Methyl halide transferase and application thereof

Abstract

An object of the present disclosure is to provide a means for efficiently producing a methyl halide.SOLUTION: In one aspect, the present disclosure provides the following inventions or inventions related thereto. ·Proteins having methylhalide transferase activity derived from Cyathea lepifera, proteins having methylhalide transferase activity derived from Sphaeropteriselifera, and methylhalide transferase proteins comprising any one of the following amino acid sequences (1) and (2): (1) the amino acid sequence set forth in SEQ ID NO: 1 or 2; and (2) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence set forth in SEQ ID NO: 1 or 2 SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methyl halide transferases and applications thereof. [Background technology]

[0002] Methyl halides (also referred to as methyl halides in this specification) are known to be industrially useful substances. For example, methyl chloride is used in the production of silicone rubber. Methyl iodide is also used as a foaming agent in pharmaceuticals. And methyl bromide is also used for fumigation.

[0003] Patent Document 1 discloses a method for producing methyl halides. Specifically, Patent Document 1 discloses heterologous genes encoding S-adenosylmethionine (SAM)-dependent methyl halide transferases (MHTs), and also discloses MHTs from various plants, including Batis maritima. The inventor of Patent Document 1 also reported the production of methyl iodide using microorganisms in Non-Patent Document 1 and Patent Documents 2 and 3. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2009 / 073560 (Special Table No. 2011-505149) Publication [Patent Document 2] European Patent Application Publication No. 2456852 [Patent Document 3] U.S. Patent No. 9,657,279 [Non-patent literature]

[0005] [Non-Patent Document 1] Bayer TS, Widmaier DM, Temme K, Mirsky EA, Santi DV, Voigt CA (2009) Synthesis of Methyl Halides from Biomass Using Engineered Microbes. J Am Chem Soc 131: 6508-6515 Summary of the Invention [Problem to be solved by the invention]

[0006] Although Patent Document 1 discloses a method for producing industrially useful methyl halides, there is a need for a more efficient production method. Therefore, an object of the present disclosure is to provide a means for efficiently producing methyl halides. [Means for solving the problem]

[0007] In order to achieve the above object, the present disclosure includes, in one aspect, the following invention. (Invention 1) A protein with methyl halide transferase activity derived from Cibotium barometz or Sphaeropteris lepifera. (Invention 2) A methyl halide transferase protein comprising any one of the following amino acid sequences (1) to (2): (1) the amino acid sequence set forth in SEQ ID NO: 1 or 2 (2) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2; (Invention 3) A protein according to invention 2, which comprises the amino acid sequence of (1). (Invention 4) A protein according to invention 2, comprising the amino acid sequence of (2) and differing from SEQ ID NO: 1 or 2 by one or more of the following modification patterns: (A) Changes within the group consisting of serine and threonine (B) Changes within the group consisting of asparagine and glutamine (C) Changes within the group consisting of aspartic acid and glutamic acid (D) Changes within the group consisting of leucine, isoleucine, valine, and alanine (E) Changes within the group consisting of cysteine ​​and methionine (Invention 5) A nucleic acid encoding the protein according to any one of inventions 1 to 4. (Invention 6) Use of the protein according to any one of Inventions 1 to 4 for producing methyl halide. (Invention 7) 1. A method for producing methyl halide, said method comprising: a step of mixing the protein according to any one of inventions 1 to 4 as an enzyme, S-adenosylmethionine as a substrate, and a halogen as a substrate, and reacting these substrates; recovering the methyl halide resulting from the reaction; A method comprising: (Invention 8) 8. The method of claim 7, wherein the methyl halide is methyl chloride. (Invention 9) 1. A method for producing silicone rubber, said method comprising: a step of reacting the methyl chloride obtained by the method according to Invention 8 with silicon to synthesize methylchlorosilanes, and isolating the methylchlorosilanes; obtaining siloxane by hydrolysis of the methylchlorosilane; polymerizing the siloxane; A method comprising: (Invention 10) 1. A method for manufacturing an automotive part, the method comprising: A step of using the silicone rubber obtained by the method according to claim 9. A method comprising: (Invention 11) 1. A method of manufacturing a motor vehicle, said method comprising: A step of using the part obtained by the method according to claim 10. A method comprising: [Effects of the Invention]

