TW201247869A - Osmolyte N- ε -acetyl- β -lysine biosynthetic genes from methanogenic archaea and application thereof - Google Patents

Abstract

The present invention relates to isolated nucleic acid molecules encoding N ε -acetyl- β -lysine biosynthetic enzymes (lysine 2, 3-aminomutase and β -lysine acetyltransferase) of methanogenic archaea. The present invention also relates to a method for procducing the N ε -acetyl- β -lysine biosynthetic enzymes in E. coli. host cells by using the isolated nucleic acid. The present invention further provides a method for in vivo and/or in vitro production of osmolyte N ε -acetyl- β -lysine.

Description

201247869 VI. Description of the invention: [Technical field to which the invention belongs] This case is a sub-case of the invention patent application No. 097146358 "Compatible acid-synthesis gene of methane archaea and its application". Earth, this hair ^ about, encoding methane ancient g plus her coffee ^ muscle (four) of the compatible quality 'B-based base - Bu, acid synthesis enzyme (lysine 2,3_amino translocation 2,3-ammomutase) And β-lysine acetonitrile ^. Intestine # heart mass production of compatible quality ^ 醯 · · lysine kinetics method. The present invention further provides a method for the in vivo and alkanax-compatible JV6-ethinyl-indole-lysine. R-square τ [Prior Art] Compatible quality is osmotic pressure protection, with characteristics of tilting, salt-resistance and drought resistance. Its biosynthetic genes are quite similar in the fermentation industry, food industry, cosmetic beauty industry, pharmaceutical, medical and agricultural fisheries. Great application potential. Compatible with f, ethyl ketone 卩 赖 lysine ^ (A ^ acetyl + lysine) is a special β-type amino acid, currently only found in the sputum archaea, in other eukaryotes, prokaryotes Found its existence. The biosynthesis pathway of compatible beryllium-ethylidene-β-lysine is converted from β-lysine by α-lysine aminotransferase (secret), followed by ρ_lysine ^B'-transferase (α6/Β) acts to synthesize ethionyl-ρ_lysine (see, for example, Lai, MC et al, 173: 5352-5358, 1991; R〇berts, MF et al,乂' Bacteriol 174: 6688-6693, 1992; A Robertson DE^ MF Robert^ 3: 1-9, 1992). ’

Pfltiger et al. (eg p/.69:6047-6055, 2003) search for autologous biosynthesis compatible TV8-acetyl-based _β-赖The genes of the amino acid enzymes are lysine 2,3-aminotransposase (fi^/A) and β-lysine acetyltransferase (^^). 'Using the northern ink collapse method to confirm that the Μ/A and M/B genes are the same operon 201247869 (operon), the 表现/operon expression will increase with the increase of the culture environment salt concentration.

増加. Using Methanococcus maripcdudis to construct ab!A anti-ablB, the mutant strain was cultured at different salt concentrations, and the content of intracellular cumulative compatibilities 'ethinyl-β-lysine was detected by NMR and tested. According to the results of the analysis, in the low salt (376.5 mM NaCl) environment, the cumulative amount of TV6-acetyl-β-lysine in the wild type cells was 〇μηιοΐ/mg, with the culture. The salinity increased to 800 mM and the accumulation of intracellular ethyl ketone _β_lysine increased by 3.7 times to 0.7 μιηοΐ/mg and increased by 7.8 times to 1.09 μιηοΐ/mg, while the mutant strain was cultured at a low level. In the salt or high-salt environment, the cumulative amount of acetyl-β-lysine was not detected in the cells, indicating that the 劼/A and Μ/Β genes are compatible with ethionyl _β_ lysine. Translation of autologous synthetic enzyme lysine 2,3-aminomutase and β-lysine acetyltmnsfemse; culture of mutants at different salinities The results of the growth test showed that the mutant strain grew at the same rate as the wild strain in the low salt (376.5 mM NaCl) environment. Growth was significantly inhibited under high salt (800 mM NaCl) environment. The salinity was 1 M NaCl. The cell damage was almost non-growth, indicating that the high osmotic pressure difference from the sputum in response to the extracellular high salt environment was cumulatively compatible. VIII-8-ethinyl-β-lysine as an osmotic pressure adaptation mechanism (PflUger, 2〇〇3, as described above). Halophilic smoldering archaea Meqi 3m?1⁄2/c►/?Mus/7orίwcfl/e«>sz'<sFDFl is an irregular bacterium 'from Sathrani et al. from Portugal Figutria da Foz containing saturated sodium hydride The salt pond was separated' and was officially named by Boone D, R in 1993 (B〇〇ne buckle α/., 1993). M FDF1 can use methanol, mono-, di- or trimethylamine as a carbon source for methanogenesis (methan〇genesis), but can not use the + (: 〇2, phthalate, acetate, A Mercaptan or dimethyl sulfoxide as growth substrate (B〇〇ne βα/·, 1993) 〇Mp〇rtea/m^FDFl can grow in an environment with a salt concentration range of 丨〇~3 〇Μ' optimum growth salt concentration It is 2.1 μ, which does not balance the intracellular and extracellular osmotic pressure by changing the cell volume in a high salt environment, so cumulative compatibilities are the main osmotic pressure regulating mechanism (LaUi < 1991, 1992). High-performance liquid chromatography (HPLC) and nuclear magnetic resonance (ICP-A) 201247869 (nuclear magnetic resonance, NMR) experiments confirmed that M / wriwca / ms / s FDF1 in the environment glycine test (glycine betaine) does not exist In the meantime, potassium ions, α-glutamic acid, β-glutamine, betaine and Λ/6-ethylindolyl _β_ are mainly accumulated in the cells in response to the high osmotic pressure difference generated by the extracellular high salt environment. Solutes such as lysine are used in response to external/parametric stresses. The intracellular conversion rate of these solutes Tum〇verrate) is very low, does not participate in protein synthesis and cellular metabolism, can accumulate in the cell for a long time, and its accumulation will increase with the increase of extracellular salt concentration, indicating that these solutes play a compatible role in FDF1 cells. Qualitative role (Lai ei 〇/., 1991; Robertson βα/., 1992, as described above). From fdFI with autologous synthetic beet, W-ethyl-β-lysine and (3_glutamine) The ability of amines as a compatibilizer for the biosynthesis of acetamino-β-lysine is the production of α-lysine via the diaminopimelate pathway, followed by lysine 2,3-aminotransferase (lysine 2,3- The aminomutase acts to convert to β-lysine, and then biosynthesizes π-ethionyl-p_lysine via (3_lysine acetyl-t-butyltransferase). Now jbu is in ψ It is said that the ancient scorpion Methanocalculus chunghsingensis KlF9705b is an irregular cocci isolated from the mariculture site near Wanggong in Changhua County, Taiwan (Lai et al., 2〇〇4), and can use h2 and (:02 as , source ^ for methanation. 尬K1F97〇5b can grow in salt concentration van JS G~2.G1V [environment, optimum growth concentration of 〇.175M. Nuclear magnetic resonance apparatus is known from the analysis (nuclear magnetic resonance, NMR) is, [mu]

