ENANTIONSELECTIVE ENZYMATIC SULFOXIDATION OF CHIRAL ARYLSULFIDES

Abstract

What is described herein refers to isolated nucleic acid fragments encoding an oxygenase subunit (StyA) and a reductase subunit (StyB), wherein the polypeptide encoded for by the nucleotide sequence for the oxygenase subunit (StyA) and the nucleotide sequence for the reductase subunit (StyB) have activity towards chiral arylsulfides.

Claims

1. An isolated nucleic acid fragment encoding an oxygenase subunit (StyA) and an isolated nucleic acid fragment encoding a reductase subunit (StyB), wherein the polypeptides encoded for by the nucleotide sequences for the oxygenase subunit (StyA) and the nucleotide sequence for the reductase subunit (StyB) together have activity towards chiral arylsulfides and wherein said nucleic acid fragments are selected from the group a) nucleic acid fragment comprising a nucleotide sequence of at least 80% sequence identity to SEQ ID NO 1 and a nucleotide sequence of at least 80% sequence identity to SEQ ID NO 2, b) nucleic acid fragment comprising a sequence complementary to SEQ ID NO 1 and a nucleic acid fragment comprising a sequence complementary to SEQ ID NO 2, c) nucleic acid fragment comprising a sequence which specifically hybridizes to said nucleic acid fragment of a) or said complementary of b).

2. The isolated nucleic acid fragment of claim 1, wherein the chiral arylsulfide towards which the polypeptides encoded for by the nucleotide sequences for the oxygenase subunit (StyA) and the nucleotide sequence for the reductase subunit (StyB) together have activity, is an arylsulfide of formula I ##STR00005## wherein X is C or N, and wherein if X is N, R.sub.1 is absent and if X is C, R.sub.1 is selected form the group consisting of a H, NO.sub.2, a halogen, NH.sub.2, an C.sub.1 to C.sub.6 alkyl, or an an C.sub.1 to C.sub.6 O-alkyl, and wherein n is 0 or 1, and wherein R.sup.2 is an C.sub.1 to C.sub.6 alkyl and wherein R.sup.3 is a halogen or H

3. The isolated nucleic acid fragment of claim 2, wherein the chiral arylsulfide towards which the polypeptides encoded for by the nucleotide sequences for the oxygenase subunit (StyA) and the nucleotide sequence for the reductase subunit (StyB) together have activity, is an arylsulfide of formula I ##STR00006## wherein X is N, and wherein R.sub.1 is absent and wherein n is 1, and wherein R.sup.2 is an C.sub.1 to C.sub.3 alkyl and wherein R.sup.3 is a halogen or H.

4. A recombinant expression vector comprising the isolated nucleic acid fragments of claim 1, wherein said recombinant expression vector is selected from the group a) recombinant expression vector comprising the isolated nucleic acid fragments of claim 1 as well as a lac repressor comprising a nucleotide sequence of at least 80% sequence identity to SEQ ID NO 3, b) recombinant expression vector comprising a nucleotide sequence of at least 80% sequence identity to SEQ ID NO 4 c) recombinant expression vector comprising a sequence complementary to SEQ ID NO 4 d) recombinant expression vector comprising a sequence which specifically hybridizes to said nucleic acid fragment of b) or said complementary of c).

5. The isolated nucleic acid fragments of claim 1, wherein the nucleotide sequence for the oxygenase subunit (StyA) codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 5 and wherein the nucleotide sequence for the reductase subunit (StyB) codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 6.

6. The isolated nucleic acid fragments of claim 1, wherein the nucleotide sequence for the oxygenase subunit (StyA) codes for the polypeptide of SEQ ID NO 5 and wherein the nucleotide sequence for the reductase subunit (StyB) codes for the polypeptide of SEQ ID NO 6.