[0008] In one aspect, the invention encompasses a protein having a specific amino acid sequence or a protein having methyl halide transferase activity of a specific species, which allows for efficient production of methyl halides. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the amino acid sequence of methyl halide transferase derived from Cibotium barometz (SEQ ID NO: 1). [Figure 2] 1 shows the amino acid sequence of methyl halide transferase derived from Sphaeropteris lepifera (SEQ ID NO: 2). [Figure 3] The amino acid sequence of methyl halide transferase from Batis maritima (SEQ ID NO: 3) is shown. [Figure 4] SDS-PAGE analysis of fusion MHT proteins. Crude extracts from E. coli expressing each fusion MHT protein and purified His-tagged proteins were analyzed by SDS-PAGE. The arrowhead indicates the band of the purified fusion MHT protein. [Figure 5] Multiple sequence alignment of the amino acid sequences of MHT proteins. Multiple sequence alignments were generated from the amino acid sequences of CibMHT2, SplMHT2, and BamMHT using the CLUSTAL O (1.2.4) multiple sequence alignment program (Madeira et al., 2022). [Figure 6]Comparison of MHT activity of fusion MHT proteins is shown. The enzyme activity values ​​of MHT activity (MCT activity, MBT activity, and MIT activity, respectively) for BamMHT, CibMHT2, and SplMHT2 in response to Cl-, Br-, and I- were compared. Each value represents the mean ± standard deviation of three independent experiments. Significant differences between each MHT activity are indicated by letters (p<0.01, ANOVA test and Tukey's multiple comparison test were used). For example, in the MCT activity graph, the letters a, b, and b are added to the bars for BamMHT, CibMHT2, and SplMHT2, respectively. This indicates a significant difference between BamMHT and CibMHT2, a significant difference between BamMHT and SplMHT2, and no significant difference between CibMHT2 and SplMHT2. DETAILED DESCRIPTION OF THE INVENTION

[0010] Specific embodiments for carrying out the present invention will be described below. The following description is intended to facilitate understanding of the invention and is not intended to limit the scope of the present invention.

[0011] 1. Methyltransferase In one embodiment, the present disclosure relates to a protein having methyltransferase activity. The enzymatic activity of the protein is S-adenosylmethionine-dependent. Specifically, the enzymatic substrates of the protein include S-adenosylmethionine and halide ions. The protein can then promote the reaction of these substrates to produce methyl halides.

[0012] In one embodiment, the protein having methyltransferase activity is a methyltransferase derived from Cibotium barometz.

[0013] In another embodiment, the protein having methyltransferase activity is a methyltransferase derived from Sphaeropteris lepifera.

[0014] In the above embodiment or another embodiment, the protein having methyltransferase activity is a methyl halide transferase protein comprising any one of the following amino acid sequences (1) to (2): (1) the amino acid sequence set forth in SEQ ID NO: 1 (2) An amino acid sequence having a sequence identity of 90% or more (preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more) with the amino acid sequence set forth in SEQ ID NO: 1.

[0015] In yet another embodiment, the protein having methyltransferase activity is a methyl halide transferase protein comprising any one of the following amino acid sequences (1) and (2): (1) the amino acid sequence set forth in SEQ ID NO: 2 (2) An amino acid sequence having a sequence identity of 90% or more (preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more) with the amino acid sequence set forth in SEQ ID NO: 2.

[0016] The protein having methyltransferase activity according to any of the above-described embodiments has higher methyltransferase activity than conventional proteins having methyltransferase activity, and therefore, these proteins enable efficient production of methyl halides.

