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  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
  • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
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  • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
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  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/84—Pichia
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
  • C12R2001/865—Saccharomyces cerevisiae

Definitions

• Ursodeoxycholic acid is a valuable bile acid frequently prescribed for the treatment of cholecystitis as it can solubilize cholesterol gallstones with fewer side effects than chenodeoxycholic acid (CDCA).
• UDCA also has anti-inflammatory properties and is applied in the therapy of cystic fibrosis and liver diseases like primary biliary cholangitis.
• the major natural source of UDCA is bile from various species of bears.
• the invention provides a method of converting LCA or 3-KCA, or a carboxylic acid ester, carboxylic amide, or carboxylate salt thereof, to UDCA or 3-KUDCA, or a carboxylic acid ester, carboxylic amide, or carboxylate salt thereof, comprising contacting the LCA or 3-KCA, or carboxylic acid ester, carboxylic amide, or carboxylate salt thereof, with a 7P-hydroxylase system in the presence of a yeast, or an extract or lysate thereof, wherein the 7P-hydroxylase system is not native to the yeast.
• FIGURE 9 depicts LCMS chromatograms from the experiment described in Example 19.
• Figure 9 A is a TIC trace of the extracted broth sample.
• Figure 9B is an Extracted Ion Chromatogram (EIC) for m/z 389.3 (3-KUDCA) of the extracted broth sample.
• Figure 9C is a TIC trace of the 3-KUDCA standard.
• Figure 9D is a TIC trace of the 3-KCA standard.
• the term “about” will compensate for variability allowed for in the chemical industry and inherent in products in this industry, such as differences in product strength due to manufacturing variation and time-induced product degradation. In one embodiment, the term allows for ⁇ 5% variability or ⁇ 10% variability.
• Coding sequence refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
• Percentage of sequence identity and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window will comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the invention provides a reaction mixture comprising (i) LCA or 3-KCA, (ii) a yeast, or an extract or lysate thereof, and (iii) a 7P-hydroxylation system.
• the invention is preferably carried out in the presence of a yeast transformed to express a non-native 7b-1 ⁇ p ⁇ H ⁇ oh system.
• the yeast is preferably selected from Saccharomyces and Pichia , and most preferably is selected from Saccharomyces cerevisiae and Pichia pastoris.
• the encoding nucleic acid sequence is selected from SEQ ID NO. 8; SEQ ID NO. 11; SEQ ID NO. 14; SEQ ID NO. 17; and SEQ ID NO. 20; or a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99%, identity with any of the foregoing sequences.
• the nucleic acid is selected from SEQ ID NO. 23; SEQ ID NO. 26; or SEQ ID NO. 29; or a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99%, identity with any of the foregoing sequences.
• the nucleic acid sequence is selected from SEQ ID NO. 32; or a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99%, identity with SEQ ID NO. 32.
• the plasmid encoding the CYP enzyme comprises SEQ ID NO. 7; SEQ ID NO. 10; SEQ ID NO. 13; SEQ ID NO. 16; or SEQ ID NO. 19; or a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99%, identity with any of the foregoing sequences.
• the plasmid encoding the CYP enzyme comprises SEQ ID NO. 22; SEQ ID NO. 25; or SEQ ID NO. 28; or a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99%, identity with any of the foregoing sequences.
• the plasmid encoding the CYP enzyme comprises SEQ ID NO. 31; or a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99%, identity with SEQ ID NO. 31.
• the CYP enzyme is a protein native to Gibberella zeae , preferably Gibber ella zeae PHI or Gibberella zeae VKM2600, most preferably Gibberella zeae VKM2600, and the organism is transformed to express such protein.
• the CPR enzyme in the 7P-hydroxylation system can be native to the organism in which the 7P-hydroxylase activity is expressed, or encoded by a CPR encoding nucleic acid sequence selected from SEQ ID NO. 2 and SEQ ID NO. 5, or a nucleic acid sequence having at least 85% 90%, 95%, 98%, or 99%, identity with any of the foregoing nucleic acid sequences.
• the CPR enzyme preferably comprises a CPR amino acid sequence selected from SEQ ID NO. 3 and SEQ ID NO. 6, or an amino acid sequence having at least 85% 90%, 95%, 98%, or 99%, identity with any of the foregoing amino acid sequences.