KlF9705b accumulates β-glutamine-rich amine and acetaminophen _β_lysine in the cell in response to the high osmotic pressure difference generated by the extracellular high salt environment, and its amount will increase with the external salt concentration (4). The intracellular rate of f (t_verrate) is very low, and it is shown that TV6-acetamido-β-lysine and β-glutamine are the main osmotic compatible compatibilities of this strain. The lysine 2,3-aminotransposase (AblA) enzyme functions to catalyze the conversion of α•lysine to β-lysine. This enzyme catalysis requires cofactors (c〇fact〇r): [4Fe_4S] duster*, S-adenosylmethionine (SAM), and pyrid〇xal 5, _ rush (PLP). (4) Intracellular regenerative (4) [4Fe_4S] 2+ reduced to activated 6 201247869 state of [4Fe-4S]1+, activated state of [4Fe-4S]1+ will release electrons and react with SAM 'cracking SAM Produces methionine and 5'-deoxyadensyl free radicals. The PLP and the ε-bank on the lysine amino acid on the AblA protein form an internal acid imine pntemai aldimine bond. In the presence of the receptor α-lysine, PLP undergoes a transaldimation reaction with the α-amino group of the receptor α_lysine to form an external aldimine bond. The 5,_de〇xyadensyi radical generated by [4Fe_4S]1+ interacts with SAM will interact with the PLP-bonded a-alpha-lysine to produce 5'_deoxyadenosine and intermediates with free radicals' intermediate products via isomerizati〇 n reaction, and hydrogen atom transfer provided by 5'-deoxyadenosine to form β_lysine (Frey and Reed, 乂ί/ν· Evymi?/· i^/αΛ Jmxs. Mo/.〇/. 66:1- 39, 1993; Chen and Milne, y. 45: 12647-12653, 2006). The lysine 2,3-aminotransposase catalyzes the formation of β-lysine by α-lysine requires the participation of [4Fe-4S] cluster, SAM and PLP 'on the amino acid sequence' and [4Fe_4S] cluster The binding site is a retention of cysteine, which has more glycine acid at the binding site to the SAM and a lysine at the pLp linkage (Chen and Frey oc/zem. 40: 596-602, 2001). It has also been pointed out that this enzyme activation requires the participation (Ruzicka, 2000), presuming that the three cysteine amino acids contained at the position near the C-terminus of the amino acid sequence are zinc-bonded sites. The location of these cofactor bindings has been found in the published serotonin 2,3_aminotransposase sequence of G51 from the A. sinensis. The lysine acetyltransferase (AblB) enzyme functions to catalyze the production of β-lysine tv6-ethyl-based-β-lysine. The enzyme acetyltransferase belongs to the GNAT (gcn_5^g-ethyltransferase) superfamily (SUperfamily), which acts to catalyze the transfer of the acetamidine coenzyme A to its primary amine. The role of the receptor can be divided into three types: (1) Wst〇ne: involved in transcriptional activation, chromatin assembly and DNA repair process, acetyltransferase mainly acts on specific lysine; (2) aminiglycosides: acetyltransferase The activity will cause antibiotic inhibition of pathogenic bacteria, which acts on aminjglyc〇SideS antibiotics, resulting in a decrease in the affinity of such antibiotics for the target; (3) aiylalkylamines: only found in vertebrates, involved in catalysis Serotonin (ser〇t〇nin) produces a process of blackening 201247869 hormone (melatonin). The amino acid sequence of the acetyltransferase can be divided into four conserved motifs according to the secondary knot, and the function of the 1 arrangement of the lipid bed or the CDAB 'phantoms B, C and D is mainly related to the structure of the protein. Stable related", A is - the segment contains a longer and highly protected (four) region, is involved in the position of acetam with a = 5, and contains a portion of the retained amino acid in this region is QHX-GXG / A (Dyda, 2000; Wolf, 1998). Up to now, and [ger, there is no research on the properties of β-lysine acetyltransferase (IV)/B): while in the published decane Taikoo M maze/ G61 β· In the lysine acetyltransferase sequence, the amino acid sequence found to be retained for a period of time is _3, such as the secret. ^ ' [Invention] In one aspect, the present invention provides a salt-tolerant methane Cc/m «KlF9705b compatible acetamidine _β· lysine (facetyl-p-lysine) biosynthetic enzyme lysine 2,3-aminotrans & lysine 2,3-aminomutase An isolated nucleic acid molecule wherein the lysine 2 3•amino group is transposed to have the amino acid sequence of SEQ ID No. 2 or a functionally equivalent variant thereof. a specific aspect of the ligated nucleic acid molecule encoding a lysine 2,3-aminotransposase comprising a nucleotide sequence selected from the group consisting of (1) SEQ ID NO: 1; and (3⁄4 having a nucleotide sequence similarity to SEQ ID NO: 1 a nucleotide sequence of a group consisting of more than 90% of the nucleotide sequences." On the other hand, the present invention provides a method for encoding a salt-tolerant sputum archaea Afe recognizes im〇ajf/cw/milk KlF9705b Compatible 醯β- lysine (A^-acetyl-P-lysine) is a synthetic nucleic acid molecule of β-lysine acetyltransferase, wherein β_ The lysine acetyltransferase has the amino acid sequence of SEQ ID No. 4 or a homologous variant thereof. In one embodiment, the isolated nucleic acid molecule encoding β-lysine acetyltransferase a core comprising a group consisting of (i) SEQ ID NO: 3; and (9) a group having a nucleotide sequence greater than 90% similar to the nucleotide sequence of SEq ID NO: 3; 201247869 In another aspect, the present invention provides a compatible probiotic synthase encoding a halophilic smoldering archaea & FDF1 An isolated nucleic acid molecule of lysine 2,3-aminoniutase, wherein the lysine 2,3-aminotransposase has an amino group of SEq ID N〇. An acid sequence or a homologous variant thereof. In one embodiment, the isolated nucleic acid molecule of the chimeric lysine 2,3-aminotransposase comprises a nucleotide sequence selected from (1) SEQ ID NO: 5; and (9) has a seq ID NO: 5 A nucleotide sequence of a group consisting of nucleotide sequences having a nucleotide sequence similarity greater than 90%. In another aspect, the present invention provides a compatibilized acetyl-P-lysine-producing enzyme β-lysine encoding Methanohabp Mus portucalensis FDF1. An isolated nucleic acid molecule of β-lysine acetyltransferase, wherein the β-lysine acetyltransferase has the amino acid sequence of SEQ ID No. 8 or a functionally equivalent variant thereof. In one embodiment, the isolated nucleic acid molecule encoding beta-lysine acetyltransferase comprises a nucleotide sequence selected from the group consisting of (1) SEQ ID NO: 7; and (8) has a nucleoside with SEq ID NO: 7. A nucleotide sequence of a group consisting of nucleotide sequences having an acid sequence similarity greater than 90%. Another aspect of the present invention provides a method for expressing a large amount of a serotonin lysine 2,3-aminotransposase using Escherichia coli, which is characterized in that a nucleic acid encoding a lysine 2,3_aminotransposase is characterized. Molecules, cloning - in appropriate expression vectors, and transforming the resulting recombinant plasmid into a large number of expressions in E. coli host cells, and purifying the lysine 2,3-aminotransposase recombinant protein. In one embodiment, the nucleic acid molecule encoding the lysine 2,3-aminotransferase is derived from the salt-tolerant decane archaea Mei/zim〇ca/cw/w>s KlF9705b genome. In another embodiment, the nucleic acid molecule encoding the lysine 2,3-aminotransposase is derived from the halophilic methanogen Aiethanohalophilus portucalensis FDFI substrate. A further aspect of the present invention provides a recombinant protein which is expressed in a large amount by means of Escherichia coli, and which is transformed into a large intestine rod g host cell towel **' and which is administered with an acid acetyltransferase. The nucleic acid molecule of a specific thiol transferase is derived from Helicobacter sinensis chunghsingensis KlF9705b ^ 核酸 lysine acetyltransferase nucleic acid molecule derived from 嗜 曱 曱: ancient

