläutert. Als Kontrollen dienten S. cerevisiae Zellen, in denen P450 und Reduktase ohne Cytochrom b5 exprimiert wurden. Anschließend erfolgte eine Bestimmung der resultierenden Enzymaktivitäten auf der Ebene der isolierten Mikrosomen und intakten Hefezellen (2.2.). Als Substrat wurde jeweils ein Verteter der Klasse der n-Alkane (Dodekan) und der Fettsäuren (Laurinsäure) eingesetzt. Die erhaltenen Ergebnisse (Tab. 1-3) zeigen eindeutig, daß Cytochrom b5 einen starken stimulierenden Effekt auf die Aktivität der untersuchten Alkan- und Fettsäure-oxidierenden P450 Formen ausübt. 2.1. Konstruktion von S. cerevisiae Stämmen zur Coexpression von C. maltosa Cytochrom b5 mit Cytochrom P450 und NADPH-Cytochrom P450 ReduktaseIn order to reconstitute enzyme systems consisting of P450 Cml or P450 Cm2 and NADPH-P450 reductase and to test the influence of cytochrome b5, these three components were co-expressed in S. cerevisiae. The system developed for this is under 2.1. explained. S. cerevisiae cells in which P450 and reductase were expressed without cytochrome b5 served as controls. The resulting enzyme activities were then determined at the level of the isolated microsomes and intact yeast cells (2.2.). A representative of the class of n-alkanes (dodecane) and fatty acids (lauric acid) was used as the substrate. The results obtained (Tab. 1-3) clearly show that cytochrome b5 has a strong stimulating effect on the activity of the investigated alkane and fatty acid oxidizing P450 forms. 2.1. Construction of S. cerevisiae strains for coexpression of C. maltosa cytochrome b5 with cytochrome P450 and NADPH-cytochrome P450 reductase
Als erster Schritt wurde durch integrative Transformation ein S. cerevisiae Stamm konstruiert, der in seinem Genom eine Kassette für die Expression des C. maltosa Cytochrom b5 trägt. Wie in Abb. 4 dargestellt, bestand die integrative Expressionskassette aus folgenden Elementen: flankierenden Teilsequenzen des TRPl-Gens für die Integration ins Hefe-Genom, einem URA3 -Markergen sowie dem GAL 10-Promotor, der Cytochrom b5 cDNA und dem ADH 1 -Terminator.As a first step, an S. cerevisiae strain was constructed by integrative transformation, which carries in its genome a cassette for the expression of the C. maltosa cytochrome b5. As shown in Fig. 4, the integrative expression cassette consisted of the following elements: flanking partial sequences of the TRPl gene for integration into the yeast genome, a URA3 marker gene as well as the GAL 10 promoter, the cytochrome b5 cDNA and the ADH 1 terminator .