• the methods of the current invention will optionally further comprise reducing the 3-KUDCA or carboxylic acid ester, carboxylic amide, or carboxylate salt thereof to UDCA or a carboxylic acid ester, carboxylic amide, or carboxylate salt thereof.
• the recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence.
• the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
• the vectors may be linear or closed circular plasmids.
• the reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points.
• a solvent e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like
• some of the reactants may be added separately, and some together at different time points.
• the hydroxylase system may be added first to the solvent.
• the enzyme preparation is added last.
• the pH of the reaction mixture may change.
• the pH of the reaction mixture may be maintained at a desired pH or within a desired pH range by the addition of an acid or a base during the course of the reaction.
• the pH may be controlled by using a solvent that comprises a buffer.
• Suitable buffers to maintain desired pH ranges are known in the art and include, for example, phosphate buffer, triethanolamine buffer, and the like. Combinations of buffering and acid or base addition may also be used.
• BMMY medium is made by dissolving 10 g yeast extract and 10 g bacto-tryptone in 700 ml dH 2 0 and sterilization by autoclaving. Just before use, 100 ml YNB stock solution, 2 ml biotin stock solution and 100 ml 100 mM potassium phosphate buffer, pH 6.0 are added.
• Plasmid pSAND102 is obtained as synthetic DNA with the sequence SEQ ID NO. 1 from a commercial provider. In brief, it contains the AOX1 promoter sequence, followed by a gene with sequence SEQ ID NO. 2, encoding a P450 reductase with sequence SEQ ID NO. 3, under control of the AOX1 promoter, followed by the AOX1 terminator sequence.
• the AOX1 promoter contains a unique Pmel restriction site to allow linearization of plasmid pSAND102
• Plasmid pSAND103 is obtained as synthetic DNA with the sequence SEQ ID NO. 4 from a commercial provider. In brief, it contains the AOX1 promoter sequence, followed by a gene with sequence SEQ ID NO. 5, encoding a P450 reductase with sequence SEQ ID NO. 6, under control of the AOX1 promoter, followed by the AOX1 terminator sequence.
• the AOX1 promoter contains a unique Pmel restriction site to allow linearization of plasmid pSAND103
• Plasmid pSAND104 is obtained as synthetic DNA with the sequence SEQ ID NO. 7 from a commercial provider. In brief, it contains the AOX1 promoter sequence, followed by a gene with sequence SEQ ID NO. 8, encoding a P450 with sequence SEQ ID NO. 9, under control of the AOX1 promoter, followed by the AOX1 terminator sequence.
• Electrocompetent cells of strain Pichia pastoris SAND 102 are transformed with plasmid pSAND104, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND 104.
• Electrocompetent cells of strain Pichia pastoris SAND 103 are transformed with plasmid pSAND104, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND 105.
• Plasmid pSAND106 is obtained as synthetic DNA with the sequence SEQ ID NO. 13 from a commercial provider. In brief, it contains the AOX1 promoter sequence, followed by a gene with sequence SEQ ID NO. 14, encoding a P450 with sequence SEQ ID NO. 15, under control of the AOX1 promoter, followed by the AOX1 terminator sequence.
• Electrocompetent cells of strain Pichia pastoris SAND 102 are transformed with plasmid pSAND106, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND 108.
• Electrocompetent cells of strain Pichia pastoris SAND 103 are transformed with plasmid pSAND107, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SANDl l l.
• EXAMPLE 7 CONSTRUCTION OF PICHIA PASTORIS STRAINS CAPABLE OF
• Pichia pastoris SAND 102 Electrocompetent cells of strain Pichia pastoris SAND 102 are transformed with plasmid pSAND108, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND112.
• EXAMPLE 8 CONSTRUCTION OF PICHIA PASTORIS STRAINS CAPABLE OF
• Plasmid pSANDl 10 is obtained as synthetic DNA with the sequence SEQ ID NO. 25 from a commercial provider. In brief, it contains the AOX1 promoter sequence, followed by a gene with sequence SEQ ID NO. 26, encoding a P450 with sequence SEQ ID NO. 27, under control of the AOX1 promoter, followed by the AOX1 terminator sequence.
• Electrocompetent cells of strain Pichia pastoris SAND 102 are transformed with plasmid pSANDl 10, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND 116.
