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OPTIMIZATION OF C-5 STEROL DESATURATION

20210222222 · 2021-07-22

    Inventors

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    Abstract

    The present invention is related to an improved method for production of 7-dehydrocholesterol (7-DHC), an important intermediate towards biotechnological production of vitamin D3 or derivatives/metabolites thereof. The invention features modified host strains expressing enzymes having improved C-5 sterol 5 desaturase activity and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.

    Claims

    1. A cholesterol-producing yeast cell comprising an enzyme having C5-sterol desaturase with at least about 45%, such as e.g. at least 50, 52, 60, 70, 80, 90, 92, 95, 98 or up to 100% identity to SEQ ID NO:2 being (heterologous) expressed in a suitable host cell for production of 7-dehydrocholesterol (7-DHC), wherein the ratio of 7-DHC to side-products including lanosterol and/or lathosterol is increased by at least about 5% compared to a non-modified host cell.

    2. A cholesterol-producing yeast cell according to claim 1, comprising an enzyme having C5-sterol desaturase activity, said yeast cell producing a sterol mix comprising at least about 84% 7-DHC, preferably comprising at least about 85, 88, 90, 92, 95, 97, 98 or up to 100% 7-DHC based on the total amount of sterols.

    3. A cholesterol-producing yeast cell according to claim 1, wherein the ratio of 7-DHC to cholesta-7-enol and/or lanosterol is in the range of about 18.

    4. A cholesterol-producing yeast cell according to claim 1, wherein the ratio of 7-DHC to cholesta-7-enol and/or lanosterol is increased by at least about 5%.

    5. A cholesterol-producing yeast cell according to claim 1 expressing a heterologous enzyme having C5-sterol desaturase activity with least about 45%, such as e.g. at least 50, 52, 60, 70, 80, 90, 92, 95, 98 or up to 100% identity to SEQ ID NO:2.

    6. A cholesterol-producing yeast cell according to claim 4 expressing a heterologous enzyme having C5-sterol desaturase activity, said enzyme being selected from the group consisting of Saccharomyces, such as Saccharomyces cerevisiae, Yarrowia, such as Y. lipolytica, Klyveromyces, such as K. lactis, Schizosaccharomyces, such as Schizosaccharomyces pombe, Pichia, such as P. pastoris, Candida, such as C. albicans, Penicillium, such as P. roqueforti, Aspergillus, such as A. nidulans, Cryptococcus, such as C. neoformans, such as Magneporte oryzae, Metarhizium, such as Metarhizium acridum, and Ustilago, such as Ustilago maydis.

    7. A cholesterol-producing yeast cell according to claim 1 in which ERG5 and ERG6 are inactivated.

    8. A cholesterol-producing yeast cell according to claim 1, wherein the yeast cell expresses a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24-reductase activity, preferably wherein the heterologous enzyme is originated from plant or vertebrate, more preferably originated from human, pig, dog, mouse, rat, horse or Danio rerio.

    9. A cholesterol-producing yeast cell according to claim 1, wherein the yeast cell expresses a heterologous enzyme having C8-isomerase activity, preferably wherein the heterologous enzyme is obtainable from Ustilago maydis, more preferably from a polypeptide having at least about 42%, such as e.g. at least 43, 44, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 75, 80, 90, 92, 95, 98 or up to 100% identity to the polypeptide according to SEQ ID NO:14.

    10. Use of a cholesterol-producing yeast cell according to claim 1 for production of sterols, preferably for the production of vitamin D3 precursors, more preferably for the production of 7-DHC.

    11. Use of a cholesterol-producing yeast cell according to claim 10, wherein the 7-DHC is further converted into vitamin D3.

    12. Use according to claim 10, wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

    13. A process for reducing the amount of cholesta-7-enol and/or lanosterol in a sterol mix produced by a yeast cell, said process comprising expression of a heterologous enzyme having C5-sterol desaturase activity, said enzyme being selected from the group consisting of Saccharomyces, such as Saccharomyces cerevisiae, Yarrowia, such as Y. lipolytica, Klyveromyces, such as K. lactis, Schizosaccharomyces, such as Schizosaccharomyces pombe, Pichia, such as P. pastoris, Candida, such as C. albicans, Penicillium, such as P. roqueforti, Aspergillus, such as A. nidulans, Cryptococcus, such as C. neoformans, such as Magneporte oryzae, Metarhizium, such as Metarhizium acridum, and Ustilago, such as Ustilago maydis, preferably selected from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe, or Saccharomyces cerevisiae.