7. A product comprising the oxygenase subunit (StyA) that codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 5 together with the reductase subunit (StyB) that codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 6 or the oxygenase subunit (StyA) that codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 5 together with a reductase subunit (StyB) that codes for the polypeptide of SEQ ID NO 6 or a oxygenase subunit (StyA) that codes for the polypeptide of SEQ ID NO 5 together with the reductase subunit (StyB) that codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 6 or the oxygenase subunit (StyA) that codes for the polypeptide of SEQ ID NO 5 together with the reductase subunit (StyB) that codes for the polypeptide of SEQ ID NO 6 for the enantioselective oxidation of a compound according to formula I ##STR00007## wherein X is C or N, and wherein if X is N, R.sub.1 is absent and if X is C, R.sub.1 is selected form the group consisting of a H, NO.sub.2, a halogen, NH.sub.2, an C.sub.1 to C.sub.6 alkyl, or an an C.sub.1 to C.sub.6 O-alkyl, and wherein n is 0 or 1, and wherein R.sup.2 is an C.sub.1 to C.sub.6 alkyl and wherein R.sup.3 is a halogen or H into the S-sulfynil-enantiomer.

8. Product according to claim 7 for the enantioselective oxidation of 2-chloro-4-(methylsulfanylmethyl)pyridine to 2-Chloro-4-((methyl-S-sulfinyl)methyl) pyridine.

9. A method for enantioselective oxidation of a compound according to formula I ##STR00008## into a S-sulfynil-enantiomer comprising providing a compound according to formula I, providing the oxygenase subunit (StyA) that codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 5 together with the reductase subunit (StyB) that codes for a polypeptide with at least 80% sequence identity to SEQ ID NO 6 as polypeptide reacting said compound according to formula I with said polypeptide for 2-48 hours, with a pH in a range of 5-9 and the temperature is in a range of 15-40 C.

10. The method according to claim 9, wherein the oxygenase subunit (StyA) and the reductase subunit (StyB) are expressed in recombinant cells provided in a cell culture medium, the compound according to formula I is directly added to the cell culture medium once the cells have reached a predetermined cell density and wherein product accumulates in the cell culture medium.

11. The method according to claim 10 wherein the compound according to formula I is added continuously or at regular time intervals at a concentration which is below the conversion rate of the polypeptide.

12. Method according to claim 10 to provide the S-enantiomer of the oxidized compound according to formula I with an ee-value of more than 95%.

13. Method according to claim 10 to provide the S-enantiomer of the oxidized compound according to formula I at a rate of 10 to 60 g/lh.

Description

FIGURES

[0150] FIG. 1 shows a schematic overview of the reaction of one embodiment of the invention. In this case the substrate is 4-chlorophenyl methyl sulfide. The substrate is added to a cell culture, the reaction takes place inside the cells and the reaction product accumulates in the cell culture medium.

[0151] FIG. 2 depicts a schematic illustration of the employed pStyAB_ADP1_lac vector.

[0152] FIG. 3 shows the specific activity of the styrene monoxygenase from A. baylyi expressed from the pStyABP_ADP1_lac vector (squares) and microbial growth (diamonds) in dependency of the cultivation time.

[0153] FIG. 4 The graph of FIG. 4 demonstrates that product accumulated to very high concentrations exceeding 62 g L1 after 2.3 h of biotransformation. No substrate accumulation was observed within the investigated time period

[0154] FIG. 5: The table of FIG. 5 demonstrates that the maximal specific production rate of the whole cell biocatalysis was surprisingly considerably higher than it could be expected from the literature.

[0155] FIG. 6 The Table of FIG. 6 demonstrates that the styrene monooxygenase A. baylyi described herein converts a variety of different substrates in vitro.

[0156] FIG. 7 The Table of FIG. 7 demonstrates that the styrene monooxygenase of A. baylyi described herein converts a variety of different substrates in vivo.

[0157] FIG. 8 shows the key parameters for the characterization and quantification of cell-free and whole-cell processes as published by Schrewe et al. 2013.

EXAMPLES

[0158] The following examples are illustrative and not limiting. One of skill will recognize a variety of non-critical parameters that can be altered to achieve essentially similar results.