[0017] The amino acid sequence identity value may be calculated using, for example, the CLUSTAL O (1.2.4) multiple sequence alignment program. For example, the value may be calculated on the Clustal Omega website using the following parameters: OUTPUT FORMAT: ClustalW with character counts DEALIGN INPUT: no MBED-LIKE CLUSTERING GUIDE-TREE: yes MBED-LIKE CLUSTERING ITERATION: yes COMBINED ITERATIONS: default(0) MAX GUIDE TREE: default MAX HMM ITERATIONS: default ORDER: aligned DISTANCE MATRIX: no OUTPUT GUIDE TREE: yes

[0018] In a further preferred embodiment, with respect to the above-mentioned sequence identity, the amino acid sequence may differ from that set forth in SEQ ID NO: 1 or SEQ ID NO: 2 by any one or more of the following substitutions: Group 1: serine, threonine Group 2: Asparagine, Glutamine Group 3: Aspartic acid, glutamic acid Group 4: alanine, valine, leucine, isoleucine (preferably valine, leucine, isoleucine) Group 5: Cysteine, methionine

[0019] For example, the eighth amino acid in SEQ ID NO: 1 shown in Figure 1 is serine. This serine may be substituted with threonine. Similarly, the ninth amino acid in SEQ ID NO: 1 shown in Figure 1 is glutamic acid. This glutamic acid may be substituted with aspartic acid.

[0020] In each group, the chemical structure differs only by about 1 to 3 carbon atoms in the side chain. Therefore, even if amino acids are changed within the range of each group, there is a high probability that the function of the protein will be maintained.

[0021] There is no particular upper limit to the number of substitutions in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. For example, the number of substitutions may be 20 or less, 15 or less, 10 or less, 5 or less, or 3 or less.

[0022] As described above, a protein having methyltransferase activity comprises the above-described amino acid sequence. This means that an amino acid sequence of any length may be added to the C-terminus and / or N-terminus of a specific amino acid sequence. The length of the added amino acid sequence is not particularly limited, and may be, for example, 50 or less, 25 or less, 15 or less, 10 or less, or 5 or less.

[0023] In a preferred embodiment, the protein with methyltransferase activity consists of the amino acid sequence defined above, i.e., no additional amino acids are added to the C-terminus and / or N-terminus of the specified amino acid sequence.

[0024] In another embodiment, any of the above proteins may be modified by deleting the start codon (the first amino acid, methionine) in the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2. This is because deleting the start codon does not affect the function of the protein, or is highly likely to do so.

[0025] In another embodiment, the present disclosure relates to a nucleic acid encoding any of the above-mentioned proteins. The nucleic acid may be DNA or RNA. The nucleic acid may be maintained by a vector (e.g., an expression vector), a plasmid, or the like, or, alternatively, or additionally, may be maintained by a host of a different species (i.e., it may be introduced as a foreign gene into a species other than the above-mentioned Takawarabi and Hikagehengo, such as yeast, E. coli, or algae). Alternatively, the nucleic acid may be maintained by being encapsulated in a lipid nanoparticle (LNP) or the like.

[0026] 2. Preparation of Methyl Halides In one embodiment, the present disclosure relates to the production of methyl halides. For example, the present disclosure relates to the use of any of the proteins described above for the production of methyl halides. In another example, the present disclosure relates to a method for producing methyl halides.

[0027] The method includes at least the following steps: A process of mixing a protein as an enzyme, S-adenosylmethionine as a substrate, and a halogen as a substrate, and reacting these substrates. recovering the methyl halide resulting from the reaction.

[0028] In the mixing and reacting step, the protein may be provided in any form. For example, the protein may be expressed by genetic engineering techniques, and then the protein may be purified and provided. In another example, the protein may be expressed by genetic engineering techniques, and then the host in which the protein has been expressed may be added to a reaction vessel.

[0029] When expressing a protein, a signal peptide may be added as appropriate to control the function of the protein at any location within the cell. For example, a signal peptide may be added that causes the protein to function in the cytoplasm, or a signal peptide that causes the protein to function in the vacuole.

[0030] The method for recovering methyl halide, the method for purifying the protein after expression, and the design of the signal peptide can be achieved by means known in the art.

[0031] The reaction conditions are not particularly limited, and the reaction may be carried out, for example, at a temperature in the range of 10 to 45° C. (e.g., room temperature). The pH is also not particularly limited, and the reaction may be carried out at a pH in the range of 6 to 8.

[0032] The type of methyl halide is not particularly limited, and may be, for example, one or more of methyl chloride, methyl iodide, methyl bromide, and methyl fluoride, with methyl chloride being preferred.