MethanohcdophUusportucalensis FOYI Liangxi is also sleepy base / Lai = other side; Fang =: Yu Yu rr", 3-aminotransposase is called 4 large acid intestinal white, transforming L-α-lysine matrix into compatible quality image B Stucco side acid. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Base lysine. In a "complex" way, by the living body (10) L_a • lysine, the conversion of the money, the base of the acid, the acid, the salt, the drought resistance and the ability to reduce the temperature P = one The specific secret is that the host is difficult to select from microorganisms, plants and animals. , in the formation of in the surface of the f'-type in vivo, the conversion of w-lysine and the blB of the nucleic acid molecule in the host cell in the common two main cells to the synthesis of La lysine, the brewing base _p _Lysine. In the body state, L_a•lysine is converted into a living body, and the acid is used to improve the salt resistance, drought resistance and high temperature resistance of the transformed or transformed organism. In another embodiment, the host organism is selected from the group consisting of a microorganism, a plant, and an animal. And a further aspect of the present invention provides a method for inducing a large amount of serotonin 1 tolerant TV6-acetamido-β-lysine (A/s_acety丨_p_〗 ysine) by T. oxysporum. In the square', the salt-tolerant sputum burns the ancient bacteria chest _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ In the anaerobic medium of M high salt (NaCl), the performance of the lysine 2,3-aminotransposase and the β-lysine acetyltransferase gene of the genus Artemisia sphaeroides was evaluated. Other features and advantages of the present invention will be apparent from the following description of the appended claims and claims. [Embodiment] Methanoca 丨cuius chunghsingensis I^lF9705b used in the winter invention is easily available to those skilled in the art, and has been deposited in the German strain center, the storage number is DSM 14539 # and the American decane. In the strain ~ ~ registered editor. Methanohalophihisportucalensis FDF1 is readily available to those of ordinary skill in the art, and is deposited in the German Stem Center (DSMZ) under the accession number DSM7471T, the American Types Center (ATCC) under the accession number BBA-912TM, and the American methanogen species. Center, the registration number is OCM 59. This study uses the published 尬 α α (10) coccw.ifes and (10) gene-named lysine 2,3-aminomutaSe, Μ / A sequence to maintain a highly retained region of the primer, The chromosomal mA of the salt-tolerant Methanocalculus chunghsingensis KlF9705b and the halophilic MethcmohabphHus portucdemis FOFI was subjected to an enzyme-linked reaction (PCR), and a partial sequence of 8 sputum and ΜρΜ/Α-0·8 kb fragments were obtained, respectively. Probes

Methanocakulus chunghsingensis anti-Methanohabphilus FDF1 gene was subjected to Southern ink collapse method (s〇uthemhybridizat analysis 'obtained the two methane archaeal compatible A^-acetyH^-lysine biosynthesis genes, respectively, life Z is McablA, McablB V human anti- MpablA, MpablQ I wind. In addition, the present invention has two methane archaea lysine 2,3_aminotransposase genes (M-/A, MeM/A), and β-lysine Transferase base ^ 201247869 (Λ / c ^ WB, Λ φβ6 / Β) 'Use the heterologous expression of E. coli, respectively, with a primer with a specific restriction enzyme cleavage site for polymerase chain reaction (pCR), after breeding Using the same restriction enzymes, the gene fragments were sent to the pET system for display in five (7) (DE3)-RIL, so that the recombinant protein can be used for in vitro synthesis of compatible iV8-ethylidene-β. _Lylamic acid. σ In addition, the present invention has utilized K1F9705b lysine 2,3_aminotransposase gene (McaWA), and β-lysine acetyltransferase gene (McaWB) to utilize Escherichia coli The common expression of the source, the McM/A gene fragment was constructed into the pET21b plastid And the Mcm/b gene fragment was constructed into the pET28a plastid' and the constructed pET21b_Mc was simultaneously introduced into the five (10) BL21 (DE3>RIL in a large number of expressions) with the /A and pET28a-M^/B plastids. The recombinant protein can be used to synthesize compatibilinyl _P_ lysine in vivo. It also recognizes (10) Μα/ορΜ(10)FDF1 lysine 2,3-aminotransposase gene (MpM/A), and β-lysine The acid acetyltransferase group 'cause (M W®)' uses a heterologous expression of E. coli to construct a gene fragment into the pET21b plastid, and the male/Β gene fragment is constructed into the pET28a plastid and will be constructed well. The pET21b-M/^/A and pET28a-M/^/B plastids were simultaneously fed into a large number of five. co/ί BL21 (DE3)-RIL, so that the recombinant protein produced can be used for in vivo synthesis and compatibility. The present invention will be described in detail with reference to the particular embodiments of the invention, which are not intended to limit the invention. The present invention is intended to include these and other variations and modifications. Embodiments Example 1. Separation and selection of salt-tolerant hospitals From bacteria

KlF9705b and the halophilic decane archaea M FDF1 ablA and ab electricity A·base ^ 12 201247869 The method for extracting decane from the ancient chromosomal DNA was modified by Jarrell (1992). 250 ml of the culture solution cultured to the middle log phase was collected into a centrifuge bottle, and centrifuged at 8000 rprn in a high-speed centrifuge (Sorvall RC5C, DuPont Co.) at 4 ° C for 15 minutes to collect the cells. The pellet obtained after centrifugation was suspended in 2 ml of lysis solution (10 mM Tris, pH 8.0, 1 mM EDTA pH 8.0; 2% SDS; 100 pg/ml proteinase K) and disrupted. The bacteria pieces were dispersed and allowed to stand on ice for 10 minutes, centrifuged at 14,000 rpm in a centrifuge (Sigma, 2K15) at 4 ° C for 10 minutes, and the supernatant was collected and stored in an equal volume of 4 ° 〇. 1^11〇1/(^11〇11〇£>〇1111^〇&111>^1(;〇11〇1 (25:24: 1) 'Gently shake evenly to centrifuge (Sigmi 2K15 ) 14000 rpm, 4 °C

Centrifuge for 10 minutes to recover the supernatant and repeat the above extraction operation once. The supernatant after centrifugation was carefully taken out and added to RNase (50 pg/ml) for one hour at 37 °C. Continue to extract twice with Phenol/chloroform/isoamyl alcohol, remove the supernatant and add half the volume of chloroform/isoamyl alcohol (24: 1). Gently shake gently to centrifuge (Sigma, 2K15) 14000 rpm, 4 ° Centrifuge for 10 minutes at C, remove the supernatant and repeat the chloroform/isoamyl alcohol extraction twice until there is no protein residue in the interface layer. The concentration of s〇dium acetate in the supernatant fraction was adjusted to 0.3 3 with 3 M sodium acetate to precipitate DNA, and 0.6-0.7 volumes of isopropanol was added and mixed uniformly to centrifuge (Sigma, 2Κ15) 14000 rpm, 4 〇C Centrifuge for 10 minutes, carefully remove the supernatant, and add a suitable amount of 70% alcohol to clean the DNA sample. Centrifuge (Sigma, 2K15) at 14000 rpm, centrifuge at 10 °C for 10 minutes, remove the supernatant, and vacuum decompressor. (Savant Speed Vac System, Savant Co.) The alcohol was drained, the DNA sample was reconstituted with sterile water, and the purity of the extracted DNA was examined by nucleic acid electrophoresis and measurement of the OD26G/28() ratio and the DNA content was calculated.

