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 host 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 with sterol acyltransferase activity having sterol acyltransferase activity comprising one of more amino acid substitution(s) at (a) position(s) corresponding to residues selected from the group consisting of 11, 281, 366, 442, 551, 554, 572, 624, 626, 627, 636, and combinations thereof in the polypeptide according to SEQ ID NO:1, preferably one or more amino acid substitution(s) corresponding to E11G and/or L281I and/or D366V and/or I442V and/or H551Y and/or H554Q and/or F572L and/or F624L and/or L626F and/or G627D and/or C636S and/or combinations thereof.

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 1.4-times 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(s) is/are selected from F624L, G627D, E11G, H554Q, I442V and combinations thereof.

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 to claim 1.

5. A host cell according to claim 4, further 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:3, preferably substitution corresponding to F592L and/or G595D.

6. 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 1.4-times compared to a host cell wherein expressing a non-modified enzyme.

7. A host cell according to claim 3, wherein ERG5 and ERG6 are inactivated.

8. A host cell according to claim 3, 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.

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 40%.

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 1.4-times compared to a process using the respective non-modified enzyme and host cell, respectively.

Description

FIGURES

[0057] FIG. 1. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #2 and #1 (see Table 1). With regards to esters of 7-DHC (“7-DHC ester”) and esters of zymosterol (“Zym ester”) two ester forms were detected as indicated by dark and light grey in the respective columns, free 7-DHC is indicated in black. (A) ratio of 7-DHC to ester formation, (B) ratio of total 7-DHC (including free and ester forms) to total zymosterol esters, (C) formation of free 7-DHC, 7-DHC esters and zymosterol esters shown by different columns. Strains were cultivated for two days with two glucose feedings in flasks without baffles. Data are mean values of 3 independent transformants each cultivated once.

[0058] FIG. 2. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #2 and #1 (see Table 1). For further details see legend to FIG. 1. Strains were cultivated for four days with one glucose feeding in flasks without baffles. Data are mean values of 2 independent transformants each cultivated once.

[0059] FIG. 3. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #9 and #1 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0060] FIG. 4. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 ion variants #24 and #20 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0061] FIG. 5. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #22 and #27 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0062] FIG. 6. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #20, #22, #27 and #22 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0063] FIG. 7. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #34, #32, #43, #24, #20, #22, and #27 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0064] FIG. 8. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #9, #35, #36, #39, and #40 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0065] FIG. 9. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #27, #33, #34, #37, and #38 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0066] FIG. 10. HPLC analysis of lipid extracts of ARE2 wild-type strain(“WT”) and Are2 variants #41, #42, #43, #24, and #20 (see Table 1). For further details see legend to FIG. 1. HPLC analysis according to standard procedure. For further details see text.

[0067] The following examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1: Generation and Screening of ARE2 Mutants

[0068] An error prone library of 10,000 yeast clones expressing variants of Saccharomyces cerevisiae acyltransferase 2 (ScAre2) were screened by thin layer chromatography (TLC) for improved 7-DHC content in sterol ester fraction (for wild-type sequences of ARE1 and ARE2 (see sequence listing). The screening method comprises the simultaneous extraction and separation of sterols from cells with slightly digested cell walls. The treated biomass was directly applied on the TLC plate and immersed into the solvent, which did the extraction and separation of sterol containing fractions in one step. In the sterol ester fraction the ratio of sterols with conjugated double bonds (as e.g. 7-DHC) was set into relation to sterols without conjugated double bonds by exploiting the different spectrophotometric properties of the compounds with the conjugated double bonds (e.g. ability to quench fluorescence, UV detection).

[0069] The best variants were re-screened in quintuplicates, sequenced, cultivated in shake flasks and analyzed in biological triplicates by HPLC-UV to determine the sterol and sterol ester compositions.