[0033] 3. Use of methyl halides The methyl halides produced by the above methods are used in various industrial fields. In one embodiment, the present disclosure relates to a method for producing products using methyl halides.

[0034] For example, methyl chloride is utilized in the production of silicone rubber. In this regard, in one embodiment, the present disclosure relates to a method for producing silicone rubber, said method comprising the steps of: a step of reacting methyl chloride with silicon to synthesize methylchlorosilanes and isolating the methylchlorosilanes; obtaining siloxanes by hydrolysis of methylchlorosilanes; and A process of polymerizing siloxane.

[0035] The methylchlorosilane may be one or more of monomethyltrichlorosilane, dimethyldichlorosilane, and trimethylmonochlorosilane. Preferably, the methylchlorosilane is dimethyldichlorosilane.

[0036] Furthermore, the silicone rubber produced by the above method is used in various industrial fields. For example, silicone rubber is used in the manufacture of automotive parts (e.g., cylinder head gaskets, engine and gearbox seals, battery modules, rechargeable batteries, headlight seals, electronic control units (ECUs), electrical and electronic connectors, spark plug boots, etc.). In this regard, in one embodiment, the present disclosure relates to a method for manufacturing an automotive part using the silicone rubber produced by the above method, and a method for manufacturing an automobile using the automotive part.

[0037] Methyl iodide is used as a foaming agent in medicines, while methyl bromide is used as a fumigation disinfectant. Methyl fluoride is used as a refrigerant. In one embodiment, the present disclosure relates to methods for producing these various products using various methyl halides. [Example]

[0038] 1. Identification of the MHT (Methyl Halide Transferase) gene in ferns

[0039] We downloaded the raw RNA-Seq data (SRR6920684) from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) from the Takakawawarabi (Cibotium barometz) and the raw RNA-Seq data (SRR8210167) from the Sphaeropteris lepifera (Sphaeropteris lepifera) (Table 1). We used the amino acid sequence of BamMHT (Ni and Hager, 1999), a methyl halide transferase from Batis maritima, as a query against the de novo transcriptome assemblies constructed using the TransPi pipeline (Rivera-Vicens et al., 2022). tblastn analysis (Gertz et al., 2006) was used to identify homologous genes.

[0040] SRA data used to construct de novo transcriptome assemblies [Table 1]

[0041] 2. Synthesis of each MHT protein in E. coli 2.1 Preparation of DNA constructs and transformation of E. coli

[0042] The DNA fragments containing each MHT protein were synthesized by Integrated DNA Technologies, Inc. Each MHT protein had a 7-amino acid residue (Met-His-His-His-His-His) (SEQ ID NO: 4) tagged with a 6xHis tag at its N-terminus and a 10-amino acid linker (Gly-Ser-Ala-Gly-Ser-Ala-Ala-Gly-Ser-Gly) (SEQ ID NO: 5) linked to a fluorescent protein (mCherry, theoretical pI / Mw: 5.62 / 26722.19) at its C-terminus. The resulting fragments were cloned downstream of the T7 promoter in the pDEST17 vector and transformed into the E. coli BL21(DE3) strain for expression in Escherichia coli.

[0043] 2.2 Expression and purification of MHT protein in E. coli

[0044] E. coli expressing each fusion MHT protein was precultured overnight at 37°C in TB medium containing 100 μg / ml ampicillin. One-fiftieth of the preculture solution was added to fresh TB medium containing ampicillin, and the mixture was cultured at 37°C for 3 hours. Isopropyl-β-D-thiogalactopyranoside (final concentration 0.5 mM) was added, and the mixture was cultured for an additional 2.5 hours at 25°C. The culture solution was collected, centrifuged, and the E. coli pellet was stored at -20°C.

[0045] The collected E. coli pellet was suspended in extraction buffer (20 mM Tris-HCl pH 8, 0.5 M NaCl, 5 mM imidazole, 0.225 mg / ml lysozyme, 2% Triton X-100, 10 mM MgCl2) in an amount equal to one-quarter of the culture medium volume, and the suspension was left at 25°C for 10 minutes. After centrifugation (16,000 g, 4°C, 10 minutes), the supernatant was used as a crude protein extract.

