Whole-cell system for cytochrome P450 monooxygenases biocatalysis

Abstract

The subject of the present invention is a whole-cell catalysis process for converting substrates of cytochrome P450 monooxygenases of eukaryotic origin into valuable biotechnological products. The subject of the present invention is also microorganisms genetically engineered to achieve those biotransformations with high rates and processes to prepare these microorganism strains.

Claims

1. A genetically engineered microorganism capable of converting cholesterol, cholesterol analogs, and derivatives thereof into steroid hormones precursors, wherein said microorganism comprises at least one DNA sequence encoding a cytochrome P450 of eukaryotic origin, an exogenous DNA sequence encoding Adx, and an exogenous DNA sequence encoding AdR, wherein said microorganism is Bacillus megaterium, wherein said cytochrome P450 of eukaryotic origin is selected from the group consisting of CYP11A1, CYP17A1, CYP11B1, CYP11B2, CYP3A4, CYP46A1, CYP27A1, CYP21A1, and CYP21A2, and wherein the gene PhaC is overexpressed by introducing into said microorganism an exogenous DNA sequence encoding PhaC.

2. The genetically engineered microorganism according to claim 1, wherein said exogenous DNA sequences have been introduced into said microorganism by means of genetic engineering techniques.

3. The genetically engineered microorganism according to claim 2, wherein said microorganism has been transformed with at least one plasmid comprising said exogenous DNA sequences.

4. The genetically engineered microorganism according to claim 1, wherein said exogenous DNA sequences are integrated into the genome of said microorganism.

5. The genetically engineered microorganism according to claim 1, wherein said microorganism further comprises a functional endogenous polymerase system capable of building polyhydroxyalkanoate bodies.

6. The genetically engineered microorganism according to claim 1, wherein said microorganism is Bacillus megaterium MS941 strain.

7. A method for producing steroid hormones precursors, comprising the steps of: a. Providing a microorganism according to claim 1, b. Culturing said microorganism under conditions allowing the expression of said exogenous DNA sequences, c. Contacting said microorganism culture with a substrate selected from the group consisting of cholesterol, cholesterol analogs, and derivatives thereof, and d. Recovering steroid hormones precursors.

8. The method according to claim 7, wherein: a. said substrate is selected from the group consisting of cholesterol, campesterol, ergostadienol, desmosterol, beta-sitosterol, generol and a mixture of oxysterols, and b. said steroid hormones precursor is pregnenolone.

9. The method according to claim 7, wherein said substrate is solubilized into -cyclodextrin and saponin.

10. A method of preparing a genetically engineered microorganism according to claim 1 capable of converting cholesterol, cholesterol analogs, and derivatives thereof into steroid hormones precursors, comprising the steps of: a. Providing a microorganism, wherein said microorganism is Bacillus megaterium; and b. Introducing by means of genetic engineering techniques into said microorganism at least one DNA sequence encoding a cytochrome P450 of eukaryotic origin, an exogenous DNA sequence encoding Adx, an exogenous DNA sequence encoding AdR, and an exogenous DNA sequence encoding PhaC, to overexpress gene PhaC, wherein said cytochrome P450 of eukaryotic origin is selected from the group consisting of CYP11A1, CYP17A1, CYP11B1, CYP11B2, CYP3A4, CYP46A1, CYP27A1, CYP21A1, and CYP21A2.

11. The method according to claim 10, wherein said microorganism comprises a functional endogenous polymerase system capable of building polyhydroxyalkanoate bodies.

12. The method according to claim 10, wherein said microorganism is Bacillus megaterium MS941 strain.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Immunostainings of CYP11A1 (A) and AdR/Adx (B); Lane 1: wildtype strain of B. megaterium MS941; lane 2: strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(2) FIG. 2: HPLC chromatogram showing the conversion of cholesterol (S for substrate) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(3) FIG. 3: HPLC chromatogram showing the conversion of 7-dehydrocholesterol (S for substrate) to 7-dehydro pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(4) FIG. 4: HPLC chromatogram showing the conversion of campesterol (S for substrate) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(5) FIG. 5: HPLC chromatogram showing the conversion of ergostadienol (S for substrate) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(6) FIG. 6: HPLC chromatogram showing the conversion of desmosterol (S for substrate) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(7) FIG. 7: HPLC chromatogram showing the conversion of mixture of oxysterols (S1, S2 and S3 for the different oxysterol substrates) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(8) FIG. 8: HPLC chromatogram showing the conversion of ergosterol (S for substrate) to hydroxy-ergosterol (P1, P2, P3 and P4 for the different hydroxylated forms of products) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(9) FIG. 9: HPLC chromatogram showing the conversion of vitamin D (S for substrate) to hydroxyl-vitamin D (P1 and P2 for the different hydroxyl-vitamin D forms) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA (Solid line) compared to B. megaterium MS941 (Dotted line).

