MODIFIED STEROL ACYLTRANSFERASES

Abstract

The present invention is related to modified sterol acyltransferase enzymes with improved activity and/or specificity towards acylation of the vitamin D3 precursor 7-dehydrocholesterol (7-DHC) to be used in biotechnological production of vitamin D3. The invention further relates to a yeast strain expressing said modified enzymes and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.

Claims

1. A modified enzyme having sterol acyltransferase activity comprising one of more amino acid substitution(s) at (a) position(s) corresponding to residues selected from 592 and/or 595 in the polypeptide according to SEQ ID NO:1, preferably substitution corresponding to F592L and/or G595D.

2. A modified enzyme according to claim 1 catalyzing the esterification of sterols comprising 7-dehydrocholesterol (7-DHC) and zymosterol, wherein the ratio of 7-DHC to zymosterol in the sterol esters is increased by at least about 15% compared to the ratio of 7-DHC to zymosterol in the catalysis using the respective non-modified enzyme.

3. A modified enzyme according to claim 1, wherein the amino acid substitution is selected from G595D.

4. A host cell, preferably a yeast, more preferably a sterol-producing yeast, even more preferably a cholesterol-producing yeast, comprising a modified enzyme according claim 1.

5. A host cell according to claim 4 used for production of a sterol mix comprising 7-DHC and zymosterol, wherein the ratio of 7-DHC to zymosterol is increased by at least about 15% compared to a host cell wherein expressing a non-modified enzyme.

6. A host cell according to claim 4, wherein ERG5 and ERG6 are inactivated.

7. A host cell according to claim 4, wherein the 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 host cell according to claim 4, wherein the host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia, preferably selected from Saccharomyces cerevisiae, Schizosaccharomyces spp., Pichia spp., Kluyveromyces spp., Hansenula spp. or Yarrowia lipolytica.

9. A process for reducing the percentage of zymosterol in a sterol mix comprising zymosterol and 7-DHC comprising cultivating a host cell according to claim 4 under suitable conditions and optionally isolating and/or purifying the 7-DHC from the sterol mix.

10. A process for increasing the percentage of 7-DHC in a sterol mix comprising 7-DHC and zymosterol comprising cultivating a host cell according to claim 4 under suitable conditions and optionally isolating and/or purifying the 7-DHC from the sterol mix.

11. A process for production of 7-DHC comprising enzymatic conversion of acetyl-CoA into a sterol mix comprising zymosterol and 7-DHC with a host cell according to claim 4, wherein the percentage of 7-DHC in the sterol mix is at least 70%.

12. A process according to claim 11, wherein the 7-DHC is further converted into vitamin D3.

13. A process according to claim 11, wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

14. Use of a modified enzyme according to claim 1 in a process for production of 7-DHC, wherein the 7-DHC is isolated from a sterol mix comprising zymosterol and 7-DHC, and wherein the ratio of 7-DHC to zymosterol is increased by at least about 15% compared to a process using the respective non-modified enzyme and host cell, respectively.

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 Y2140 was constructed starting from a wildtype CEN.PK background strain. Into this wildtype strain an erg5 disruption cassette was transformed that contained a codon-optimized gene for a sterol 24-reductase from zebrafish flanked by a PGK1 promoter and a CYC1 terminator in combination with TRP1. Subsequently, an erg6 disruption cassette was transformed that contained the gene for a sterol 24-reductase from rat flanked by a TDH3 promoter and a PGK1 terminator in combination with LEU2. All mentioned strains are MATα, and harbor an overexpressed copy of the truncated constitutively active HMG-CoA reductase gene (tHMG1).

TABLE-US-00001 TABLE 1 Saccharomyces cerevisiae strains. Y2159 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Classical and erg6Δ::TDH3p-S24R1-PGK1t-URA3 standard erg4Δ::Hyg.sup.R TDH3p-tHMG1 molecular and genetic techniques Y2017 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 replacement erg4Δ::PGK1p-Scer-ARE1-CYC1t-LEU2 with LEU2 TDH3p-tHMG1 cassette Y2157 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 replacement erg4Δ::PGK1p-Scer-are1 F592L-CYC1t-LEU2 with LEU2 TDH3p-tHMG1 cassette Y2159 erg5Δ::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6Δ::TDH3p-S24R1-PGK1t-URA3 replacement erg4Δ::PGK1p-Scer-are1 G595D-CYC1t-LEU2 with LEU2 TDH3p-tHMG1 cassette

TABLE-US-00002 TABLE 2 plasmids used for construction of ARE mutations. “Scer” means Saccharomyces cerevisiae. SEQ Plasmid Backbone Insert Oligos or source ID NO pHyD459 pHyD445 Scer-ARE1 LEU2 insertion pMB7584 pHyD459 Scer-are1 MO10013 & MO10014, 16 & 17 F592L MO10016 & MO10017 19 & 20 pMB7585 pHyD459 Scer-are1 MO10013 & MO10015 16 & 18 G595D

Example 2: Construction of ARE1-WT Plasmid pHyD459

[0054] WT 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 Kpnl-Agel cloning.

Example 3: Cloning of ARE1 Mutant Genes

[0055] 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 a 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 the sequence listing.

Example 3: Introduction of ARE1 WT and Mutant Genes into Saccharomyces cerevisiae

[0056] To the test the impact of the mutant ARE1 genes in 7-DHC production in comparison to the WT gene, strain Y2140 was transformed with three different constructs:

[0057] (1) a SalI fragment of plasmid pHyD459 which is an erg4 disruption construct harboring the WT ARE1 gene under the control of the PGK1 promoter and LEU2.on construct harboring the WT ARE1 gene under the control of the PGK1 promoter and LEU2;

[0058] (2) a SalI fragment of plasmid pMB7584 which is an erg4 disruption construct harboring the are1 F592L gene under the control of the PGK1 promoter and LEU2are1 F592L gene under the control of the PGK1 promoter and LEU2;

[0059] (3) a SalI fragment of plasmid pMB7585 which is an erg4 disruption construct harboring the are1 G595D gene under the control of the PGK1 promoter and LEU2are1 G595D gene under the control of the PGK1 promoter and LEU2.

[0060] Transformants were selected on (minimal media) at 30° C. and screened for hygromycin sensitivity. 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 in Example 4 below.

Example 4: HPLC Analysis of Sterols in ARE Mutant Strains

[0061] 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.

[0062] For extraction of sterols from the cultures 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 (see Table 3).

TABLE-US-00003 TABLE 3 ratios of 7-DHC to selected sterol intermediates in control and ARE1 and/or ARE2 mutant strains. “Lano-/ latho” means a mix of lanosterol and lathostherol, “zym” means zymosterol. Numbers are in mg/ml of sterols. Ratio Ratio 7- lano-/ Cholesta- 7-DHC DHC to Mutant 7-DHC latho 8-enol zym to zym lano/latho ARE1 wt 1060 70 99 92 12 15 are2-F624L 1090 35 122 80 14 31 are2-G627D 1141 50 140 82 14 23 are1-F592L 1240 62 143 105 14 20 are1-G595D 1448 104 152 77 19 14

[0063] As the result of a screen of various S. cerevisiae Are1 mutants, the inventors found a number of Are1 variants that, when expressed, produce 7-DHC with a higher overall productivity, less accumulation of sterol side products (zymosterol, lathosterol, lanosterol, cholesta-8-enol, etc), or both.