4^ Research using the published Methanococcoides burtonii and marpa/wito gene-named lysine 2,3-aminomutase (rib/A) sequence highly retained region design primer ablA bm-F fi'-GAAGATCCTCTTTCCGAAGAT-3', ablAbm-R 5,-GGTGATAA CACCTTCATAATT-3', obtained by polymerase chain reaction (PCR) of our laboratory-purified salt-tolerant M 13 201247869 炊(10);y KlF9705b and halophilic M-like & FDF1 chromosomal DNA The sequence Mca6/A-0.8 kb and the MpM/A-0.8 kb fragment were subsequently made into probes, respectively, for Southemhybridization analysis from KlF9705b and M I<DF1 genomes, respectively. Will

KlF9705b and M FDF1 chromosomal DNA were treated with restriction enzyme #1, respectively. Electrophoresis analysis was performed. The colloid was soaked in a colloidal shock plate with 0.25 N HC1 for 20 minutes, then the colloid was soaked with a denaturation buffer (0.5 NNaOH, 1.5 M NaCl) and shaken. After 20 minutes, the gel was soaked in a neutralization buffer (1.5 MNaCl, 1 MTris) and shaken for 20 minutes. The nucleic acid was then cleaved to a HybondTM-N+ Nylon membrane (Amersham) by vacuum spinning to immobilize the nucleic acid on the membrane with a short-wave ultraviolet nucleus. The probe prepared by the DIG DNA Labeling and Detection Kit was used for hybridization and immunodetection. As a result, two synthetic genomic DNAs of T.-acetyl-p-lysine compatible with the broth were obtained, which were named McaWA-1320 bp (listed in SEQ ID NO: 1) and Μ^ό/Β- 858 bp (sequences are listed in SEQ ID NO: 3), and ΛφΜ/Α-1314Βρ (sequences are listed in SEQ ID NO: 5) and ΛφΜ/Β-831 bp (sequences are listed in SEQ ID NO: 7, 8)' The relative positions of these genes are shown in Figure 1. The amino acid sequence of the 2,3_aminotransferase of the two strains of the genus Artemisia sphaeroides was analyzed, and the 2,3-aminotransfer of lysine of the two strains of decane archaea obtained by the present invention was found. The amino acid sequence of the enzyme gene 盥c/ay along the   lysine 2,3_aminotransferase amino acid sequence has a (1). combined with [4Fe-4S] duster The positional region contains the retained amino acid cystdne (CXXXCXXC); (2). The binding site with the SAM is DAPG/HGGGKIPV; (3). The binding site with the PLP is the lysine (K) in the SAM binding region; in addition, (4) Containing three halves at a position near the c-terminus, the amine acid 'presumably is the position to bind to zinc (see 2). The results of sequence analysis revealed that the secret t gene obtained from the Jiaxiagu g of the present invention is an acid-testing 2,3-aminotransposase gene. 201247869 The characteristics of the Jiji transferase enzyme can be divided into four according to the structure.

=mQtif) 'The order of its production is (3)_A_B, where the model has a longer and highly reserved area, is involved in a_ coenzyme A ί Y //λ and contains a portion of the retained amino acid in this region is 7 As a result, we found that the retention sequence of the β-lysine transferase-silica sequence and GNAT suPe-ily enzyme-one of the two strains of the sputum archaea we obtained was G/K_K/L_G 视福_k/g(10)2) . 'We have obtained the lysine from the methane archaea, and we can use these lysines to transfer acid to the acid by the α-lysine, and then hide the acid from the (tetra) amino acid. R σ to W acetyl-P-lysine can also confirm that the two gene products do have lysine 2,3·amino transposition oxime and β-lysine B The activity of thiol transferase. The two aminoglycans of the sucralose transferase were analyzed by the amino acid sequence of 甲ίί thiol transferase. The results showed that the amino acid sequences of blA and Mt. The degree is 47~80%; the similarity of the amino acid sequence of the tender and other ancient bacteria fibers is 24~52%' and the compatible nature of the salt-tolerant methanogens is synthesized by 6-ethenyl-lysine τϋηθ Genes are quite different. These two strains of burnt ancient lungs to protect iif low 'and have more negatively charged amino acids' can be exchanged with hydroxide ions, ί two surfaces for hydration layer, and maintain the hydrophobic surface Sex, in the formation of high-two bismuth; 唁 salty, salt-tolerant biological protein and salt into the halophilic, salt-tolerant biological protein can resist the organic solvent hall i'乂ϋ brother. The lysine 2 > aminotransferase and the ρ-; succinyltransferase have the characteristics of salt tolerance and high solvent resistance, and are more suitable for industrial and extracellular production applications. The implementation of ϋ ί ί ί ί ί 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί ί The obtained fragment was ligated with the plastid pGEM_7zf, and the obtained plastid was screened and confirmed by sequencing to have the gene of lysine 2,3-aminotransferase and β-lysine acetyltransferase. After performing a polymerase chain reaction, the restriction ends can be nicked at each end of the ruthenium (