[0046] Fusion MHT protein was purified from crude protein extracts using a His-Bind Quick Column (Novagen) resin in a batch system according to the manufacturer's protocol. The purified fusion MHT protein was desalted and concentrated using a centrifugal ultrafiltration filter (10 kD, GVS). The purified fusion MHT protein solution was adjusted to 0.1 M Tris-acetate (pH 7.5), 1 mM DTT, and 50% glycerol. The purified fusion MHT protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue (CBB) for confirmation (Figure 4). The concentration of the purified fusion MHT protein was determined by the Bradford method using bovine serum albumin (BSA) as a standard.

[0047] 2.3 Analysis of methyl halide transferase activity

[0048] Halide ions (Cl - , I - , Br - MHT activity against the substrates was assessed by quantifying S-adenosyl-L-homocysteine ​​(SAH), a common reaction product after methylation of each substrate, which was fluorescently labeled with chloroacetaldehyde (CAA) (Albu et al., 2013).

[0049] A total of 75 μl of a mixture of 0.1 M Tris-acetate (pH 7.5), 0.2 mM S-adenosyl-L-methionine (SAM, Sigma-Aldrich), and a halide (300 mM KCl, 50 mM KI, or 100 mM KBr) and a known amount (approximately 0.1 μg) of each fusion MHT protein was incubated at 30°C for a set time. The reaction was stopped by adding an equal volume of 1 M HClO4 and mixing thoroughly. After centrifugation (16,000 xg, 4°C, 10 min), 120 μl of the supernatant was used for fluorescent labeling. The 120 μl supernatant was mixed thoroughly with 68.5 μl of 1 M sodium acetate (pH 5), 20.2 μl of 4 M NaOH, 39.4 μl of ultrapure water, and 1.88 μl of 40% CAA, and incubated at 60°C for 1.5 hours. After the reaction, the reaction mixture was diluted 20 times with ultrapure water.

[0050] The fluorescently labeled solution was analyzed using a HPLC (Shimadzu) equipped with an ODS-80Ts column (4.6 × 150 mm, TOSOH) and a fluorescence detector (L-7485, Hitachi). Separation was performed using eluent A (8 mM sodium octanesulfonate, 20 mM NaH2PO4, pH 3) and eluent B (100% methanol). The column temperature was 25°C, and the flow rate was 1 ml min-1. The concentration of eluent B was 32% for the first 3 minutes, then increased from 32% to 60% over the next 1 minute, held at 60% for 1 minute, and then decreased from 60% to 32% over the next 1 minute. The column was equilibrated for 10 minutes. The fluorescence detector was set to excitation at 270 nm and emission at 410 nm. The retention time of SAH was determined using a standard sample of SAH (Sigma-Aldrich). The amount of SAH synthesis was plotted against reaction time for each MHT activity (methyl chloride transferase activity (MCT activity), methyl bromide transferase activity (MBT activity), methyl iodide transferase activity (MIT activity)) of each fusion MHT protein, and the enzyme activity was calculated based on the data in the linear range from the start of the reaction on the reaction curve.

[0051] 3.Results: From the de novo transcriptome assemblies constructed from the SRA data of ferns (C. barometz and S. lepifera), we identified transcripts encoding proteins with high amino acid sequence identity to BamMHT from B. maritima. The MHT identified from C. barometz was named CibMHT2, and the MHT identified from S. lepifera was named SplMHT2 (Figs. 1-3).

[0052] At the amino acid level, CibMHT2 and SplMHT2 showed approximately 88% identity, BamMHT and CibMHT2 approximately 47%, and BamMHT and SplMHT2 approximately 47% identity (Fig. 5).

[0053] Crude extracts were prepared from E. coli expressing each fusion MHT protein, and the fusion MHT protein was purified using the added His-tag, confirming that fusion MHT proteins with the desired molecular weight were obtained (Figure 4).