[0070] Plasmids containing the best variants were isolated and re-transformed into a cholesta-5,7,24-trienol producing Saccharomyces cerevisiae strain 10A (are1 are2 erg5 erg6::24R; for construction see Example 1 in WO2017108799). Mutations of variants with multiple amino acid exchanges were separated by introducing respective mutation into ARE2 by site-directed mutagenesis (silent mutations were not taken into account) to find out which mutation caused the desired effect. Strains were cultivated and analyzed by HPLC.

Example 2: HPLC-UV Analysis Standard Procedure

[0071] Pre-cultures—10 ml YPD with geneticin (100 μg/mL)—were inoculated with our strains of interest (3 transformants per Are2 variants) and grown at 30° C. to appropriate density (24 to 48 h). For better comparison, three different transformants with the wild-type ARE2 plasmid were also inoculated, which were transformed at the same time as the variants. Main cultures of 50 mL YPD with geneticin were inoculated to OD.sub.600 0.1 in 250 mL shake flasks without baffles and were cultivated for 3 days with 3 times glucose feeding (glucose was added to 2% final concentration after approx. 30, 45, and 60 h) with 200 rpm and 80% humidity at 30° C. 200 OD units of biomass were harvested (centrifuged for 5 min with 1600×g and supernatant was removed) in 15 mL Greiner tubes and stored at −20° C. until analysis.

[0072] For extraction, the 200 OD cell pellet was thawed, resuspended in 1 mL zymolyase solution (5 mg/mL zymolyase 20T in 50 mM KPi, pH 7, with 1 M D-sorbitol) and incubated for 15 min at 37° C. (750 rpm on thermomixer). The zymolyase solution was removed after centrifugation (2500×g, 5 min) and 3.73 mL of absolute EtOH were added to the pellet (resuspended with 1 mL by pipetting up and down carefully, then adding additional 2.73 mL). 267 μL of internal standard (cholesteryl acetate, 1 mg/mL in EtOH) were added, the cell suspension was vortexed and heated to 70° C. for 1 h with mixing (750 rpm on thermomixer). After some minutes of leaving the tubes to cool down to room temperature, the cell debris was pelleted (2500×g, 10 min at room temperature) and 3 mL of the supernatant were transferred into Pyrex tubes which were brought to dryness under N2. The lipids were taken up in 200 μL of ethyl acetate (vortexed and mixed with 750 rpm on a thermomixer at 40° C. for 15 min). The solution was centrifuged once more (2500×g, 5 min) and transferred into a glass vial with inlay for the subsequent HPLC-UV analysis.

[0073] Lipid extracts were analyzed by HPLC with UV detection at two wavelengths (210 nm and 280 nm). Zymosterol compounds were detected at 210 nm, 7-DHC compounds were quantified at 280 nm. [0074] Solvent: 80% EtOH 20% MeOH 0.1% TFA [0075] Column: YMC-Pack Pro C18 RS [0076] Method: injection volume: 10 μL [0077] injector thermostat: 40° C. [0078] flow: 0.6 mL/min [0079] column thermostat: 20° C. [0080] UV detection: 210 nm, 280 nm (sterols with conjugated double bonds)

[0081] Standard mixtures of 7-DHC, zymosterol, cholesteryl acetate and squalene in 3 different concentrations (0.5, 1.0, and 2.0 mg/mL of each substance) were analyzed as well and standard curves were generated for each substance to calculate the concentration in μg/μL sterol in extract or μg/OD.sub.600.

Example 3: Evaluation of ARE2 Variants with Regards to Activity and/or Specificity

[0082] For direct comparison, the wild-type ARE2 plasmid was re-transformed along with the plasmids expressing Are2 variants (see Ex. 1) into strain 10A and the resulting strains were analyzed (see Ex. 2) in the same run. The results of the HPLC analyses are summarized in Table 1 and FIGS. 1-10. Values in the table give the fold change between strains expressing the mutant/variant compared to the wild type. The first value indicates improvements regarding the ratio between the ester fraction and the free 7-DHC fraction while the second value shows the improvement concerning the ratio of 7-DHC and zymosterol in the ester fractions. The third value is a comparison of total 7-DHC content in biomass of mutants and wild type. Some of the listed variants showed especially improvement in the ester level while others showed improvements in the 7-DHC/zymosterol ratio in the ester fraction.