I, mel-McablQ-Noil, Nhel-MpablA-XhcA tile Nhel-Mpam-XhcA). After colonization with the pGEM®-T Easy vector, transformation was performed on the competent E. coli JM101' onto a LA phte containing IPTG/X_gal and Ampicimn for screening. After selection by blue and white, the white (four) is selected and cultured, and the plastid is extracted, and the enzyme is cut by restriction enzymes (鸠61, from ^ and 〇1), and the fragment having the restriction enzyme cleavage site is screened and recovered. And then the expression vector after cutting with the same restriction enzymes: Ding 211) at 16. (: After 16 hours of cooperation, the cells were transformed into competent cell E. coli JM101, and the drug resistance gene Ampr was used as a screening marker. After further confirmation by restriction enzyme cleavage, the confirmed plastid ^ET21b_McabiA, pET21b-MpablA' pET21b-McablB and pET21b-male ablB) were re-transformed into the competent cell E. coli BL21 (DE2)-RIL, and the protein expression was induced by IPTG. The expression of recombinant protein was analyzed by protein electrophoresis. From the results shown in Fig. 3, wMblA, McAWB and the field exhibited a large amount of recombinant protein expression in Escherichia coli under the induction of IPTG. Moreover, these recombinant proteins expressed by the large intestine-producing bacteria are present as soluble proteins. Example 3. The heterologous E. coli host heterologously expresses the co-genesis of the methane archaea. The pGEM®-T-A7^I>M secret/Β_ΛΜ plastid obtained in Example 2 was cleaved with a restriction enzyme (Mel and chest I), and a fragment having a restriction enzyme cleavage site was selected and screened. Then, it was combined with Wei Lai pET28a in the _ hidden class, and then transferred to the competent cell E. coli toilet to resist the drug base, Kan%4_ mark. More recent - after the step of cutting the lion cut is confirmed, the confirmed plastids (pET21b-尬ablA and pET28a she a_ simultaneously transformed into the competent cell large intestine lake BL21 (DE2> RIL towel, with (four) lead her view eight The performance of the recombinant gene protein was analyzed by protein electrophoresis with the 201247869 fitAblB protein. The pGEM®-T obtained in Example 2 was uncovered, and the e-bee was used to cut the plastids to limit and gamma. The restriction cleavage site = the _ after the ____, the performance of the qing, tearing the machine for 16 hours 're-transformed to the competent cell E. coli leg (1), with the resistance group I ^ Kan as the _ mark, More closely - after the enzyme is confirmed, the ffl (pET21b姊ablA and pET28a姊ablB) will be transformed into the competent cell colon g g21 _2) l, with IPTG — MMblA and handsome AblB protein. Co-expression, paste protein electrophoresis analysis of recombinant fine protein conditions 0 The results of Figure 4 show that ' McAblA, her AblB, ΜΜ· and release awb have a large number of recombinant proteins in E. coli induced by IPTG. Example 4 .Northern ink collapse analysis The synthetic gene of A(-acetyl-p-ly sine) is affected by salt stress. The halophilic methane archaea 77 (?___> In an anaerobic medium containing different salinities (1.2, 1.65, 2.1, 2.5 and 2.9 MNaCl), the cells were cultured until the middle log phase to collect the cells by centrifugation. Or, the M FDF1 was cultured at 1.2 MNaCl to the middle log phase. Anaerobic operation, adding high-salt (2丄3.0, 3.8 and 4.6 M NaCl) anaerobic culture solution to the final salt concentration of 165, 2丄2.5 and 2.9 M NaCl and continue to culture at different time points The cells were collected by centrifugation, and then the RNA was extracted with a Rare RNA kit (Zhenxing Biotech Co., Ltd.), electrophoresed using TBE colloid, and RNA was spun using semi_dry electr〇bl〇tter system (Galileo Bioscience). On the HybondTM-N+ Nylon membrane, the nucleic acid was immobilized on a mold with a short-wave UV holder, and the homogenized A^-acetyl-p-lysine biosynthesis gene (71φαΖ)/Α was used as a probe for Northern ink collapse analysis. Scan the film into a map with the scanner IamgeScannerTM II (Amersham Co.) and use TINA The software software analyzes the signal size on the membrane (Versi〇n 2 〇9e, 17 201247869 raytest IsotopenmepgerSte). The relative transcript of a gene is calculated by dividing the target signal value by the 16S rRNA signal value relative to the target signal. The experimental results in Fig. 5 show that the halophilic A. facilis MFDF1 will increase with the increase of the concentration of the external salt, and the expression of the intracellular A^-acetyl-p-lysine biosynthesis gene will also increase. It can be inferred that the performance of lysine 2,3-aminotransferase (lysine 2,3-amin〇mutaSe) and β-lysine acetyltmnsfemse gene of methane archaea As the salt concentration increases, the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Other specific bears are that the features can be combined in any combination. Replace it with ^. Therefore, except for the same, equal or similar purpose, only the characteristics revealed by the - series equivalent thief-like feature ship indication'

From the foregoing description, the features of the present invention, and without departing from the scope of the invention, the subject matter of the present invention can be readily adapted to various different uses and various changes and modifications of the invention. Contains other specific aspects. The situation. Therefore, it is also included in the scope of the patent application 201247869 [Simple description of the diagram] Figure 1 shows the genetic sequence of the ablA and ablB genomes and regulatory regions in M-like & KlF9705b and Μ 吋wca/ hunger FDF1. Fig. 2 shows the results of comparative analysis of the amino acid sequence of the arsenic archaea acetyl-p-lysine. (A) is a sequence alignment of the lysine 2,3-aminotransposase amino acid sequence of archaea (C and osinW/ww such as 7m> 2a/e (LAM), The alignment analysis was obtained by the Qustai W program (SDSC Biology Workbench), wherein (8): ligand of iron atom; (b): lysine residue of SAM binding domain and PLP-binding position; (c): combination of words (B) is a sequence alignment analysis of the retention motif A of the primary sequence of the N-acetyltransferase associated with the GCN-5 of the Saccharomyces cerevisiae Hatl. (9) The expression of AblA and AblB of 诒KlF9705b and Μpom/ciz/famine FDF1 in Escherichia coli BL(DE3)-RIL. The E. coli transformants arriving in the middle log phase were treated with IPTG 4 (II), 8 (12, respectively) , 22 (13) hours to induce AblA and AblB expression, and separated by ΐ2·5°/〇SDS-PAGE. Μ: rainbow marker. Arrow indicates induced AblA (blue) and AblB (red) protein Figure 4 shows the common performance of A blA and AWB of M chunghsingemis KlF9705b anti-HP〇rtucalensis FDF1 in Escherichia coli BL(DE3)-RIL. The transgenic plants were treated with IPTG for 2 (ι), 4 (12), and 8 (13) hours, respectively, to induce AblA and AblB to be expressed together and separated by 12.5% SDS-PAGE. M: rainbow marker. Arrow indicates induction (blue) and AblB (red) proteins. Figure 5 shows the degree of transcription of M grown in the specified salt concentration medium, analyzed by the northern collapse. (a) The result of heterozygous probes; b) One quarter of the total RNA is separated by denaturing agar gel, stained with EtBr and directly detected by uv light; (c) Relative transcript calculated by TINA software. [Key Symbol Description] 201247869 Sequence Listing <110>Zhongxing University<12,0> Compatible saccharide of methicillin-β-lysine synthesis gene and its application <1608 <170> Patentln Version 3.4 <2101 <2Π>1320

<212>DNA <2:丨3> 盐盐甲甲菌菌 Afe/Zzawoca/cw/Ms K1 F9705b <4001 at^gaattat tttcacaaaa atacaaatcc ggagaatatc acaactcgat cggagaggat 60 gcaccccagt ggagggactg gcgctggcag atcgcccata ccgtccggag cctctcgatg 120 cttgagaagg ttttcggcat caccttcccc cccgaagagc gggagaagct gcaggagacg 180 atagataagt ttccccctgc tgcaacaccc tactatctct cgctcatcaa gaccgaggat 240 tatgccaatg atcccatctt ccggcaggcc gttccggttc ctgatgaaat gagggtggaa 300 gagtgtgagc ttgaagatcc ccttgccgaa gatagcgaca gcccggttcc ggggatcaca 360 caccggtatc cggatcgcgt cctcttcctt gtctcaaatg tctgcgcaat gtactgccgt 420 cattgtacac ggaagcggaa agtcggagat cgggacagaa tcccgacatg ggaggagatg 480 gaggtgggta tcacctatat tcgtgagcat cccgaggtcc gggatgtcct cctctcgggc 540 ggtgatccac tcatgctccc cgatgatctc cttgatagga Tcctcaccca acttcgggcg 600 caccggtatc cggatcgcgt cctcttcctt gtctcaaatg tctgcgcaat gtactgccgt 420 cattgtacac ggaagcggaa agtcggagat cgggacagaa tcccgacatg ggaggagatg 480 gaggtgggta tcacctatat tcgtgagcat cccgaggtcc gggatgtcct cctctcgggc 540 g gtgatccac tcatgctccc cgatgatctc cttgatagga tcctcaccca acttcgggcg 600 attccgcatg tcgaggtgat ccggatcggg tcgagaaccc ctgtcgtcct tccattcaga 660 ataactgatg gtctggtgaa tgtcctcaaa aaacatcagc cgatctggct caatacccat 720 tttaatcatc cccaggagat caccccgagt gctgagaagg cacttgcgaa actcgcggat 780 gccgggattc cactcggcaa ccagtcggtc ctcctcgccg gagtgaatga ctgcccgagg 840 atcatgaagt cgctcgtcca gaagctcgtc aaaaacaggg tccggccgta ctatctctac 900 cagtgtgatc tctctgaggg tctctcgcac ttccggaccc cggtcgggaa ggggattgag 960 atcatggaga acctcatcgg ccatacaagc ggctttgcgg ttccaaccta tgtcatcgac 1020 gcacctggtg gcggcgggaa gatcccggtg atgccgacat atctcatatc gtactccacc 1080 aacaaggtga tcctcaggaa ctttgaaggg gtgatcacca cctaccggga gcctgacaac 1140 tatacctcga tcttctgtga tcgcaactgc aaggactgtt cactccagct gaaactcgag 1200 gccggagacg agcagcatgt cgtcgggatc gcaaagctcc tctcagatta tgatgaagcg 1260 201247869 1320 acggcactcg tccctgccga ctcggaacgg atggcacgga gagaggatga agaggagtga < 210 > 2 < 211 >439