[0054] Analysis of the MHT activity of each fusion MHT protein revealed that CibMHT2 from C. barometz exhibited approximately 7-fold higher MCT activity, approximately 9-fold higher MBT activity, and approximately 3-fold higher MIT activity than BamMHT from B. maritima, which was previously known to exhibit high MHT activity. SplMHT2 from S. lepifera exhibited approximately 7-fold higher MCT activity, approximately 6-fold higher MBT activity, and equivalent MIT activity compared to BamMHT (Figure 6).

[0055] Specific embodiments of the invention have been described above. The above embodiments are merely illustrative examples, and the present invention is not limited to these embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Furthermore, unless otherwise specified, for a particular method, the order of some steps may be interchanged, and additional steps may be added between two specific steps. The scope of the present invention is defined by the claims.

[0056] References Albu C, Litescu SC, Radu GL, Y. Aboul-Enein H (2013) Validated HPLC-Fl method for the analysis of S-adenosylmethionine and S-adenosylhomocysteine biomarkers in human blood. J Fluoresc 23: 381-386 Bayer TS, Widmaier DM, Temme K, Mirsky EA, Santi DV, Voigt CA (2009) Synthesis of methyl halides from biomass using engineered microbes. J Am Chem Soc 131: 6508-6515 Gertz EM, Yu Y-K, Agarwala R, Schaffer AA, Altschul SF (2006) Composition-based statistics and translated nucleotide searches: Improving the TBLASTN module of BLAST. BMC Biol 4: 41 Madeira F, Pearce M, Tivey ARN, Basutkar P, Lee J, Edbali O, Madhusoodanan N, Kolesnikov A, Lopez R (2022) Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50: W276-W279 Ni X, Hager LP (1998) cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme. Proc Natl Acad Sci 95: 12866-12871 Ni X, Hager LP (1999) Expression of Batis maritima methyl chloride transferase in Escherichia coli. Proc Natl Acad Sci 96: 3611-3615 Rivera-Vicens RE, Garcia-Escudero CA, Conci N, Eitel M, Worheide G (2022) TransPi-a comprehensive TRanscriptome ANalysiS PIpeline for de novo transcriptome assembly. Mol Ecol Resour 22: 2070-2086 Voigt CA, Bayer TS (2012) Cell-based systems for production of methyl formate, EP2456852A. Voigt CA, Bayer TS (2017) Biological systems for production of commercially valuable compounds, US9657279B.

Claims

1. A protein with methyl halide transferase activity derived from Cibotium barometz or Sphaeropteris lepifera.

2. A methyl halide transferase protein comprising any one of the following amino acid sequences (1) to (2): (1) The amino acid sequence set forth in SEQ ID NO: 1 or 2 (2) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2;

3. The protein of claim 2, which consists of the amino acid sequence of (1).

4. 3. The protein of claim 2, comprising the amino acid sequence of (2) and differing from SEQ ID NO: 1 or 2 by one or more of the following modification patterns: (A) Alterations within the group consisting of serine and threonine (B) Alterations within the group consisting of asparagine and glutamine (C) Alterations within the group consisting of aspartic acid and glutamic acid (D) Alterations within the group consisting of leucine, isoleucine, valine, and alanine (E) Alterations within the group consisting of cysteine ​​and methionine

5. A nucleic acid encoding the protein according to any one of claims 1 to 4.

6. Use of the protein according to any one of claims 1 to 4 for producing methyl halides.

7. 1. A method for producing methyl halide, said method comprising: a step of mixing the protein according to any one of claims 1 to 4 as an enzyme, S-adenosylmethionine as a substrate, and a halogen as a substrate, and reacting these substrates; recovering the methyl halide resulting from the reaction; A method comprising:

8. 8. The method of claim 7, wherein the methyl halide is methyl chloride.

9. 1. A method for producing silicone rubber, said method comprising: a step of reacting the methyl chloride obtained by the method according to claim 8 with silicon to synthesize methylchlorosilanes, and isolating the methylchlorosilanes; obtaining siloxane by hydrolysis of the methylchlorosilane; polymerizing the siloxane; A method comprising:

10. 1. A method for manufacturing an automotive part, the method comprising:

10. A step of using the silicone rubber obtained by the method of claim 9. A method comprising:

11. 1. A method of manufacturing a motor vehicle, said method comprising: Using the part obtained by the method of claim 10. A method comprising:

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