    14. A process for the production of a sterol mix, preferably a vitamin D3-precursor, more preferably a sterol mix with at least about 84% 7-DHC, in a yeast cell comprising: (a) inactivation of ERG5 and ERG6, (b) expressing of a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24-reductase activity on cholesta-7,24-dienol, zymosterol or trienol, preferably plant or vertebrate sterol Δ24-reductase, more preferably vertebrate sterol Δ24-reductase, (c) expression of a heterologous enzyme having C5-sterol desaturase activity, said enzyme being selected from the group consisting of Saccharomyces, such as Saccharomyces cerevisiae, Yarrowia, such as Y. lipolytica, Klyveromyces, such as K. lactis, Schizosaccharomyces, such as Schizosaccharomyces pombe, Pichia, such as P. pastoris, Candida, such as C. albicans, Penicillium, such as P. roqueforti, Aspergillus, such as A. nidulans, Cryptococcus, such as C. neoformans, such as Magneporte oryzae, Metarhizium, such as Metarhizium acridum, and Ustilago, such as Ustilago maydis, preferably selected from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe, or Saccharomyces cerevisiae, (d) cultivating said yeast cell under conditions suitable for sterol production; wherein the ratio of 7-DHC to cholesta-7-enol and/or lanosterol present in the sterol mix is more than about 17.2.

    Description

    EXAMPLES

    Example 1: General Methods, Strains and Plasmids

    [0067] All basic molecular biology and DNA manipulation procedures described herein were generally performed according to Sambrook et al. (1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York) or Ausubel et al. (1998. Current Protocols in Molecular Biology. Wiley: New York). Genotyps of the used S. cerevisiae strains and plasmids are listed in Table 1 and 2. Saccharomyces cerevisiae 7-DHC producing strain Y2159 was constructed as described in Example 4. All listed strains are MATα.

    TABLE-US-00001 TABLE 1 Saccharomyces cerevisiae strains. Y2159 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 See erg6Δ::TDH3p-S24R1-PGK1t-URA3 Example 4 erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 Y2346 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 insertion erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-S. cerevisiae- INT66 locus ERG3-PGK1t-HYG.sup.R Y2322 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 insertion erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-P. pastoris- INT66 locus ERG3-PGK1t-HYG.sup.R Y2316 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 insertion erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-P. roqueforti- INT59 locus ERG3-PGK1t-HYG.sup.R Y2337 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 insertion erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-S. pombe-ERG3- INT66 locus PGK1t-HYG.sup.R

    TABLE-US-00002 TABLE 2 plasmids used for cloning of ERG3 homologs. Plasmid Backbone Insert Oligos or source pMB7722 pMB7622 S. cerevisiae-ERG3 Synthesized fragment pMB7700 pMB7622 P. pastoris-ERG3 Synthesized fragment pMB7721 pMB7622 P. roqueforti-ERG3 Synthesized fragment pMB7701 pMB7622 S. pombe-ERG3 Synthesized fragment

    Example 2: Cloning of Various ERG3 Homologs into S. cerevisiae Y2159

    [0068] All ERG3 cassettes were constructed as follows. Open reading frames were codon optimized based on the deduced amino acid sequence and synthesized with 5′-BamHI (GGATCCatg . . . ) sites and 3′-EcoRI sites). These were cloned by inserting BamHI-EcoRI-digested ERG3 fragments into BamHI-EcoRI-digested pMB7621, which allows targeting to the intergenic locus INT66 on the right arm of chromosome XIII between the RKR1 and GAD1 genes (ca. position 769,000).

    [0069] Besides S. cerevisiae ERG3 (SEQ ID NO:7; plasmid pMB7677), the genes synthesized comprise ERG3 homologues (codon-optimized) from Pichia pastoris (SEQ ID NO:9; plasmid pMB7732), Penicillium roqueforti (SEQ ID NO:10; plasmid pMB7721), and Schizosaccharomyces pombe (SEQ ID NO:11; plasmid pMB7681), see sequence listing.

    [0070] To test the impact of the different ERG3 genes in 7-DHC production, strain Y2159 was transformed with four different Sfil-generated fragments, representing one of the four species detailed above, at the INT66 locus using hygromycin resistance (HygR) as a selectable marker, and the strong constitutive TDH3-promoter as a controlling element.