<212>PRT <2\3> salt-tolerant sputum archaea Methmoca/cuiuschmghsingensis KlF9705b <4002

Met Glu Leu Phe Ser Gin Lys Tyr Lys Ser Gly Glu Tyr His Asn Ser 15 10 15 lie Gly Glu Asp Ala Pro Gin Trp Arg Asp Trp Arg Trp Gin lie Ala 20 25 30

His Thr Val Arg Ser Leu Ser Met Leu Glu Lys Val Phe Gly lie Thr 35 40 45

Fhe Pro Pro Glu Glu Arg Glu Lys Leu Gin Glu Thr lie Asp Lys Phe 50 55 60

Pro Pro Ala Ala TTir Pro Tyr Tyr Leu Ser Leu lie Lys Thr Glu Asp 65 70 75 80

Tyr Ala Asn Asp Pro lie Phe Arg Gin Ala Val Pro Val Pro Asp Glu 85 90 95

Met Arg Val Glu Glu Cys Glu Leu Glu Asp Pro Leu Ala Glu Asp Ser 100 105 110

Asp Ser Pro Val Pro Gly lie Thr His Arg Tyr Pro Asp Arg Val Leu 115 120 125

Phe Leu Val Ser Asn Val Cys Ala Met Tyr Cys Arg His Cys Thr Arg 130 135 140

Lys Arg Lys Val Gly Asp Arg Asp Arg lie Pro Thr Trp Glu Glu Met 14:5 150 155 160

Glu Val Gly lie Thr Tyr He Arg Glu His Pro Glu Val Arg Asp Val 165 170 175

Leu Leu Ser Gly Gly Asp Pro Leu Met Leu Pro Asp Asp Leu Leu Asp 180 185 190

Arg lie Leu Thr Gin Leu Arg Ala He Pro His Val Glu Val lie Arg 195 200 205 lie Gly Ser Arg Thr Pro Val Val Leu Pro Rie Arg lie Thr Asp Gly 210 215 220

Leu Val Asn Val Leu Lys Lys His Gin Pro lie Trp Leu Asn Thr His 225 230 235 240

Fhe Asn His Pro Gin Glu lie Thr Pro Ser Ala Glu Lys Ala Leu Ala 245 250 255

Lys Leu Ala Asp Ala Gly lie Pro Leu Gly Asn Gin Ser Val Leu Leu 260 265 270

Ala Gly Val Asn Asp Cys Pro Arg lie Met Lys Ser Leu Val Gin Lys 2 201247869 275 280 285

Leu Val Lys Asn Arg Val Arg Pro Tyr Tyr Leu Tyr Gin Cys Asp Leu 290 295 300

Ser Glu Gly Leu Ser His Phe Arg Thr Pro Val Gly Lys Gly lie Glu 305 310 315 320 lie Met Glu Asn Leu lie Gly His Thr Ser Gly Phe Ala Val Pro Thr 325 330 335

Tyr Val He Asp Ala Pro Gly Gly Gly Gly Lys lie Pro Val Met Pro 340 345 350

Thr Tyr Leu lie Ser Tyr Ser Thr Asn Lys Val lie Leu Arg Asn Rie 355 360 365

Glu Gly Val lie Thr Thr Tyr Arg Glu Pro Asp Asn Tyr Thr Ser lie 370 375 380

Phe Cys Asp Arg Asn Cys Lys Asp Cys Ser Leu Gin Leu Lys Leu Glu 385 390 395 400

Ala Gly Asp Glu Gin His Val Val Gly lie Ala Lys Leu Leu Ser Asp 405 410 415

Tyr Asp Glu Ala Thr Ala Leu Val Pro Ala Asp Ser Glu Arg Met Ala 420 425 430

Arg Arg Glu Asp Glu Glu Glu 435 <2103 <211>858

≪ 212 > DNA < 213 > tolerance, said archaea Methanocalculuschunghsingemis KAF97Q5b < 4003 atgaagagga gtgatccggt catcagggtg ggccgaagcc tcatccagca tggaagctat 60 aacaatcgga tctacctgat gaaactcgat cctgaggata ccgccctcat tcttggttgg 120 atccgggaga cgcttgaaga agaggggtat agcaaggtct tcgcaaagat cccgggtccg 180 gctgcacctg catttgagag ggaggggttc ggcgttgagg cgagaatccc cggctacttc 240 agaaatggtg atacctgtct cttcatgggg aagtacaccg atccctcgcg gagggagiat 300 aatgaggaag gggtagccgt agcactcggg actgccttag aacgggcagg ttcaggccat 360 aatccattaa aggagggata tctgatccat gaggcagacc tttccgatgc tgaggcgctt 420 gcagagctct atggaacggt ctttccgagc tacccgtttc cgatagatga tcccggcttc 480 atacagacct cgatagagag cggggagacc cgtttcttta tggtgatgaa cggagagacc 540 ctcctcgccg cctcgtcggc agaacttgac cctgattccg gaacggtcga gatgactgac 600 tttgcgaccc tcccggaggc gagggggctc ggtgttgccg gtgctctcct gagacatatg 660 gaaggggttg tacgggatga Cggctatcat cttgcctaca ccatctgccg tggcgaggag 720 cctgcggtga atatcctctt tgcacgtgga gggtaccagt ttgccggcac cctcccgaa c 780 aatacccaga ttggaggagg atttgagagt atgaatgtct ggtaccgatc ccttcttacg 840 aacccttcgc ctggttaa 858 3 201247869 <210>4 <2I1>285

<2;[2>PRT <213> Saskatchewan ψ'跋j archaea Methanocalcu丨uschmghsingensisKAF9705b <4Q0>4