Zur Gewinnung und Klonierung dieser einzelnen Elemente wurde wie folgt vorgegangen:The procedure for obtaining and cloning these individual elements was as follows:
Die Teilsequenzen des TRPl-Gens wurden durch PCR aus genomischer DNA amplifiziert. Als Primerpaare dienten:The partial sequences of the TRPl gene were amplified by PCR from genomic DNA. The following were used as primer pairs:
TRP-Al: 5'-TCTAGCCATGGTGACTATTGAGCACG-3' / TRP-A3CR: 5*- ACAGGTACCGATATCAATGCCGTAATC-3' undTRP-Al: 5'-TCTAGCCATGGTGACTATTGAGCACG-3 '/ TRP-A3CR: 5 * - ACAGGTACCGATATCAATGCCGTAATC-3' and
TRP-B4: 5'-TATTGCGGCCGCTTAGATTAAATGG-37 TRP-B2CR: 5'-CTAGGAATTCGGCACACAGTGG-3'TRP-B4: 5'-TATTGCGGCCGCTTAGATTAAATGG-37 TRP-B2CR: 5'-CTAGGAATTCGGCACACAGTGG-3 '
Die amplifizierten Fragmente entsprechen den Regionen 49-390 bzw. 670-1425 innerhalb der Sequenz TRP1 Gens (zugrundeliegende Sequenz: V01341, Gene Bank). Diese Fragmente wurden in das bereits beschriebene Plasmid "pBM21-Ex2" ((Zimmer, T., Kaminski, K., Scheller, U., Vogel, F., and Schunck, W.-H. DNA and Cell Biology 14 (1995) 619-628) unter Verwendung der Restriktionsorte PmaCl I EcoRV (TRP-A) bzw. Notl EcoRI (TRP-B) eingeführt (resultierendes Plasmid: "Ex2-TRAB").The amplified fragments correspond to regions 49-390 and 670-1425 within the sequence TRP1 gene (underlying sequence: V01341, Gene Bank). These fragments were inserted into the already described plasmid "pBM21-Ex2" ((Zimmer, T., Kaminski, K., Scheller, U., Vogel, F., and Schunck, W.-H. DNA and Cell Biology 14 (1995 ) 619-628) using the restriction sites PmaCl I EcoRV (TRP-A) or Notl EcoRI (TRP-B) (resulting plasmid: "Ex2-TRAB").
Das URA3-Markergen wurde durch Restriktion mit Pvu I / Nru I aus dem Plasmid pSEY304 (Bankaitis, V.A., Johnson, L.M., and Emr, S.D. Proc.Natl.Acad.Sci. USA 83 (1986) 9075- 9079) isoliert und in den Eco RV-Ort des Plasmids Ex2-TRAB einkloniert (resultierendes Plasmid: Ex2-TRAB/URA).The URA3 marker gene was isolated from the plasmid pSEY304 (Bankaitis, VA, Johnson, LM, and Emr, SD Proc.Natl.Acad.Sci. USA 83 (1986) 9075-9079) by restriction with Pvu I / Nru I and in cloned the Eco RV site of the plasmid Ex2-TRAB (resulting plasmid: Ex2-TRAB / URA).
Die Cytochrom b5 cDNA wurde aus dem Plasmid pCR 2.1™ (vgl. 1.3.) durch Restriktion mit Sal I und Bam HI isoliert und in das mit den gleichen Restriktasen geöffnete Plasmid Ex2-TRAB/URA umkloniert (resultierendes Plasmid: pEx2/Cmb5, siehe Abb. 4). Die verwendete Cytochrom b5 cDNA entspricht der SEQ ID No: 2.The cytochrome b5 cDNA was obtained from the plasmid pCR 2.1 ™ (see 1.3.) By restriction isolated with Sal I and Bam HI and cloned into the plasmid Ex2-TRAB / URA opened with the same restrictases (resulting plasmid: pEx2 / Cmb5, see FIG. 4). The cytochrome b5 cDNA used corresponds to SEQ ID No: 2.
Die so konstruierte integrative Expressionskassette wurde dann aus dem Plasmid pEx2/Cmb5 durch Restriktion mit Pma CI und Nhe I isoliert und in den S. cerevisiae Stamm YS 18 (isogen zu GRF18 aber ura3; Sengstag and Hinnen, Gene 62 (1988) 223-228) transformiert. Dabei wurden sowohl Transformanten erhalten, bei denen die Expressionskassette durch homologe Rekombination in das TRPl-Gen der Wirtzelle eingebaut wurde (TRP" ,URA+ , im weiteren als YSCmb5Cl bezeichnet), als auch solche, die aus einem zufälligen Einbau an einem anderen, nicht näher charakterisierten Ort des Hefe-Genoms resultierten (TRP+ ,URA+ , im weiteren als YSCmb5C10 bezeichnet).The integrative expression cassette thus constructed was then isolated from the plasmid pEx2 / Cmb5 by restriction with Pma CI and Nhe I and into the S. cerevisiae strain YS 18 (isogenic to GRF18 but ura3; Sengstag and Hinnen, Gene 62 (1988) 223-228 ) transformed. Both transformants were obtained in which the expression cassette was incorporated into the TRPl gene of the host cell by homologous recombination (TRP " , URA + , hereinafter referred to as YSCmb5Cl), and those which resulted from an accidental incorporation into another were not Characterized location of the yeast genome resulted (TRP + , URA + , hereinafter referred to as YSCmb5C10).
