OPTIMIZATION OF C-8 STEROL ISOMERIZATION

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 yeast strains expressing enzymes having improved C-8 sterol isomerase activity leading to increased ratios of 7-DHC in the sterol mix.

Claims

1. A cholesterol-producing yeast cell comprising an enzyme having C8-sterol isomerase activity, said yeast cell producing a sterol mix comprising at least about 80% 7-dehydrocholesterol (7-DHC), preferably comprising at least about 82, 85, 88, 90, 92, 95, 97, 98 or up to 100% 7-DHC based on the total amount of sterols.

2. A cholesterol-producing yeast cell according to claim 1, wherein the ratio of 7-DHC to cholesta-8-enol is in the range of about 20.

3. A cholesterol-producing yeast cell according to claim 1, wherein the ratio of 7-DHC to cholesta-8-enol is increased by at least about 2-fold.

4. A cholesterol-producing yeast cell according to claim 1 expressing a heterologous enzyme having C8-sterol isomerase activity with 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:6.

5. A cholesterol-producing yeast cell according to claim 4 expressing a heterologous enzyme having C8-sterol isomerase 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, Magneporte, such as Magneporte oryzae, Metarhizium, such as Metarhizium acridum, and Ustilago, such as Ustilago maydis.

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

7. 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.

8. A cholesterol-producing yeast cell according to claim 1, wherein the yeast cell expresses a heterologous enzyme having C5-desaturase activity, preferably wherein the heterologous enzyme is obtainable from Pichia pastoris, more preferably from a polypeptide having 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:14.

9. 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.

10. Use of a cholesterol-producing yeast cell according to claim 9, wherein the 7-DHC is further converted into vitamin D3.

11. Use according to claim 9, wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

12. A process for reducing the amount of cholesta-8-enol in a sterol mix produced by a yeast cell, said process comprising expression of a heterologous enzyme having C8-sterol isomerase 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, Magneporte, such as Magneporte oryzae, Metarhizium, such as Metarhizium acridum, and Ustilago, such as Ustilago maydis, preferably selected from Ustilago maydis, Schizosaccharomyces pombe, Candida albicans, or Saccharomyces cerevisiae.

13. A process for the production of a sterol mix, preferably a vitamin D3-precursor, more preferably a sterol mix with at least 80% 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 424-reductase, more preferably vertebrate sterol Δ24-reductase, (c) expression of a heterologous enzyme having C8-sterol isomerase 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, Magneporte, such as Magneporte oryzae, Metarhizium, such as Metarhizium acridum, and Ustilago, such as Ustilago maydis, preferably selected from Ustilago maydis, Schizosaccharomyces pombe, Candida albicans, or Saccharomyces cerevisiae, (d) cultivating said yeast cell under conditions suitable for sterol production; wherein the ratio of 7-DHC to cholesta-8-enol present in the sterol mix is more than about 8.7.

Description

EXAMPLES

Example 1: General Methods, Strains and Plasmids

[0053] 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 Example 4 erg6Δ::TDH3p- S24R1-PGK1t-URA3 erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted insertion erg6Δ::TDH3p-S24R1-PGK1t-URA3 construct at INT59 locus erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 INT59::HSP26p- S. cerevisiae- ERG2-TDH3t-NAT.sup.R erg5Δ::PGK1p-524R2-CYC1t-TRP1 Targeted insertion erg6Δ::TDH3p-524R1-PGK1t-URA3 construct at INT59 locus erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 INT59::HSP26p-U. maydis- ERG2-TDH3t-NAT.sup.R erg5Δ::PGK1p-524R2-CYC1t-TRP1 Targeted insertion erg6Δ::TDH3p-524R1-PGK1t-URA3 construct at INT59 locus erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 INT59::HSP26p-C. albicans- ERG2-TDH3t-NAT.sup.R erg5Δ::PGK1p-524R2-CYC1t-TRP1 Targeted insertion erg6Δ::TDH3p-524R1-PGK1t-URA3 construct at INT59 locus erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 INT59::HSP26p-S. pombe- ERG2-TDH3t-NAT.sup.R