1) Identification and Characterization of a Novel Styrene Monooxgenase

[0159] Gene sequences of known SMOs and monooxygenases were screened for promoter and or ribosome entry sites in order to detect potentially functional enzymes. In the next step the different SMOs were expressed e.g. in E. coli. Moreover, structural models of the different SMOs were created and the different SMOs were screened for activity with the potential substrate 2-chloro-4-(methylsulfanylmethyl)pyridine. Surprisingly, it was found that the isolated nucleic acid fragments encoding an oxygenase subunit (StyA) and a reductase subunit (StyB) respectively described herein was able to generate the (S)-enantiomer of -2-chloro-4-(methylsulfinylmethyl)pyridine in high enantiomeric excess.

[0160] In detail, the isolated nucleic acid fragments encoding an oxygenase subunit (StyA) and a reductase subunit (StyB), respectively described herein from A. baylyi were expressed and it was demonstrated that chiral sulfoxides are generated using each of the substrates given in the table below. In order to do so, the StyA unit from Acinetobacter sp. ADP1 was cloned into a pET-vector inducible with IPTG and expressed in E. coli BL21 pLysS grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l or 20 g/l NaCl, 100 g/ml ampicillin, 50 g/ml Chloramphenicol). For the expression a 5 ml overnight culture was used as inoculum for a fermenter with 31 medium or a flask with 500 ml medium. The cells were cultured at 37 C. until the culture reached an optical density of 0.6 which was the threshold for induction of recombinant gene expression using IPTG (0.1 mM). The culture was performed for another 18 h at 20 C. During this time the medium turned blue, if the expression was successful due to indigo formation. The cells were harvested and the cellular walls were disrupted using a French press. The protein was located in the soluble part of the cell extract, which was separated via centrifugation (45 min at >20000g). The target protein was purified for the subsequent characterization.

[0161] The StyB unit from Acinetobacter baylyi sp. ADP1 was expressed in a similar fashion.

[0162] In order to test the activity the StyB unit (reductase) was employed in excess to supply the StyA unit (oxygenase) with sufficient reduced FAD. Therefore, NADH was produced by a format-dehydrogenase from format and NAD in the respective enzymatic reaction.

[0163] The enzyme conversion rate was probed every minute for 15 min for the kinetic analysis. If possible, i.e. soluble, 2 mM substrate were employed. In case of clouding 1 mM substrate was employed. The substrate was dissolved in ethanol. Reactions in the probes were stopped with a 1:1 mixture of ice-cold methanol and acetonitrile and afterwards analyzed using HPLC.

[0164] The educt and the product were analyzed on an Agilent 1100 HPLC device.

[0165] The results are shown in the table below:

TABLE-US-00002 Substrat yield %) ee % Abs. confi. PhSCH3 48 99.3 S p(Cl)PhSCH3 34 95.5 S p(Br)PhSCH3 58 98.1 S p(F)PhSCH3 55 99.0 S p(CH3)PhSCH3 42 97.6 S PhSCHCH2 40 99.7 S

2) Whole Cell Biocatalysis Using the Novel SMO

[0166] In order to further increase production rates it was tried to generate the product, i.e. the S-enantiomer of -2-chloro-4-(methylsulfinylmethyl)pyridine using a whole cell bio-catalysis in a minimal medium employing live cells even though the substrate is potentially toxic. Ideally, slowly growing or no-growing cells, i.e. resting but metabolically active cells, were used. To further enhance the enantiomeric excess (ee) value a novel vector, the pCom10:lac Vector, was developed.

[0167] In detail, the styrene monooxygenase subunits StyA and StyB were expressed in E. coli JM101 cultivated in M9 minimal medium from the pStyAB_ADP1_lac vector. To find the maximum specific activity samples were taken after previously defined time points after addition of the inducer (1 mM IPTG). Cells were retrieved from the actively growing culture and used for the determination of the resting cell activity. As shown in FIG. 3 the specific activity increased steadily from the time point of induction and reached a maximum of 45030 U/gcdw 4.8 h after induction.

[0168] The success of the biotransformation was assessed based on substrate conversion, product formation, and enantiomeric excess (ee) determined by chiral HPLC and GC.