Mel. Lys Arg Ser Asp Pro Val lie Arg Val Gly Arg Ser Leu lie Gin 15 10 15

His Gly Ser Tyr Asn Asn Arg He Tyr Leu Met Lys Leu Asp Pro Glu 20 25 30

Asp Thr Ala Leu lie Leu Gly Trp lie Arg Glu Thr Leu Glu Glu Glu 35 40 45

Gly Tyr Ser Lys Val Phe Ala Lys lie Pro Gly Pro Ala Ala Pro Ala 50 55 60

Phe Glu Arg Glu Gly Hie Gly Val Glu Ala Arg lie Pro Gly Tyr Phe 65 70 75 80 hr\l Asn Gly Asp Thr Cys Leu Phe Met Gly Lys Tyr Tlir Asp Pro Ser 85 90 95

Arg Arg Glu Tyr Asn Glu Glu Gly Val Ala Val Ala Leu Gly Thr Ala loo 105 no

Leu Glu Arg Ala Gly Ser Gly His Asn Pro Leu Lys Glu Gly Tyr Leu 115 120 125 lie His Glu Ala Asp Leu Ser Asp Ala Glu Ala Leu Ala Glu Leu Tyr 130 135 140

Gly Thr Val Phe Pro Ser Tyr Pro Fhe Pro lie Asp Asp Pro Gly Phe 14:5 150 155 160 lie Gin Thr Ser lie Glu Ser Gly Glu Thr Arg Phe Phe Met Val Met 165 170 175

Asn Gly Glu Thr Leu Leu Ala Ala Ser Ser Ala Glu Leu Asp Pro Asp 180 185 190

Ser Gly Thr Val Glu Met Thr Asp Phe Ala Thr Leu Pro Glu Ala Arg 195 200 205

Gly Leu Gly Val Ala Gly Ala Leu Leu Arg His Met Glu Gly Val Val 210 215 220

Arg Asp Asp Gly Tyr His Leu Ala Tyr Thr lie Cys Arg Gly Glu Glu 22:5 230 235 240

Pro Ala Val Asn lie Leu Phe Ala Arg Gly Gly Tyr Gin Phe Ala Gly 245 250 255

Thr Leu Pro Asn Asn Thr Gin lie Gly Gly Gly Phe Glu Ser Met Asn 260 265 270

Val Trp Tyr Arg Ser Leu Leu Thr Asn Pro Ser Pro Gly 275 280 285 201247869 <2105 <211>1314

≪ 212 > DNA < 2] .3 > order salt, Wyatt archaea Methanohalophihisportucalensis FDF \ < 400 > 5 atgacacagc acacacaagc acaaaaagaa atagctaaaa aaatcgattc tgatactgtt 60 att: gccaact ggaaggattg gagatggcaa ctcaagcatt ctattggtga tattgataca 120 tugagactt tacttggcat caaattcaaa cccggggaaa aggataaact caaacaaact 180 ctggaaaaat tcccattate cgtaactcca tactaccttt ccctcataga tgccgaggat 240 ttcaggaatg atcccatatt cctgcaggcc tttccttcac caaaggagct ggacatcgat 300 gaagaegate ttgaagatcc tctttctgag gatgaggaca gtcctgttga aggaatcaca 360 cacaggtatc cggacagggt actattccat ataagcaaca catgttcgat gtactgcagg 420 cactgtacac gtaagagaaa agtgggagat gtagattcca ttcctacaaa ggatgcagtc 480 tetgaaggae tggaatacat caggaatacc ccacaggtac gagatgtcct gctttcgggc 540 ggagatcctt ttatgttgcc tgatgettat ctggactgga tcctggcaaa attgcgggaa 600 attcctcatg ttgagattat Tegtateggt acaaggatgc ccgtggtgct tccttacagg 660 gtaactgatg atcttgtaaa aatactcaaa aagcatcatc cgctctggat caatactcac 720 ttcaaccatc cacgtgaagt aaeggeatet tccagggaag ccctgcgcaa aettgeagat 780 gcaggaattc ccctcggaaa ccagacggtg ttactttcag gagtgaatga ctgccacagg 840 attatgaaaa ggcttgtaca gaaacttgtg caaaaccgtg taegteetta ttatctctac 900 cagtgtgacc ttteggaagg cctgtcccat ttccgcactc cggttggcaa gggtatagaa 960 ataatggaac acctcatcgg ccacacaagt ggttttgcag ttcctaccta tgttatcgat 1020 gctccccatg gaggaggtaa gatacctgtc atgccttcct atettatate ctggtccacc 1080 aacagggtta ttttgcgtaa ctatgagggt gtaatcacct catataaaga accggattcc 1140 tatgageega tatactgtga ccgtaaatgc gaagaatgta aactccagct caaacttgac 1200 Gatgeagetg aatataaatc tacaggtatt gcaaaactgc ttgccgataa tgatgaagtt 1260 gtaagtcttg taccggaaga taccgaaaga atggagagaa gggacgttga gtga 1314 <2106 <2.11>437

<2.12>PRT <2\3> 靖盘ψ,炭古错 Methanohalophiiuspomtcaiensis FDF\ <4006

Met Thr Gin His Thr Gin Ala Gin Lys Glu lie Ala Lys Lys lie Asp 15 10 15

Ser Asp Thr Val lie Ala Asn Trp Lys Asp Trp Arg Trp Gin Leu Lys 20 25 30

His Ser lie Gly Asp lie Asp Thr Phe Glu Thr Leu Leu Gly lie Lys 35 40 45

Hie Lys Pro Gly Glu Lys Asp Lys Leu Lys Gin Thr Leu Glu Lys Phe . 5 201247869 50 55 60

Pro Leu Ser Val Thr Pro Tyr Tyr Leu Ser Leu lie Asp Ala Glu Asp 65 70 75 80

Phe Arg Asn Asp Pro lie Phe Leu Gin Ala Phe Pro Ser Pro Lys Glu 85 90 95

Leu Asp He Asp Glu Asp Asp Leu Glu Asp Pro Leu Ser Glu Asp Glu 100 105 110

Asp Ser Pro Val Glu Gly lie Thr His Arg Tyr Pro Asp Arg Val Leu 115 120 125

Hie His lie Ser Asn Thr Cys Ser Met Tyr Cys Arg His Cys Thr Arg 130 135 140

Lys Arg Lys Val Gly Asp Val Asp Ser lie Pro Thr Lys Asp Ala Val 145 150 155 160

Set Glu Gly Leu Glu Tyr lie Arg Asn Thr Pro Gin Val Arg Asp Val 165 170 175

Leu Leu Ser Gly Gly Asp Pro Phe Met Leu Pro Asp Ala Tyr Leu Asp 180 185 190

Trp lie Leu Ala Lys Leu Arg Glu He Pro His Val Glu lie lie Arg 195 200 205

He Gly Thr Arg Met Pro Val Val Leu Pro Tyr Arg Val Thr Asp Asp 210 215 220

Leu Val Lys lie Leu Lys Lys His His Pro Leu Trp lie Asn Thr His 225 230 235 240

Phe Asn His Pro Arg Glu Val Thr Ala Ser Ser Arg Glu Ala Leu Arg 245 250 255

Lys Leu Ala Asp Ala Gly lie Pro Leu Gly Asn Gin Thr Val Leu Leu 260 265 270

Ser Gly Val Asn Asp Cys His Arg lie Met Lys Arg Leu Val Gin Lys 275 280 285 k:u Val Gin Asn Arg Val Arg Pro Tyr Tyr Leu Tyr Gin Cys Asp Leu 290 295 300