Die so konstruierten und für die weiteren Versuche ausgewählten Stämme YSCmb5Cl und YSCmb5C10 tragen jeweils eine stabil in das Hefegenom integrierte Expressionskassette, die eine Galaktose-induzierbare Expression des C. maltosa Cytochrom b5 gestattet. Dabei zeichnet sich der Stamm YSCmb5C10 durch eine etwa 2.5-fach höhere Cytochrom b5 Expression im Vergleich zu YSCmb5Cl aus.The strains YSCmb5Cl and YSCmb5C10 constructed in this way and selected for the further experiments each carry an expression cassette which is stably integrated into the yeast genome and which enables galactose-inducible expression of the C. maltosa cytochrome b5. The YSCmb5C10 strain is characterized by an approximately 2.5-fold higher cytochrome b5 expression compared to YSCmb5Cl.
Ausgehend von YSCmb5Cl und YSCmb5C10 wurden dann Hefe-Stämme erzeugt, die eine gleichzeitige Expression von Cytochrom P450, NADPH-P450 Reduktase und Cytochrom b5 ermöglichen. Dazu erfolgte eine Transformation der YSCmb5-Stämme mit den autonom replizierenden Plasmiden YEp51Cml-R bzw. YEp51Cm2-R. Diese Plasmide wurden bereits früher beschrieben: Zimmer, T., Kaminski, K., Scheller, U., Vogel, F., and Schunck, W.-H. DNA and Cell Biology 14 (1995) 619-628 und DE 195 07 546 AI). Sie tragen zwei Expressionskassetten, die jeweils unter Kontrolle des GAL10 Promotors zur Expression der gewünschten P450-Komponente (P450 Cml bzw. P450 Cm2 aus C. maltosa) und der NADPH-P450 Reduktase aus C. maltosa genutzt werden.Starting from YSCmb5Cl and YSCmb5C10, yeast strains were then generated which enable the simultaneous expression of cytochrome P450, NADPH-P450 reductase and cytochrome b5. To this end, the YSCmb5 strains were transformed with the autonomously replicating plasmids YEp51Cml-R and YEp51Cm2-R. These plasmids have been described previously: Zimmer, T., Kaminski, K., Scheller, U., Vogel, F., and Schunck, W.-H. DNA and Cell Biology 14 (1995) 619-628 and DE 195 07 546 AI). They carry two expression cassettes, which are used under the control of the GAL10 promoter to express the desired P450 component (P450 Cml or P450 Cm2 from C. maltosa) and the NADPH-P450 reductase from C. maltosa.