(10) FIG. 10: HPLC chromatogram showing the conversion of beta-sistosterol (S for substrate) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(11) FIG. 11: HPLC chromatogram showing the conversion of stigmasterol (S for substrate) to hydroxyl-stigmasterol (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(12) FIG. 12: HPLC chromatogram showing the conversion of generol 100 (S1, S2 and S3 for three of the different sterols contained in general 100) to pregnenolone (P for product) by strain of B. megaterium MS941 transformed with pSMF2.1_SCCAA.

(13) FIG. 13: Comparison of CYP11A1 in vivo activity in absence and presence of polyhydroxybutyrate-bodies (KO: PhaC knockout strain of B. megaterium MS941; WT: wildtype of B. megaterium MS941 strain) after 24 and 48 hours of reaction.

(14) FIG. 14: Restoration of substrate conversion activity in a PHB-depleted strain (MS941_deltaphaC) overexpressing CYP11A1 and its redox partners; comparison of CYP11A1 in vivo activity in absence (Structure Knockout strain(MS941_deltaphaC)+CYP11A1) and presence of polyhydroxybutyrate-bodies (Wildtype strain+CYP11A1) and in a strain wherein PHB-bodies have been reconstituted by transforming the strain with a plasmid encoding the structure operon (Structure Knockout strain(MS941_deltaphaC)+CYP11A1+reconstitution of structures(pMGBm19_RBCP).

(15) FIG. 15: Map of expression vector pSMF2.1_SCCAA.

(16) FIG. 16: Comparison of pregnenolone yields produced in different microorganisms expression different cytochrome P450.

BRIEF DESCRIPTION OF THE SEQUENCES

(17) SEQ ID NO: 1: Plasmid sequence of pSMF2.1_SCCAA.

(18) SEQ ID NO: 2: Amino acid sequence of CYP11A1.

(19) SEQ ID NO: 3: Amino acid sequence of AdR.

(20) SEQ ID NO: 4: Amino acid sequence of Adx.

EXAMPLES

Example 1: Construction of B. megaterium Expression Plasmid pSMF2.1 SCCAA

(21) For the cloning of the respective genes, the following restriction enzymes were used: CYP11A1: Spel/Mlul AdR: Kpnl/Sacl Adx: BsRGI/Sphl

(22) Plasmid pSMF2.1_SCCAA is derived from Bacillus megaterium shuttle vector pKMBm4 (obtained from the group of Prof. Dr. Dieter Jahn, T U Braunschweig, Stammen S. et al. (2010)). pKMBm4 was modified as as described below:

(23) A) the Pacl-restriction site was deleted by mutagenesis,

(24) B) a new multiple cloning site was inserted, (Bleif et al., 2012)

(25) C) fragments encoding adrenodoxin, adrenodoxin reductase and CYP11A1 were inserted.

(26) All three genes are under the control of the strong-inducible promoter PXyIA. Each gene contains its own ribosomal binding site.

(27) In E. coli, the beta-lactamase is expressed, conferring ampicillin resistance.

(28) In B. megaterium, the tetracycline-resistance protein is expressed, conferring tetracycline resistance.

(29) Plasmid map and sequence of pSMF2.1_SCCAA are respectively provided in FIG. 15 and SEQ ID N 1.

(30) Bacillus megaterium strain MS941 were obtained from the group of D. Jahn/Technische Universitat Braunschweig (Wittchen K. D. und Meinhardt F., 1995) derived from DSM319/Deutsche Stammsammlung von Mikroorganismen und Zellkulturen.

(31) B. megaterium cells were transformed according to the PEG-mediated protoplast transformation by Barg H. et. al (2005).

Example 2: Cultivation Conditions for Recombinant B. megaterium and Samples Treatment

(32) Either LB-(25 g/L), TB-(24 g/L yeast extract, 12 g/L tryptone, 0.4% glycerol, 10 mM potassium phosphate buffer) or EnPresso Tablet medium have been used for the cultivation of B. megaterium. All reagents are listed in table 1 below.

