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 process for production of 7-dehydrocholesterol (7-DHC) comprising enzymatically converting acetyl-CoA into a sterol mix comprising zymosterol and 7-DHC with a host cell, wherein the percentage of 7-DHC in the sterol mix is at least 70%, and wherein the host cell is a recombinant host cell that expresses a modified enzyme selected from EC 2.3.1.26 having sterol acyltransferase activity comprising one or two amino acid substitutions at positions corresponding to residues 592 and 595 in the polypeptide according to SEQ ID NO:1, wherein the substituted amino acid corresponding to residue 592 in the polypeptide according to SEQ ID NO:1 is leucine and the substituted amino acid corresponding to residue 595 in the polypeptide according to SEQ ID NO:1 is aspartic acid.

2. The process of claim 1, wherein the 7-DHC is further converted into vitamin D3.

3. The process of claim 1, wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

4. The process of claim 1, wherein the modified enzyme comprises a modified yeast ARE1 or ARE2 polypeptide.

5. The process of claim 4, wherein the ratio of 7-DHC to zymosterol in the sterol mix is increased by at least 15% compared to a sterol mix produced by the corresponding process using the corresponding host cell that expresses the corresponding non-modified enzyme selected from EC 2.3.1.26 having sterol acyltransferase activity.

6. The process of claim 4, wherein the modified enzyme comprises a modified yeast ARE1 or ARE2 polypeptide, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces spp., Pichia spp., Klyuveromyces spp., Hansenula spp. and Yarrowia lipolytica.

7. The process of claim 6, wherein the modified enzyme comprises a modified Saccharomyces cerevisiae ARE1 or ARE2 polypeptide.

8. The process of claim 7, wherein the modified enzyme comprises the amino acid sequence of SEQ ID NO: 1 comprising the F592L and/or G595D substitution or the amino acid sequence of SEQ ID NO: 3 comprising the F624L and/or G627D substitution.

9. The process of claim 4, wherein the host cell is a recombinant cholesterol-producing yeast cell.

10. The process of claim 4, wherein the host cell is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces spp., Pichia spp., Kluyveromyces spp., Hansenula spp. and Yarrowia lipolytica.

Description

EXAMPLES

Example 1: General Methods, Strains and Plasmids

(1) 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 MAT, and harbor an overexpressed copy of the truncated constitutively active HMG-CoA reductase gene (tHMG1).

(2) TABLE-US-00001 TABLE 1 Saccharomyces cerevisiae strains. Y2159 erg5::PGK1p-S24R2-CYC1t-TRP1 Classical and erg6::TDH3p-S24R1-PGK1t-URA3 standard erg4::Hyg.sup.R TDH3p-tHMG1 molecular and genetic techniques Y2017 erg5::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6::TDH3p-S24R1-PGK1t-URA3 replacement erg4::PGK1p-Scer-ARE1-CYC1t-LEU2 with LEU2 TDH3p-tHMG1 cassette Y2157 erg5::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6::TDH3p-S24R1-PGK1t-URA3 replacement erg4::PGK1p-Scer-are1 F592L-CYC1t-LEU2 with LEU2 TDH3p-tHMG1 cassette Y2159 erg5::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6::TDH3p-S24R1-PGK1t-URA3 replacement erg4::PGK1p-Scer-are1 G595D-CYC1t-LEU2 with LEU2 TDH3p-tHMG1 cassette

(3) 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

(4) 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 erg4::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

(5) 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

(6) 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: (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; (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; (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.

(7) 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

(8) 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 l ethanol was added to each well as a feed. At 72 hours post-inoculation the cells were sampled for sterol content.

(9) 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).

(10) 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

(11) 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.