Letztlich wurden für die Untersuchung des stimulierenden Effekts von Cytochrom b5 auf die Aktivität Alkan- und Fettsäure-oxidierender P450 Enzyme folgende S. cerevw/αe-Stämme erhalten:Ultimately, the following S. cerevw / αe strains were obtained for the investigation of the stimulating effect of cytochrome b5 on the activity of alkane and fatty acid oxidizing P450 enzymes:
YSCmb5Cl/CmlR und YSCmb5vC10/CmlR - zur Coexpression von Cytochrom b5, P450 Cml und NADPH-P450 Reduktase;YSCmb5Cl / CmlR and YSCmb5vC10 / CmlR - for coexpression of cytochrome b5, P450 Cml and NADPH-P450 reductase;
YSCmb5Cl/Cm2R und YSCmb5C10/Cm2R - zur Coexpression von Cytochrom b5, P450 Cm2 und NADPH-P450 Reduktase;YSCmb5Cl / Cm2R and YSCmb5C10 / Cm2R - for coexpression of cytochrome b5, P450 Cm2 and NADPH-P450 reductase;
YS18/CmlR - Kontrollstamm zur Expression von P450 Cml und NADPH-P450 Reduktase ohne Cytochrom b5YS18 / CmlR - control strain for the expression of P450 Cml and NADPH-P450 reductase without cytochrome b5
YS18/Cm2R - Kontrollstamm zur Expression von P450 Cm2 und NADPH-P450 Reduktase ohne Cytochrom b5YS18 / Cm2R - control strain for the expression of P450 Cm2 and NADPH-P450 reductase without cytochrome b5
2.2. Einfluß von Cytochrom b5 auf die P450-katalysierte Oxidation von Dodekan und Laurinsäure2.2. Influence of cytochrome b5 on the P450-catalyzed oxidation of dodecane and lauric acid
Für diese Untersuchungen wurden die oben genannten S. cerevisiae Stämme (vgl. 2.1.) wie unter 1.5. beschrieben kultiviert und die Expression der jeweiligen Komponenten (P450, Reduktase und Cytochrom b5) durch Zugabe von Galaktose induziert. Die Zellen wurden 24 h nach Galaktose-Zugabe geerntet, mechanisch aufgeschlossen und die Gewinnung der Mikrosomen erfolgte durch differentielle Zentrifugation (siehe 1.5).For these investigations, the S. cerevisiae strains mentioned above (see 2.1.) As in 1.5. described cultivated and the expression of the respective components (P450, reductase and cytochrome b5) induced by adding galactose. The cells were harvested 24 hours after the addition of galactose, mechanically disrupted and the microsomes were obtained by differential centrifugation (see 1.5).
Die Reaktionsansätze (1 ml) enthielten in 100 mM K-K-Phosphatpufer pH 7.25, 10 mM KC1, 5 mM MgCl2, ca. 0.2 mg mikrosomales Protein, 100 nmol NADPH, ein NADPH- regenerierendes System bestehend aus 3.1 μMol Glucose-6-Phosphat und 1 U Glucose-6- Phosphat-Dehydrogenase sowie als Substrat entweder [1-14C] Dodekan (1000 nMol, lxlO6 dpm) oder [1-14C] Laurinsäure (250 nMol, 5xl05 dpm). Die Reaktionen wurden durch Zugabe von NADPH gestartet, bei einigen Experimenten wurde gleichzeitig NADH (500 nmol) zugesetzt. Die Inkubation erfolgte 30 min bei 30°C. Die weitere Aufarbeitung (Chloroform/Methanol-Extraktion und Dünnschicht-Chromatographie) sowie die Quantifizierung der Reaktionsprodukte mit einem TLC-Radio-Scanner erfolgte wie früher beschrieben (Scheller, U., Zimmer, T., Kärgel, E. and Schunck, W.-H. Arch. Biochem. Biophys. 228 (1996) 245-254).The reaction mixtures (1 ml) contained in 100 mM KK phosphate buffer pH 7.25, 10 mM KC1, 5 mM MgCl 2 , approx. 0.2 mg microsomal protein, 100 nmol NADPH, a NADPH regenerating system consisting of 3.1 μmol glucose-6-phosphate and 1 U glucose-6-phosphate dehydrogenase, as well as the substrate either [1- 14 C] dodecane (1000 nmol, lxlO 6 dpm) or [1- 14 C] lauric acid (250 nmol, 5xl0 5 dpm). The reactions were started by adding NADPH; in some experiments NADH (500 nmol) was added at the same time. Incubation took place at 30 ° C for 30 min. Further processing (chloroform / methanol extraction and thin-layer chromatography) and quantification of the reaction products using a TLC radio scanner were carried out as previously described (Scheller, U., Zimmer, T., Kärgel, E. and Schunck, W. -H. Arch. Biochem. Biophys. 228 (1996) 245-254).