[0169] In detail, the specific activity was quantified as follows: StyA and StyB were expressed in E. coli JM101 carrying the pStyAB_ADP1_lac vector. Cells were cultivated in M9 minimal medium and induced at an OD of 0.6-0.8. After 3-5 hours the cell were harvested, washed in PP buffer and resuspended in the same buffer containing glucose Then 750 l of cells (diluted if needed) were taken and incubated in 2 ml tubes on a thermomixer set to 30 C. and 1500 rpm. Cell suspensions were pre-warmed for 5 min and then 2 mM of the substrate was added from 100-fold concentrated isopropanol stock solutions. After desired time points, 40 L of 20% (w/v) perchloric acid was added to quench the reaction. The whole-cell biocatalyst is inactivated directly upon addition of perchloric acid that leads to a pH shift from pH 7.4 to 2.0 The addition of perchloric acid had no effect on the product itself, however, the protein immediately precipitates and was separated via centrifugation.

[0170] Successful separation of the product i.e. the S-enantiomer of 2-chloro-4-(methylsulfinylmethyl)pyridine was achieved on a Dionex UltiMate 3000 HPLC system (Thermo Scientific) equipped with a non-polar HPLC column with C18 matrix (Accucore C18, 3150 mm, 2.6 m particle size, Thermo Scientific). The oven was set to 30 C. and the analytes were eluted isocratically (0-6 min) 80% 10 mM ammonium phosphate buffer and 20% ACN and a subsequent (6-18 min) gradient to 90% ACN. Afterwards the column was re-equilibrated 2 min at 80% 10 mM ammonium phosphate buffer and 20% ACN. 2 L of the respective sample were injected. The organic compounds were detected by absorption using diode array detector (DAD) at a wavelength of 210 nm. The sample preparation for analytical standards was performed as follows: 5 L of 100-fold concentrated isopropanol stock solutions of educt or product were added to 495 L potassium phosphate buffer (pH 7.4, 0.1 M) and subsequently 500 L of a 1 to 1 (v/v) mixture of ACN and MeOH were added. The sample was centrifuged after mixing and directly subjected to HPLC analysis.

[0171] In the next step a preparation on technical scale was conducted.

[0172] A technical scale bioreactor (3 L total volume) experiment was performed to produce the product on a larger scale. For this, E. coli JM101 (pStyAB_ADP1_lac) was cultivated in a 3 L stirred tank reactor equipped with two Rushton impellers. The working volume was set to 2 L. The cells were cultivated in M9 minimal medium in batch mode overnight (12 h, 1.5% (w/v) glucose) followed by a glucose limited fed-batch phase (6 h). The pH was controlled at 7.2 by titration with 15% phosphoric acid and 25% (v/v) ammonium hydroxide. The gene expression of the subunits StyA and StyB was induced 1 h after start of the fed batch phase with IPTG. The growth rate was set to 0.18 h.sup.1 in order prevent acetate formation and to reach a cell density of approximately 20 gcdw L.sup.1 after 6 h of glucose feed. Subsequently, the cells were harvested by centrifugation and resuspended in 2 L 0.1 M potassium phosphate buffer (pH 7.4). The specific activity of the whole cell biocatalyst was determined in separate resting cell assays. Only 1.5% glucose was added to the bioreactor to provide the living cells with energy. A constant educt feed (0.82 g min1) i.e. a feed with educt (2-chloro-4-(methylsulfanylmethyl)pyridine was started 5 min later. This educt feed ensured that the available specific activity of the whole cell biocatalyst was so high that the added educt was immediately converted into the less toxic product product i.e. the S-enantiomer of -2-chloro-4-(methylsulfinylmethyl)pyridine. Samples for biomass, product/substrate and glucose/acetate were retrieved every 15 minutes.

[0173] This approach led to an efficient product formation without detectable substrate accumulation until the solubility of the product was exceeded.

[0174] As shown in FIG. 4 product accumulated to surprisingly high concentrations exceeding 62 g L.sup.1 after 2.3 h, i.e. 30 g L.sup.1 per hour of biotransformation. No substrate accumulation was observed within the investigated time period. Product was analyzed as described above.