(33) Pre-Culture:

(34) Inoculation of 50 mL medium containing 10 g/mL tetracycline were performed with cells from a plate or glycerol stock.

(35) Main Culture:

(36) Inoculation of 50 mL medium containing 10 g/mL tetracycline were performed with 500 L sample of the pre-culture.

(37) Induction of Protein Expression:

(38) The main culture was grown until an optical density of 0.4 has been reached. Protein expression was induced after addition of 0.25 g xylose dissolved in 1 mL distilled water.

(39) Substrate Solubilization:

(40) Steroids were dissolved in a 45% 2-hydroxypropyl--cyclodextrin/4% Quillaja saponin-solution. 2.5 ml of the solution were added to the culture directly after protein induction leading to a final Quillaja saponin concentration of 0.2% and a final concentration of 2-hydroxypropyl--cyclodextrin of 2.25%.

(41) Sample Treatment for HPLC-Analysis:

(42) Steroids without intrinsic absorption were converted into their .sub.4-3-keto-derivatives, to allow photometric detection at 240 nm.

(43) 1 mL Culture samples was boiled in water for 1 min. 20 l cholesterol oxidase-solution (5 mg cholesterol oxidase and 5 mg Na-cholate dissolved in 5 ml 50 mM HEPES buffer pH 7, containing 0.05% Tween-20) was added and the sample was incubated at 37 C. with 1000 rpm shaking for 1 hour. For HPLC analysis, the sample was extracted twice with 1 mL ethylacetate and the extract was dissolved in the appropriate solvent.

(44) TABLE-US-00001 TABLE 1 List of reagents Reagent Manufacturer 2-Hydroxypropyl--cyclodextrin Sigma-Aldrich (332607) Ampicillin Roth Cholesterol oxidase (Nocardia sp.) CALBIOCHEM EnPresso tablet medium Biosilta Ethylacetate Sigma-Aldrich Glycerol Grssing HEPES Roth K.sub.2HPO.sub.4 Grssing KH.sub.2PO.sub.4 Grssing LB BD Na-cholate SERVA Quillaja saponin Sigma-Aldrich (S7900) Restriction enzymes NEB Tetracycline SERVA Tryptone BD Tween-20 Roth Yeast extract BD

Example 3: Conversion of Cholesterol, Cholesterol Analogs and Derivatives into Pregnenolone by Genetically Engineered B. megaterium MS941 Expressing Bovine CYP11A1 and its Redox Partners Adx and AdR

(45) Bacillus megaterium strain MS941 has been genetically modified to express the steroidogenic enzyme CYP11A1 and its redox partners Adx and AdR. B. megaterium has been transformed with the expression vector pSMF2.1_SCCAA (FIG. 1) comprising genes for CYP11A1, AdR and Adx under the control of the strongly-inducible promoter PXyIA as described in example 1 and encoding respectively for proteins of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. So far, the mitochondrial cytochrome P450 CYP11A1 has not been expressed in Bacillus megaterium.

(46) The expression of all three proteins has been confirmed by immunostaining (FIG. 1).

(47) The following substrates have been solubilized with 2-hydroxypropyl--cyclodextrin and Quillaja saponin and converted in-vivo with the aforementioned recombinant strain: Cholesterol, 7-Dehydrocholesterol, Campesterol, Ergosta-5,24-dienol, Desmosterol, mixture of oxysterols, Ergosterol, Vitamin D, Beta-Sitosterol, Stigmasterol, Generol. (FIGS. 2-12).

(48) As a conclusion, this system allows the whole-cell biocatalysis of the hydrophobic steroids cholesterol, its plant-derived analogs and vitamin D and has the ability to take up high concentrations of highly water-insoluble compounds and convert them at high rates. The product yield of this system is superior to other pregnenolone-producing systems (FIG. 16).

Example 4: Role of Polyhydroxybutyrate-Bodies (PHB-Bodies) into the Conversion of Cholesterol, Cholesterol Analogs and Derivatives into Pregnenolone by Genetically Engineered B. megaterium MS941 Expressing Bovine CYP11A1 and its Redox Partners Adx and AdR

(49) Fluorescent cholesterol analog 25-NBD cholesterol has been shown to localize to the carbon-storage serving PHB-bodies in living B. megaterium-cells (data not shown). In order to localize CYP11A1 in B. megaterium-cells, a fusion protein was expressed in living B. megaterium-cells consisting of the fluorescent protein eGFP and CYP11A1. Similar to 25-NBD cholesterol, CYP11A1eGFP was localized in the PHB-bodies, whose identity was confirmed by nile red staining (data not shown).