Die Ergebnisse sind in Tab. 1 und Tab. 2 zusammengefaßt. Sie zeigen, daß Cytochrome b5 einen starken stimulierenden Effekt auf die Aktivität der untersuchten P450 Enzyme hat.The results are summarized in Tab. 1 and Tab. 2. They show that cytochrome b5 has a strong stimulating effect on the activity of the investigated P450 enzymes.
Beispielsweise wurde die Geschwindigkeit der NADPH-abhängigen, P450 Cm2 katalysierten Oxidation von Laurinsäure durch die Anwesenheit von Cytochrom b5 etwa 4-fach gesteigert (siehe Tab. 1). Bei Einsatz eines Gemisches von NADPH und NADH erfolgte eine weitere Erhöhung, so daß durch Cytochrom b5 eine insgesamt mehr als 7-fache Stimulierung erreicht wurde. Der synergistische Effekt von NADPH und NADH trat nur in Anwesenheit von Cytochrom b5 auf (siehe Tab. 1). Die Aktivität von P450 Cml, das im Gegensatz zum P450 Cm2 bevorzugt n-Alkane oxidiert, wurde ebenfalls deutlich durch Cytochrom b5 stimuliert. Das coexprimierte Cytochrom b5 bewirkte eine mehr als 4-fache Steigerung der P450 Cml katalysierten Oxidation von Dodekan (Tab. 2). Zusätzlich wurde gezeigt, daß eine Stimulierung der P450-Aktivität auch durch Zusatz von gereinigtem Cytochrom b5 zu Mikrosomen erreicht werden kann, die nur P450 und NADPH-P450 Reduktase enthielten (Tab. 2). Unter den gewählten Reaktionsbedingungen waren 1-Dodekanol und 12- Hydroxylaurinsäure die Hauptprodukte der P450-abhängigen Dodekan bzw. Laurinsäure- Oxidationen. Eine signifikante Weiteroxidation bis zur entsprechenden Dikarbonsäure trat vor allem bei hohen Enzymaktivitäten auf.(vgl. Tab. 1).For example, the rate of NADPH-dependent, P450 Cm2 catalyzed oxidation of lauric acid was increased approximately 4-fold by the presence of cytochrome b5 (see Table 1). When a mixture of NADPH and NADH was used, there was a further increase, so that a total of more than 7-fold stimulation was achieved by cytochrome b5. The synergistic effect of NADPH and NADH only occurred in the presence of cytochrome b5 (see Table 1). The activity of P450 Cml, which in contrast to P450 Cm2 preferentially oxidizes n-alkanes, was also clearly stimulated by cytochrome b5. The coexpressed cytochrome b5 caused a more than 4-fold increase in the P450 Cml-catalyzed oxidation of dodecane (Table 2). In addition, it was shown that stimulation of the P450 activity can also be achieved by adding purified cytochrome b5 to microsomes that only contained P450 and NADPH-P450 reductase (Table 2). Under the chosen reaction conditions, 1-dodecanol and 12-hydroxylauric acid were the main products of the P450-dependent dodecane and lauric acid oxidations, respectively. Significant further oxidation to the corresponding dicarboxylic acid occurred above all with high enzyme activities (see Table 1).
In einer weiteren Versuchsreihe wurde die Fähigkeit intakter Hefezellen zur Oxidation von Laurinsäure untersucht. Dazu wurden je 2 ml Aliquote Galaktose-induzierter Kulturen der oben genannten Hefestämme eingesetzt. Diese wurden mit [1-14C] Laurinsäure (500 nMol, lxlO6 dpm; gelöst in 10 μl Ethanol) versetzt und unter Schütteln für 30 min bei 30°C inkubiert. Die Extraktion und Quantifizierung der Reaktionsprodukte erfolgte wie oben für die mikrosomalen Ansätze beschrieben.In another series of experiments, the ability of intact yeast cells to oxidize lauric acid was investigated. For this purpose, 2 ml aliquots of galactose-induced cultures of the above-mentioned yeast strains were used. These were labeled with [1- 14 C] lauric acid (500 nmol, lxlO 6 dpm dissolved in 10 ul ethanol) and incubated min at 30 ° C with shaking for 30 seconds. The reaction products were extracted and quantified as described above for the microsomal approaches.