• Plasmid pSANDl 11 is obtained as synthetic DNA with the sequence SEQ ID NO. 28 from a commercial provider. In brief, it contains the AOX1 promoter sequence, followed by a gene with sequence SEQ ID NO. 29, encoding a P450 with sequence SEQ ID NO. 30, under control of the AOX1 promoter, followed by the AOX1 terminator sequence.
• Electrocompetent cells of strain Pichia pastoris SAND 102 are transformed with plasmid pSANDl 11, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND118.
• Electrocompetent cells of strain Pichia pastoris SAND 103 are transformed with plasmid pSANDl 11, plated onto YPD agar containing 100 pg/ml nourseothricin and 100 pg/ml zeocin and incubated at 30°C until colonies become visible.
• the resulting strain is named Pichia pastoris SAND 119.
• EXAMPLE 11 CONSTRUCTION OF PICHIA PASTORIS STRAINS CAPABLE OF EXPRESSING SEP ID NO. 32 (FGSG 02672V21 _
• EXAMPLE 13 LCA CONVERSION USING WHOLE CELLS OF PICHIA PASTORIS STRAINS PICHIA PASTORIS SAND 104 - PICHIA PASTORIS SANDHI GROWN ON BMG MEDIUM
• EXAMPLE 14 3-KCA CONVERSION USING WHOLE CELLS OF PICHIA PASTORIS STRAINS PICHIA PASTORIS SAND 104 - PICHIA PASTORIS SAND 121 GROWN ON YPD MEDIUM
• Cells are harvested by centrifugation at 4000 xg for 5 minutes and resuspended in 30 ml 50 mM potassium phosphate buffer, pH 7.5 containing 2 mM aminolevulenic acid and 1 mM 3- KCA. The cell suspension is incubated at 30°C shaking at 200 RPM with addition of methanol (1% v/v) every 24 hours for 3 days.
• Synthetic Galactose Minimal Medium contains 6.7 g/L yeast nitrogen base without amino acids, 20 g/L galactose and 1.3 g/L amino acid dropout powder and is sterilized by autoclaving.
• Synthetic Galactose Minimal Agar Medium contains 20 g/L agar.
• 100 ng DNA was added to 40 m ⁇ of the competent cells and transferred to a 2 mm gap electroporation cuvette, precooled on ice.
• Cells were electroporated on a BTRX ECM 630 decay wave electroporation system, using 1500 V, 200 W, 25 pF settings.
• 1 mL cold 1 M sorbitol was added immediately, and the mixture was transferred to a sterile Eppendorf tube.
• Cells were regenerated at 30 °C, shaking at 250 RPM for at least 30 minutes. Cells were then spread onto YPD agar plates containing appropriate antibiotics, then incubated at 30 °C for 2 days or until colonies became visible.
• Plasmid pSAND102 was linearized by digestion with the restriction enzyme Pmel. Linearized pSAND102 was used to transform Pichia pastoris SANDIOI by electroporation using standard methods. The resulting strain was labelled Pichia pastoris SAND 102.
• Bioconversion culture broths stored at -20 °C as described in example 17, were thawed and centrifuged at 4500 RPM for 15 minutes. The resulting supernatant of 100 mL was decanted and extracted three times with an equal volume of ethyl acetate containing 0.1% formic acid, stirring for 45 minutes. The organic phases were pooled and evaporated under vacuum to yield a crude weighing 179 mg.
• NMR spectroscopy in d4-m ethanol of this sample was undertaken and compared to a commercially obtained sample of UDCA (Sigma- Aldrich) which was run at the same time.
• NMR spectra were recorded on a Bruker 500 MHz DCH Cryoprobe Spectrometer at 298 K operating at 500.05 MHz and 125.75 MHz for 1H and 13C respectively.
• the UDCA commercially available standard NMR spectra was consistent with the sample NMR spectra (see figure 5, figure 6, figure 7 and figure 8).
• EXAMPLE 20 CONSTRUCTION OF SACCHAROMYCES CEREVISIAE STRAINS CAPABLE OF EXPRESSING SEP ID NO. 2 AND SEP ID NO. 32
• Plasmid pSAND113 to express a gene encoding a P450 with sequence SEQ ID NO. 33, under control of the Gall promoter, and a gene encoding a P450 reductase with sequence SEQ ID NO. 3, under control of the GallO promoter, was constructed as follows.
• EXAMPLE 21 BIOCONVERSION OF LCA TO UDCA BY SACCHAROMYCES CEREVISIAE SAND 122

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