TABLE-US-00002 TABLE 2 plasmids used for cloning of ERG2 homologs. Plasmid Backbone Insert Oligos or source pMB7677 pMB7621 S. cerevisiae-ERG2 Synthesized fragment pMB7732 pMB7621 U. maydis-ERG2 Synthesized fragment pMB7683 pMB7621 C. albicans-ERG2 Synthesized fragment pMB7681 pMB7621 S. pombe-ERG2 Synthesized fragment

Example 2: Cloning of Various ERG2 Homologs into S. cerevisiae Y2159

[0054] All ERG2 cassettes were constructed as follows. Open reading frames were codon-optimized based on the deduced amino acid sequence and synthesized with 5′-XbaI (TCTAGAACAAAatg . . . ) sites and 3′-PstI sites). These were cloned by inserting XbaI-PstI-digested ERG2 fragments into XbaI-PstI-digested pMB7621, which allows targeting to the intergenic locus INT59 on chromosome XI between the SRP40 and PTR2 genes (ca. position 615,000).

[0055] Besides S. cerevisiae ERG2 (SEQ ID NO:1; plasmid pMB7677), the genes synthesized comprise ERG2 homologues (codon-optimized) from Ustilago maydis (SEQ ID NO:9; plasmid pMB7732), Candida albicans (SEQ ID NO:10; plasmid pMB7683), and Schizosaccharomyces pombe (SEQ ID NO:11; plasmid pMB7681), see sequence listing.

[0056] To test the impact of the different ERG2 genes in 7-DHC production, strain Y2159 was transformed with four different SfiI-generated fragments, representing one of the four species detailed above, at the INT59 locus using nourseothricin resistance (NatR) as a selectable marker, and the strong constitutive HSP26 promoter as a controlling element.

[0057] Transformants were selected on YPD agar with 200 mg/L nourseothricin 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

[0058] 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 OD.sub.600 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.

[0059] Sterols from the cultures were extracted and assayed as follows. Eighty microliters of whole broth were 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 ERG2 homologs. Strain Ratio 7-DHC to zymosterol SC2159-parent 22.9 U. maydi ERG2 28.4 ratios of 7-DHC to cholesta-8-enol in control and strains carrying ERG2 homologs. Strain Ratio 7-DHC to Cholesta-8-enol SC2159-parent 8.7 U. maydis ERG2 38.1 C. albicans ERG2 21.9 S. pombe ERG2 21 S. cerevisiae ERG2 24.4

TABLE-US-00004 TABLE 4 ratios of 7-DHC to mix of lanosterol and lathosterol in control and strains carrying ERG2 homologs. Strain Ratio 7-DHC to lanosterol/lathosterol SC2159-parent 12.9 U. maydis ERG2 17.1 S. pombe ERG2 13.6

Example 4: Construction of Y2159

[0060] Wild-type S. cerevisiae ARE1 was synthesized by DNA2.0, incorporating an XbaI site at the 5′ end (TCTAGAACAAAatg . . . ) and a PstI site at the 3′end. This was cloned into an erg4Δ::Hyg.sup.R deletion plasmid using unique XbaI and PstI sites. LEU2 was subsequently used to replace the HygR moiety via a KpnI-AgeI cloning. The result was plasmid pHyD459.

[0061] S. cerevisiae ARE1 mutant variant pMB7584 (F592L) was generated by ligating a BsrGI-BsaI-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-BsaI-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-00005 TABLE 5 plasmids used for construction of ARE mutations. “Scer” means Saccharomyces cerevisiae. Plasmid Backbone Insert Oligos or source SEQ ID NO pHyD459 pHyD445 Scer-ARE1 LEU2 insertion pMB7584 pHyD459 Scer-are1 F592L MO10013     MO10014, 16     17 MO10016     MO10017 19     20 pMB7585 pHyD459 Scer-arel G595D MO10013     MO10015 16     18