Die erhaltenen Ergebnisse sind in Tab. 3 zusammengefaßt. Sie zeigen, daß bereits auf der Ebene der intakten Zellen ein stimulierender Effekt des Cytochrom b5 klar nachweisbar ist. Dieser war im Fall von P450 Cm2 besonders stark ausgeprägt. So wies der Stamm mit der höchsten Cytochrom b5 Coexpression (YSCmb5C10/Cm2R) eine etwa 4-fach höhere Aktivität auf, als der entsprechende Stamm, der nur P450 Cm2 und die NADPH-P450 Reduktase exprimiert (YS18/Cm2R). Dabei wurde nicht nur eine verstärkte Bildung der 12- Hydroxylaurinsäure sondern auch der Dodekandisäure beobachtet (Tab. 3). Dieses Ergebnis zeigt, daß Cytochrom b5 sowohl die primäre Hydroxylierungsreaktion, als auch die nachfolgenden Oxidationsschritte bis zur entsprechenden Dikarbonsäure stimuliert. Tab. 1. Effekt des coexprimierten Candida maltosa Cytochrom b5 (Cmb5, SEQ ID No.2) auf die Laurinsäure-oxidierende Aktivität der mikrosomalen Cytochrom P450 Enzyme P450 Cm1 und P450 Cm2.The results obtained are summarized in Tab. 3. They show that a stimulating effect of cytochrome b5 can already be clearly demonstrated at the level of the intact cells. This was particularly pronounced in the case of P450 Cm2. For example, the strain with the highest cytochrome b5 coexpression (YSCmb5C10 / Cm2R) had about 4 times higher activity than the corresponding strain, which only expresses P450 Cm2 and the NADPH-P450 reductase (YS18 / Cm2R). Not only was an increased formation of 12-hydroxylauric acid observed, but also dodecanedioic acid (Table 3). This result shows that cytochrome b5 stimulates both the primary hydroxylation reaction and the subsequent oxidation steps to the corresponding dicarboxylic acid. Tab. 1. Effect of the coexpressed Candida maltosa cytochrome b5 (Cmb5, SEQ ID No.2) on the lauric acid oxidizing activity of the microsomal cytochrome P450 enzymes P450 Cm1 and P450 Cm2.
Figure imgf000018_0001
Figure imgf000018_0001
Die Produkte wurden durch Cochromatogrphie mit authentischen Standardsubstanzen identifiziert: 12-OH-LA, 12-Hydroxylaurinsäure; DDDA, 1,12-Dodekandisäure Tab. 2. Effekt von coexprimiertem bzw. gereinigtem Candida maltosa Cytochrom b5 (Cmb5, SEQ ID No.2) auf die Dodekan-oxidierende Aktivität der mikrosomalen Cytochrom P450 Enzyme P450 Cm1 und P450 Cm2.The products were identified by cochromatography with authentic standard substances: 12-OH-LA, 12-hydroxylauric acid; DDDA, 1,12-dodecanedioic acid Tab. 2. Effect of coexpressed or purified Candida maltosa cytochrome b5 (Cmb5, SEQ ID No.2) on the dodecane oxidizing activity of the microsomal cytochrome P450 enzymes P450 Cm1 and P450 Cm2.