3) Variety of Substrates that can be Employed

[0175] In addition it was surprisingly found that the polypeptides described herein does not only accept 2-chloro-4-(methylsulfanylmethyl)pyridine as substrate, but converts a variety of different substrates with excellent enantioselectivity. The observed efficiency of the biocatalyst with respect to activity and extremely high ee values was unexpected, since the biocatalysts that have been shown to form enantiopure sulfoxides from the corresponding sulfides so far were strongly dependent on the arylsulfide applied (Rioz-Martnez et al., 2010, Adam et al., 2005). In detail, the results presented in FIG. 6 and FIG. 7 demonstrate that the claimed enzyme converts a variety of different substrates with excellent enantioselectivity both in vitro and in vivo using biotransformation.

REFERENCES

[0176] Adam W, Heckel F, Saha-mo C R, Taupp M, Meyer J, Schreier P. 2005. Opposite Enantioselectivities of Two Phenotypically and Genotypically Similar Strains of Pseudomonas frederiksbergensis in Bacterial 71:2199-2202. [0177] Bhler B, Witholt B, Hauer B, Schmid A. 2002. Characterization and application of xylene monooxygenase for multistep biocatalysis. Appl. Environ. Microbiol. 68:560-568. [0178] Doig S D, Avenell P J, Bird P A, Gallati P, Lander K S, Lye G J, Wohlgemuth R, Woodley J M. 2002. Reactor operation and scale-up of whole cell Baeyer-Villiger catalyzed lactone synthesis. Biotechnol. Prog. 18:1039-1046. [0179] Julsing M K, Kuhn D, Schmid A, Bhler B. 2012a. Resting cells of recombinant E. coli show high epoxidation yields on energy source and high sensitivity to product inhibition. Biotechnol. Bioeng. 109:1109-1119. [0180] Julsing M K, Schrewe M, Cornelissen S, Hermann I, Schmid A, Bler B. 2012b. Outer membrane protein AlkL boosts biocatalytic oxyfunctionalization of hydrophobic substrates in Escherichia coli. Appl. Environ. Microbiol. 78:5724-5733. [0181] Lindmeyer M, Meyer D, Kuhn D, Bhler B, Schmid A. 2015. Making variability less variable: matching expression system and host for oxygenase-based biotransformations. J. Ind. Microbiol. Biotechnol. 42:851-866. [0182] Rioz-Martnez A, De Gonzalo G, Pazmio D E T, Fraaije M W, Gotor V. 2010. Enzymatic synthesis of novel chiral sulfoxides employing Baeyer-villiger monooxygenases. European J. Org. [0183] Sadauskas, M., Vaiteknas, J., Gasparaviit, R., Meksy, R. (2017) Genetic and Biochemical Characterization of Indole Biodegradation in Acinetobacter sp. Strain O153 Appl. Environ. Microbiol. doi:10.1128/AEM.01453-17 [0184] Schrewe M, Julsing M K, Bhler B, Schmid A. 2013. Whole-cell biocatalysis for selective and productive CO functional group introduction and modification. Chem. Soc. Rev. 42:6346-77 [0185] Schrewe M, Magnusson A O, Willrodt C, Bhler B, Schmid A. 2011. Kinetic Analysis of Terminal and Unactivated CH Bond Oxyfunctionalization in Fatty Acid Methyl Esters by Monooxygenase-Based Whole-Cell Biocatalysis. Adv. Synth. Catal. 353:3485-3495. [0186] Tischler, D., Eulberg, D., Lakner, S., Kaschabek S., van Berkel W., Schlmann, M. (2009) Identification of a Novel Self-Sufficient Styrene Monooxygenase from Roodococcus opacus 1CP, JOURNAL OF BACTERIOLOGY, August 2009, p. 4996-5009 [0187] Tischler D, Grning J A D, Kaschabek S R, Schlmann, M. (2012) One-component 690 styrene monooxygenase: an evolutionary view on a rare class of flavoproteins. Appl. 691 Biochem. Biotechnol. 167:931-944 [0188] Volmer J, Schmid A, Bhler B. 2017. The application of constitutively solvent-tolerant P. taiwanensis VLB120 C ttV for stereospecific epoxidation of toxic styrene alleviates carrier solvent use. Biotechnol. J.:1600558.