Ser Glu Gly Leu Ser His Phe Arg Thr Pro Val Gly Lys Gly lie Glu 305 310 315 320 lie Met Glu His Leu lie Gly His Thr Ser Gly Phe Ala Val Pro Thr 325 330 335

Tyr Val lie Asp Ala Pro His Gly Gly Gly Lys lie Pro Val Met Pro 340 345 350

Ser Tyr Leu lie Ser Trp Ser Thr Asn Arg Val lie Leu Arg Asn Tyr 355 360 365

Glu Gly Val lie Thr Ser Tyr Lys Glu Pro Asp Ser Tyr Glu Pro lie 370 375 380

Tyr Cys Asp Arg Lys Cys Glu Glu Cys Lys Leu Gin Leu Lys Leu Asp 385 390 395. 400 201247869

Asp Ala Ala Glu Tyr Lys Ser Thr Gly He Ala Lys Leu Leu Ala Asp 405 410 415

Asn Asp Glu Val Val Ser Leu Val Pro Glu Asp Thr Glu Arg Met Glu 420 425 430

Arg Arg Asp Val Glu 435 <2107

<211>83I

i tt ^ agtgata taatcgaaaa tataggcagc tccctgatcc agcacggtac gtacaacaat 60 cgtatttacc ttatgaaact ggatactggc gatctgccgg atattatacc tgaaatggat 120 ca ^ tiggcag aacaacgcgg ctacagcaag atatttgcaa aagtccctgc agattccaga 180 gtcgcttttg aagcaaaggg atatataaag; < 212 > DNA < 2] 3 > addicted Sao ancient mirror MethanohalopMusportucaiemisFOF \ < m & gt gaggctggtg ttcccggcta ttattccggc 240 aagaaagaag ccctgttcat gtgtaaatat ttctcagagc acagacagga ggacacacag 300 gccgaggatg accggatcat caccactgcc agaggtaagg caggagacat catgaaacct 360 gaattacccg aaggtttcct tatacggata tgtacggaag cggatgtgcc tgcaatggca 420 gagatctacg ccgaagtgtt ccctacatat ccttttecta tccaggaccc ggcctacctg 480 aaagagacca tggaagataa tgttgtatat ttcgcagttg aaaatgaagg taacatagtt 540 gccctttcct cctgcgagat cgattttgaa aaccggaatg eggaaatgae cgactttgcc 600 acactccctc agttcagggg aatgggtctg tccacttatc Tcctctcaaa gatggaaaca 660 gaaatgaata agaaagggat ccttatcttt tatacaattg cccgggccgc gtcctatggt 720 atgaatactg tttttgcaag gcttgggtat gaatttaccg gaaaacttgt tcaaaatacc 780 aatatat Ccg gtaaccttga aaatatgaat gtatggtaca agcgcatctg a 831 CH>8 <!11>276

<il2>PRT <213> Eating salt archaea MethanohabphilusporiucalensisFOF] <400>8

Met Ser Asp lie lie Glu Asn lie Gly Ser Ser Leu lie Gin His Gly 1 5 · 10 15 Ήιγ Tyr Asn Asn Arg lie Tyr Leu Met Lys Leu Asp Thr Gly Asp Leu 20 25 30

Pro Asp lie lie Pro Glu Met Asp Gin Leu Ala Glu Gin Arg Gly Tyr 35 40 45

Ser Lys He Rie Ala Lys Val Pro Ala Asp Ser Arg Val Ala Phe Glu 50 55 60

Ala Lys Gly Tyr lie Lys Glu Ala Gly Val Pro Gly Tyr Tyr Ser Gly 7 201247869 65 70 75 80

Lys Lys Glu Ala Leu Phe Met Cys Lys Tyr Phe Ser Glu His Arg Gin 85 90 95

Glu Asp Thr Gin Ala Glu Asp Asp Arg lie lie Thr Tlir Ala Arg Gly 100 105 110

Lys Ala Gly Asp lie Met Lys Pro Glu Leu Pro Glu Gly Phe Leu lie 115 120 125

Arg lie Cys Thr Glu Ala Asp Val Pro Ala Met Ala Glu lie Tyr Ala 130 135 140

Glu Val Fhe Pro ΤΤπ Tyr Pro Phe Pro lie Gin Asp Pro Ala Tyr Leu 145 150 155 160

Lys Glu Thr Met Glu Asp Asn Val Val Tyr Phe Ala Val Glu Asn Glu 165 170 175

Gly Asn lie Val Ala Leu Ser Ser Cys Glu lie Asp Phe Glu Asn Arg 180 185 190

Asn Ala Glu Met Thr Asp Phe Ala Thr Leu Pro Gin Phe Arg Gly Met 195 200 205

Gly Leu Ser Tlir Tyr Leu Leu Ser Lys Met Glu Thr Glu Met Asn Lys 210 215 220

Lys Gly lie Leu lie Hie Tyr Thr lie Ala Arg Ala Ala Ser Tyr Gly 225 230 235 240

Met Asn Thr Val Phe Ala Arg Leu Gly Tyr Glu Phe Thr Gly Lys Leu 245 250 255

Val Gin Asn Thr Asn lie Ser Gly Asn Leu Glu Asn Met Asn Val Τφ 260 265 270

Tyr Lys Arg lie 275

Claims (1)

201247869 VII. Scope of application for patents: 1. A compatible τν6-acetamido-β-lysine (TV^acetyl-p-lysine)-derived enzyme-based enzyme encoding the salt-tolerant A. sylvestris Aiethanohabphihis portuccdensis FDF1 An isolated nucleic acid molecule of lysine 2,3-aminomutase, wherein the lysine 2,3-aminotransposase has the amino acid sequence of SEQ ID No. 6. 2. An isolated nucleic acid molecule encoding a lysine 2,3-aminotransposase according to claim 5, which comprises the nucleotide sequence of SEQ ID NO: 5. 3. A post-compilation of the salt of the genus Methanohalophihis poriucalemis FDF1 compatible with acetyl-β-lysine (A^-acetyl-p-lysine) biosynthetic enzyme β-lysine transfer An isolated nucleic acid molecule of the enzyme (β-lysine acetyltransferase), wherein the β-lysine acetyltransferase has the amino acid sequence of SEQ ID No. 8. 4. The isolated nucleic acid molecule encoding a beta-lysine acetyltransferase according to claim 7 of the patent application, comprising the nucleotide sequence of SEQ ID NO: 7. A method for producing a compatible A^-acetyl-p-lysine in vivo, which is characterized by using the susceptibility according to items 2 and 4 of the patent application scope. Salted methane archaea her ω, ie 2, FDF1 lysine 2,3-aminotransposase and β-lysine acetyltransferase nucleic acid molecule 'selected in appropriate expression vector, and will be obtained The recombinant plasmid is transformed into an E. coli host cell to express a large amount of recombinant protein to convert the L-α-lysine matrix into a compatible TV6-acetamido-β-lysine. 6. A method for inducing a large amount of biosynthesis-compatible Λ^-acetyl-p-lysine (A--acetyl-p-lysine) in arsenic archaea, characterized by halophilic decane The archaea Methanohalophilus portucalensis FDF1 is cultured in an anaerobic medium containing a final salt concentration of 1.5 to 3.0 Μ high salt (NaCl) to enhance the lysine 2,3_aminotransferase and β-lysine B of the methane archaea The performance of the thiol transferase gene.