Figure imgf000019_0001
Figure imgf000019_0001
Tab. 3. Effekt des coexprimierten Candida maltosa Cytochrom b5 (Cmb5, SEQ ID No.2) auf die Laurinsäure-oxidierende Aktivität der Cytochrom P450 Enzyme P450 Cm1 und P450 Cm2 in intakten Saccharomyces cerevisiae Zellen.Tab. 3. Effect of the coexpressed Candida maltosa cytochrome b5 (Cmb5, SEQ ID No.2) on the lauric acid oxidizing activity of the cytochrome P450 enzymes P450 Cm1 and P450 Cm2 in intact Saccharomyces cerevisiae cells.
Figure imgf000020_0001
Figure imgf000020_0001
Die Produkte wurden durch Cochromatogrphie mit authentischen Standardsubstanzen identifiziert: 12-OH-LA, 12-Hydroxylaurinsäure; DDDA, 1,12-Dodekandisäure The products were identified by cochromatography with authentic standard substances: 12-OH-LA, 12-hydroxylauric acid; DDDA, 1,12-dodecanedioic acid
Legende zu den Abbildungen:Legend for the pictures:
Abb. 1 : Nucleinsäuresequenz und abgeleitete Aminosäuresequenz für das Cytochrom b5 der alkanverwertenden Hefe Candida maltosa.Fig. 1: Nucleic acid sequence and derived amino acid sequence for the cytochrome b5 of the alkane-utilizing yeast Candida maltosa.
Abb. 2: Vergleich der Aminosäuresequenz des Cytochrom b5 aus Candida maltosa mit der bekannter Cytochrom b5 Proteine. (A)- AHgnment mit der Sequenz des Cytochrom b5 aus Saccharomyces cerevisiae (Swiss Prot Accession number: P40312); (B)- AHgnment mit der Sequenz des menschlichen Cytochrom b5 (mikrosomale Form, Swiss Prot Accession number: P00167).Fig. 2: Comparison of the amino acid sequence of the cytochrome b5 from Candida maltosa with the known cytochrome b5 proteins. (A) - AHgnment with the sequence of the cytochrome b5 from Saccharomyces cerevisiae (Swiss Prot Accession number: P40312); (B) - AHgnment with the sequence of the human cytochrome b5 (microsomal form, Swiss Prot Accession number: P00167).
Abb. 3: Heterologe Expression der Candida maltosa Cytochrom b5 cDNA in Saccharomyces cerevisiae. Dargestellt sind die Differenzspektren (reduzierte gegen oxidierte Probe) zum Nachweis von Cytochrom b5 in verschiedenen Zellfraktionen: A: Homogenat; B: 10 000 g Überstand; C: 100 000 g Überstand; D: 100 000 g Sediment (Mikrosomen). #1- Zellfraktionen des Stammes GRF18/YEp51-b5 (Expression des C. maltosa Cytochrom b5); #2- Zellfraktionen des Kontrollstammes GRF18/YEp51; #0- Grundlinie zu #1.Fig. 3: Heterologous expression of the Candida maltosa cytochrome b5 cDNA in Saccharomyces cerevisiae. The difference spectra (reduced versus oxidized sample) for the detection of cytochrome b5 in different cell fractions are shown: A: homogenate; B: 10,000 g of supernatant; C: 100,000 g of supernatant; D: 100,000 g sediment (microsomes). # 1 cell fractions of strain GRF18 / YEp51-b5 (expression of the C. maltosa cytochrome b5); # 2- cell fractions of the control strain GRF18 / YEp51; # 0- baseline to # 1.
Abb.4: Karte des Plasmids pEx2/Cmb5. Dieses Plasmid enthält die konstuierte Expressionskassette zur heterologen Expression des Cytrochrom b5 aus Candida maltosa in Saccharomyces cerevisiae. Fig.4: Map of the plasmid pEx2 / Cmb5. This plasmid contains the constructed expression cassette for the heterologous expression of the cytrochrome b5 from Candida maltosa in Saccharomyces cerevisiae.