    [0071] Transformants were selected on YPD agar with 200 mg/L hygromycin after 3 days at 30° C. Strains resulting from these transformations are listed in Table 1 above. These strains were subsequently assayed for their 7-DHC productivity and overall 7-DHC sterol purity as described below.

    Example 3: HPLC Analysis of Sterols from Transformed Strains

    [0072] Strains were cultivated as follows. Strains to be tested were initially plated onto YPD agar and incubated for 48 hours at 30° C. Two milliliters YPD pre-cultures were inoculated from these plates and grown on a roller wheel for 24 hours at 30° C. In a 24-well microtiter plate, 0.8 mL of YPD+10 g/L ethanol were inoculated from the preculture to a final OD600 of 0.5. Microtiter plates were grown at 30° C. in a humidified environment and shaking at 800 rpm on a shaker with an orbit of 3 mm. At 24 and 48 hours post-inoculation, 16 μl ethanol was added to each well as a feed. At 72 hours post-inoculation the cells were sampled for sterol content.

    [0073] Sterols from the cultures were extracted and assayed as follows. Eighty microliters of whole broth was pipetted into a 2-mL Precellys tube with glass beads. Eight hundred microliters of saponification solution (5% KOH in ethanol) was added, and samples were placed into a Precellys 24 Homogenizer and agitated at 6500 rpm for 3 cycles at 15 seconds per cycle. Sixty microliters of glacial acetic acid were then added and the tubes were centrifuged for 1 minute at top speed. The supernatant was assayed via HPLC for sterol content. The results are shown in Table 3, 4, and 5.

    TABLE-US-00003 TABLE 3 ratios of 7-DHC to zymosterol in control and strains carrying ERG3 homologs. Ratio 7-DHC to Strain zymosterol SC2159 - parent 18.1 P. pastoris ERG3 18.8

    TABLE-US-00004 TABLE 3 ratios of 7-DHC to cholesta-8-enol in control and strains carrying ERG3 homologs. Ratio 7-DHC to Strain cholesta-8-enol SC2159 - parent 11.7 P. pastoris ERG3 12.1

    TABLE-US-00005 TABLE 4 ratios of 7-DHC to mix of lanosterol and lathosterol in control and strains carrying ERG3 homologs. Ratio 7-DHC to Strain lanosterol/lathosterol SC2159 - parent 17.2 P. pastoris ERG3 22.9 P. roqueforti ERG3 19.8 S. pombe ERG3 18.1

    Example 4: Construction of Y2159

    [0074] WT S. cerevisiae ARE1 was synthesized by DNA2.0, incorporating an Xbal site at the 5′ end (TCTAGAACAAAatg . . . ) and a PstI site at the 3′end. This was cloned into an erg4A::HygR deletion plasmid using unique Xbal and PstI sites. LEU2 was subsequently used to replace the HygR moiety via a Kpnl-Agel cloning. The result was plasmid pHyD459.

    [0075] S. cerevisiae ARE1 mutant variant pMB7584 (F592L) was generated by ligating a BsrGI-Bsal-cleaved PCR product generated from ARE1 (oligos according to SEQ ID NO:16 & 17) with a double-stranded oligo derived by annealing SEQ ID NO:19 and 20 into BsrGI-PstI-cleaved pHyD459. Similarly, S. cerevisiae ARE1 mutant variant pMB7585 (G595D) was generated by ligating a BsrGI-Bsal-cleaved PCR product generated from ARE1 (oligos according to SEQ ID NO:16 & 18) with a double-stranded oligo derived by annealing SEQ ID NO:21 and 22 into BsrGI-PstI-cleaved pHyD459. The oligos as well as further sequences used herein are listed in Table 5.

    TABLE-US-00006 TABLE 5 plasmids used for construction of ARE mutations. “Scer” means Saccharomyces cerevisiae. Oligos or Plasmid Backbone Insert source SEQ ID NO pHyD459 pHyD445 Scer-ARE1 LEU2 insertion pMB7584 pHyD459 Scer-are1 MO10013 custom-character 16 custom-character  17 F592L MO10014, 19 custom-character  20 MO10016 custom-character MO10017 pMB7585 pHyD459 Scer-are1 MO10013 custom-character 16 custom-character  18 G595D MO10015