TABLE-US-00001 TABLE 1A Summary of relative ester formation of Are2 variants based on several independent experiments. “Relative ester formation” means the x-times increase in the percentage of 7-DHC based on the total amount of sterols generated using the indicated amino acids exchange instead of a wild-type ARE2.; “7-DHC-ester/zym-ester” means ratio of esters from 7-DHC towards esters of zymosterol (“zym”); “7-DHC-total/zym-total” is the ratio of total 7-DHC (free and esters) towards total zymosterol (free and esters). For more explanation, see text. Relative 7-DHC- 7-DHC- ester ester/ total/ # AA exchange(s) formation zym-ester zym-total 1 H554Q 1.2 1.0-2.0 3.0-4.2 9 H554Q-F572L 1.2 1.7-3.4 3.9-8.5 15 L281I 1.5 24 I442V-L626F 3.5 1.0-1.3 1.7-2.4 27 E11G-D366V-C636S 2.2 1.2-1.5 2.3-3.4 32 E11G-D366V-F624L-C636S 1.3 5.3 12.7 33 E11G-D366V-F624L-C636S 1.5 6.0 15.4 34 E11G-D366V-G627D-C636S 1.4 7.1-7.3 16.417.8 37 E11G-D366V-I442V-F624L- 1.6 5.4 13.9 C636S 38 E11G-D366V-I442V-G627D- 1.3 7.1 20.5 C636S 41 I442V-F624L-L626F 1.9 4.8 10.4 42 I442V-L626F-G627D 2.5 3.4  6.7 43 I442V-G627D 1.9 5.5-5.9 11.76-11.7 

TABLE-US-00002 TABLE 1B Summary of specificity of Are2 variants based on several independent experiments. The number indicates the x-times increase in the percentage of 7-DHC compared to the percentage of zymosterol in the sterol mx generated using the indicated amino acids exchange instead of a wild-type ARE2. For more explanation, see text. # AA exchange(s) Specificity 1 H554Q 1.4 2 V286V-H551Y-F572L-S633S 1.7 9 H554Q-F572L 1.8 20 F624L 3.9 22 G627D 4.4 32 E11G-D366V-F624L-C636S 3.1 33 E11G-D366V-F624L-C636S 3.8 34 E11G-D366V-G627D-C636S 4.4 35 E11G-F624L 3.2 36 E11G-G627D 4.2 37 E11G-D366V-I442V-F624L-C636S 3.4 38 E11G-D366V-I442V-G627D-C636S 4.5 39 H554Q-F572L-F624L 12.2 40 H554Q-F572L-G627D 5.0 41 I442V-F624L-L626F 3.1 42 I442V-L626F-G627D 2.2 43 I442V-G627D 3.5

TABLE-US-00003 TABLE 1C Summary of total 7-DHC production of Are2 variants based on several independent experiments. The number indicates the x-times increase in the total amount of produced 7-DHC generated using the indicated amino acids exchange instead of a wild-type ARE2. For more explanation, see text. Total 7-DHC # AA exchange(s) production 1 H554Q 1.0 20 F624L 1.0 22 G627D 1.0 24 I442V-L626F 1.3 27 E11G-D366V-C636S 1.2 32 E11G-D366V-F624L-C636S 1.4 33 E11G-D366V-F624L-C636S 1.4 34 E11G-D366V-G627D-C636S 1.3 35 E11G-F624L 1.1 36 E11G-G627D 1.1 37 E11G-D366V-I442V-F624L-C636S 1.4 38 E11G-D366V-I442V-G627D-C636S 1.1 39 H554Q-F572L-F624L 1.0 40 H554Q-F572L-G627D 1.3 41 I442V-F624L-L626F 1.4 42 I442V-L626F-G627D 1.4 43 I442V-G627D 1.4