Optimization of C-5 sterol desaturation

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 expressing (i) a heterologous C5-sterol desaturase with at least 90% sequence identity to SEQ ID NO: 2, 4, 6 or 8 and (ii) a heterologous C8-isomerase with at least 90% sequence identity to SEQ ID NO:_14, wherein the yeast cell is capable of producing a sterol mix comprising 7-dehydrocholesterol (7-DHC), lanosterol, and lathosterol, wherein the ratio of 7-DHC to lanosterol and lathosterol combined is at least 5% higher than the ratio of 7-DHC to lanosterol and lathosterol combined in a sterol mix produced by a reference yeast cell that does not express the heterologous C5-sterol desaturase and C8-isomerase.

2. The cholesterol-producing yeast cell according to claim 1, wherein at least 84% of the sterol mix produced is 7-DHC.

3. The cholesterol-producing yeast cell according to claim 1, wherein 7-DHC is produced at a ratio to lanosterol of 18 or more.

4. The cholesterol-producing yeast cell according to claim 1, wherein the heterologous C5-sterol desaturase is from S. cerevisiae, Y. lipolytica, K. lactis, S. pombe, P. pastoris, C. albicans, P. roqueforti, A. nidulans, C. neoformans, M. oryzae, M. acridum, or U. maydis.

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

6. The cholesterol-producing yeast cell according to claim 1, wherein the yeast cell further expresses a heterologous plant or vertebrate sterol 424-reductase.

7. A process for production of 7-DHC, comprising cultivating the yeast cell according to claim 1 under conditions suitable for sterol production.

8. The process according to claim 7, wherein the 7-DHC is further converted into vitamin D3.

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

10. A process for production of a sterol mix, said process comprising cultivating the yeast cell according to claim 4 under conditions suitable for sterol production.

11. A process for production of a sterol mix, comprising cultivating the yeast cell according to claim 6 under conditions suitable for sterol production, wherein (a) ERG5 and ERG6 are inactivated in the yeast cell, (b) the heterologous sterol ?24-reductase is a vertebrate sterol ?24-reductase, and (c) the heterologous C5-sterol desaturase is from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe, or Saccharomyces cerevisiae.

12. The cholesterol-producing yeast cell according to claim 1, wherein (a) the heterologous C5-sterol desaturase is from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe, or Saccharomyces cerevisiae, (b) ERG5 and ERG6 are inactivated in the yeast cell, and (c) the yeast cell expresses a heterologous vertebrate sterol ?24-reductase.

13. A process for production of a sterol mix comprising cultivating the yeast cell according to claim 12 under conditions suitable for sterol production.

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 Y2159 was constructed as described in Example 4. All listed strains are MAT?.

(2) TABLE-US-00001 TABLE 1 Saccharomyces cerevisiae strains. Y2159 erg5?::PGK1p-S24R2-CYC1t-TRP1 See Example erg6?::TDH3p-S24R1-PGK1t-URA3 4 erg4?::PGK1p-Scer-are1 G595D-CYC1t-LEU2 TDH3p-tHMG1 Y2346 erg5?::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6?::TDH3p-S24R1-PGK1t-URA3 insertion erg4?::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-S. cerevisiae- INT66 locus ERG3-PGK1t-HYG.sup.R Y2322 erg5?::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6?::TDH3p-S24R1-PGK1t-URA3 insertion erg4?::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-P. pastoris- INT66 locus ERG3-PGK1t-HYG.sup.R Y2316 erg5?::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6?::TDH3p-S24R1-PGK1t-URA3 insertion erg4?::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-P. roqueforti- INT59 locus ERG3-PGK1t-HYG.sup.R Y2337 erg5?::PGK1p-S24R2-CYC1t-TRP1 Targeted erg6?::TDH3p-S24R1-PGK1t-URA3 insertion erg4?::PGK1p-Scer-are1 G595D-CYC1t-LEU2 construct at TDH3p-tHMG1 INT66 TDH3p-S. pombe-ERG3- INT66 locus PGK1t-HYG.sup.R

(3) 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

(4) 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).

(5) 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.

(6) 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.

(7) 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

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

(9) 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.

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

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

(12) TABLE-US-00005 TABLE 4 ratios of 7-DHC to mix of lanosterol and lathosterol in control and strains carrying ERG3 homologs. Strain Ratio 7-DHC to 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

(13) WT S. cerevisiae ARE1 was synthesized by DNA2.0, incorporating an XbaI site at the 5 end (TCTAGAACAAAatg . . . ) and a PstI site at the 3end. This was cloned into an erg4?::HygR 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.

(14) 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.

(15) TABLE-US-00006 TABLE 5 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