Claims

Patentansprüche claims
1. Nukleinsäure-Sequenzen aus alkanverwertenden Candida Hefen, die ein Cytochrom b5- Polypeptid kodieren sowie ihre Fragmente, Varianten und Mutationen.1. Nucleic acid sequences from alkane-utilizing Candida yeasts which encode a cytochrome b5 polypeptide as well as their fragments, variants and mutations.
2. Nukleinsäure-Sequenzen nach Anspruch 1, dadurch gekennzeichnet, daß sie aus Candida maltosa stammen.2. Nucleic acid sequences according to claim 1, characterized in that they originate from Candida maltosa.
3. Nukleinsäure-Sequenzen nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß sie vollständig komplementär sind.3. Nucleic acid sequences according to claim 1 or 2, characterized in that they are completely complementary.
4. Nukleinsäure-Sequenzen nach Anspruch 1 bis 3, gekennzeichnet durch die SEQ ID No. 1 oder deren Varianten.4. Nucleic acid sequences according to claims 1 to 3, characterized by SEQ ID No. 1 or its variants.
5. Nukleinsäure-Sequenzen nach Anspruch 1 bis 3, gekennzeichnet durch die SEQ ID No. 2 oder deren Varianten.5. Nucleic acid sequences according to claims 1 to 3, characterized by SEQ ID No. 2 or their variants.
6. Cytochrom b5 -Polypeptide, die von den Nukleinsäure-Sequenzen gemäß einem der Ansprüche 1 bis 5 kodiert werden.6. cytochrome b5 polypeptides which are encoded by the nucleic acid sequences according to any one of claims 1 to 5.
7. Cytochrom b5-Polypeptide gekennzeichnet durch die SEQ ID No. 3, Varianten davon, die Deletionen oder Modifikationen aufweisen und Cytochrom b5 -Aktivitäten aufweisen.7. Cytochrome b5 polypeptides characterized by SEQ ID No. 3, variants thereof which have deletions or modifications and have cytochrome b5 activities.
8. Plasmide, die eine Nukleinsäure-Sequenz gemäß einem der Ansprüche 1 bis 5 enthalten.8. plasmids which contain a nucleic acid sequence according to any one of claims 1 to 5.
9. Vektoren, die Nukleinsäure-Sequenz gemäß einem der Ansprüche 1 bis 5 enthalten.9. Vectors containing the nucleic acid sequence according to any one of claims 1 to 5.
10. Wirtszellen, die einen Vektor gemäß Anspruch 9 enthalten.10. host cells containing a vector according to claim 9.
11. Antikörper, die spezifisch an die Polypeptide gemäß Anspruch 7 binden.11. Antibodies that bind specifically to the polypeptides according to claim 7.
12. Verwendung der Nukleinsäuresequenzen oder Polypeptide gemäß einem der Ansprüche 1 bis 7 zur Stimulierung der Aktivität von Cytochrom P450-Enzymen. 12. Use of the nucleic acid sequences or polypeptides according to one of claims 1 to 7 for stimulating the activity of cytochrome P450 enzymes.
13. Verwendung der Nukleinsäuresequenzen oder Polypeptide gemäß einem der Ansprüche 1 bis 7 in Verfahren zur Oxidation von langkettigen Alkylverbindungen (>C10).13. Use of the nucleic acid sequences or polypeptides according to one of claims 1 to 7 in processes for the oxidation of long-chain alkyl compounds (> C10).
14. Verwendung der Nukleinsäuresequenzen oder Polypeptide gemäß einem der Ansprüche 1 bis 7 in Verfahren zur Herstellung von langkettigen Dicarbonsäuren durch Oxidation von n-Alkanen (>C10) und Fettsäuren (>C10). 14. Use of the nucleic acid sequences or polypeptides according to one of claims 1 to 7 in processes for the production of long-chain dicarboxylic acids by oxidation of n-alkanes (> C10) and fatty acids (> C10).
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