(50) The PHB-body producing polymerase PhaC has been deleted from the genome of B. megaterium by homologous recombination. Briefly, a deletion cassette was constructed, consisting of flanking regions of the PhaC gene. This construct was cloned on a plasmid containing a temperature-sensitive origin of replication. After two homologous recombination events, the curing of the plasmid was achieved by incubation at the non-permissive replication temperature. The deletion of the gene was verified by PCR. In order to verify the role of the PHB-bodies in CYP11A1 aggregation and substrate storage, the resulting absence of PHB-bodies in the B. megaterium-cells has been monitored by nile red-staining (data not shown). This morphological change leaded to a drastic decrease in whole-cell conversion activity of the PhaC-knockout strain transformed with pSMF2.1_SCCAA (FIG. 13).

Example 5: Modulation of Polyhydroxybutyrate-Bodies (PHB-Bodies) into a Genetically Engineered B. megaterium MS941 Strain Expressing Bovine CYP11A1 and its Redox Partners Adx and AdR

(51) a-Conversion of Cholesterol, Cholesterol Analogs and Derivatives into Pregnenolone

(52) Substrate conversion activity of the structure-depleted CYP11A1 expressing strain has been restored by transforming it with a plasmid encoding the structure operon encoding for PhaR, PhaB, PhaC and PhaP genes under the control of the original promoter of PhaR, PhaB and PhaC genes (pMGBm19_RBCP plasmid, FIG. 14). The complemented strain exhibited an increase of product yield by more than 100% after 48 hours compared with the structure-depleted strain. The conversion activity was still significantly lower compared with the wildtype strain, since the recombinant expression of the structure operon likely leaded to a reduced expression of CYP11A1 and its redox partners (due to the co-expression of the additional proteins).

(53) These data convincingly illustrates the importance of the granule structures for the whole-cell system.

(54) b-Storage Capacity of Bacillus megaterium MS941 by Modulation of Pha System Genes.

(55) The following strategies are applied:

(56) 1) Phasin gene (in case of Bacillus megaterium phaP) is overexpressed to modify the size of the granules. Phasin gene is inserted into a vector under control of a xylose inducible promoter. Bacillus megaterium is transformed with the resulting plasmid. Yield of substrate conversion into product in the strain overexpressing phaP is measured as described in example 2 and in FIGS. 2-12.

(57) 2) Pha depolymerase gene is knocked-out to hinder the depolymerization of the granules. Pha depolymerase genome locus is amplified via PCR by introducing a deletion of the locus leading to a nonfunctional gene. This nonfunctional DNA fragment is inserted into a knock out vector in order to replace the original genomic functional gene with a nonfunctional one in an gene replacement experiment. Yield of substrate conversion into product in the strain knocked out in the depolymerase gene is measured as described in example 2 and in FIGS. 2-12.

(58) 3) Bacillus megaterium metabolism is engineered to increase the production of Acetyl-CoA, the main precursor of the metabolism for the granule production, by inserting 3-Ketothiolase gene (phaA) into a vector under control of a xylose inducible promoter. Bacillus megaterium is transformed with the resulting plasmid. Yield of substrate conversion into product in the strain overexpressing phaA is measured as described in example 2 and in FIGS. 2-12.

(59) 4) Genes involved in granule synthesis (3-Ketothiolase (phaA), Acetoacetyl-CoA reductase (phaB), Phasin (phaP) and pha synthase (phaC/phaR) are overexpressed. The above-mentioned genes are inserted into a vector under control of a xylose inducible promoter. Bacillus megaterium is transformed with the resulting plasmid. Yield of substrate conversion into product in the strain overexpressing phaA, phaB, phaP, phaC and phaR is measured as described in example 2 and in FIGS. 2-12.

(60) 5) More active pha synthases from other organisms are introduced into Bacillus megaterium MS941 strain to produce more polymer. Pha genes, from Ralstonia eutropha, Pseudomonas aeruginosa and Allochromatium vinosum are used. Exogenous pha synthases genes are inserted into a vector under control of a xylose inducible promoter. Bacillus megaterium is transformed with the resulting plasmid. Yield of substrate conversion into product in the strain overexpressing pha synthase(s) from other microorganisms is measured as described in example 2 and in FIGS. 2-12.

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