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WO2000065061A2 - Sequences d'acide nucleique issues de levures de candida, codant des polypeptides b5 cytochromes - Google Patents

Sequences d'acide nucleique issues de levures de candida, codant des polypeptides b5 cytochromes 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 (fr
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Wolf-Hagen Schunck
Alexei Chernogolov
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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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

Definitions

  • 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

L'invention concerne des séquences d'acide nucléique issues de levures de Candida, de préférence de Candida maltosa, qui codent des polypeptides b5 cytochromes. L'invention concerne également les polypeptides b5 cytochromes correspondants et leur utilisation pour augmenter l'activité de systèmes P450 cytochromes, notamment pour stimuler l'activité de systèmes P450 cytochromes à effet hydroxylant de l'alcane et de l'acide gras lors de la préparation d'acides dicarboxyliques à chaîne longue (≥C10).
PCT/DE2000/001246 1999-04-24 2000-04-18 Sequences d'acide nucleique issues de levures de candida, codant des polypeptides b5 cytochromes Ceased WO2000065061A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008413A3 (fr) * 2000-07-26 2002-11-14 Cognis Corp Gene b5 de cytochrome et proteine de candida tropicalis et procedes correspondants

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FR2679249B1 (fr) * 1991-07-15 1993-11-26 Centre Nal Recherc Scientifique Souches de levure avec integration stable de genes heterologues.
JPH07289252A (ja) * 1994-04-28 1995-11-07 Sumitomo Chem Co Ltd チトクロムｐ４５０の一原子酸素添加活性の向上方法
DE19507546C2 (de) * 1995-03-03 2001-05-03 Max Delbrueck Centrum Verfahren zur regioselektiven Hydroxylierung von langkettigen Alkanen, Fettsäuren und anderen Alkylverbindungen
CA2293737A1 (fr) * 1997-07-21 1999-01-28 E.I. Du Pont De Nemours And Company Souches de levure transformees et leur utilisation dans la production de carboxylates aliphatiques a terminaison unique et a terminaison double

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008413A3 (fr) * 2000-07-26 2002-11-14 Cognis Corp Gene b5 de cytochrome et proteine de candida tropicalis et procedes correspondants
US6503734B1 (en) 2000-07-26 2003-01-07 Cognis Corporation Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

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