TWI450964B - Osmolyte nε-acetyl-β-lysine biosynthetic genes from methanogenic archaea and application thereof - Google Patents

Description

Compatible N-ε-acetyl-β-lysine biosynthesis gene of methane archaea and its application

This case is the divisional case of the invention patent application of No. 097146358 "Compatible N ^ε -Ethyl-β-lysine synthesis gene of methane archaea and its application".

The present invention relates to a compatible N ^ε -acetyl-β-lysine biosynthesis enzyme encoding methanogenic archaea (lysine 2,3- lysine 2,3-aminotransferase (lysine 2,3- Aminomutase) and a single-stranded nucleic acid molecule of β-lysine acetyltransferase. The present invention also relates to the use of such nucleic acid molecules to produce a compatible N ^ε -acetyl group in an Escherichia coli host. A method of synthesizing an enzyme with basal-β-lysine. The present invention further provides a method for producing a methane archaeal compatible N ^ε -acetyl-β-lysine in vivo and/or in vitro.

Compatible with osmotic pressure protection factor, it has the characteristics of moisturizing, salt-resistant and drought-resistant. Its biosynthetic genes have considerable application potential in the fermentation industry, food industry, cosmetic beauty industry, pharmaceutical, medical and agricultural fishery. Compatible mass ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) for a particular amino acid type beta], now only been found in methanogenic archaea in other eukaryotes, It has not been found in prokaryotes. The biosynthesis pathway of the compatible N ^ε -acetamido-β-lysine is converted from β-lysine by α-lysine via lysine 2,3-aminotransposase ( abl A) Acid, followed by the synthesis of N ^ε -ethinyl-β-lysine by the action of β-lysine acetyltransferase ( abl B) (see, for example, Lai, MC et al., J. Bacteriol . 173: 5352-5358, 1991; Roberts, MF et al, J. Bacteriol . 174: 6688-6693, 1992; and Robertson DE and MF Roberts, BioFactors 3: 1-9, 1992).

Pfl Ger et al. ( Appl. Environ. Microbiol . 69 : 6047-6055, 2003) from methane archaic creature Methanosarcina mazei G In the whole genome sequence of 1 , the gene for the self-generated synthetic N ^ε -acetyl-β-lysine enzyme is lysine 2,3-aminotransposase ( abl A). And beta-lysine acetyltransferase ( abl B). Using the northern blotting method to confirm that the two genes abl A and abl B are the same operon, the expression of the abl operon will increase with the increase of the salt concentration in the culture environment. And using Methanococcus maripaludis JJ to construct abl A and abl B mutants, the mutants were cultured at different salt concentrations, and the content of intracellular cumulative compatible N ^ε -acetyl-β-lysine was detected by NMR and tested. The growth condition was confirmed by NMR analysis. The accumulation of N ^ε -ethyl thio-β-lysine in the wild type cells was 0.14 μmol/mg in the low salt (376.5 mM NaCl) environment. As the culture salinity increased to 800 mM and 1 M NaCl, the cumulative amount of intracellular N ^ε -acetyl-β-lysine increased by 3.7 times to 0.7 μmol/mg and increased by 7.8 times to 1.09 μmol/mg. However, when the mutant strain was cultured in a low salt or high salt environment, the accumulation of N ^ε -ethyl thiol-β-lysine was not detected in the cells, indicating that the abl A and abl B genes are compatible N. ^ε -Ethyl-β-lysine autologous biosynthesis lysine 2,3-aminomutase and β-lysine acetyltransferase (β- Translated by lysine acetyltransferase; the growth test results of the mutant strains cultured under different salinities showed that the growth rate of the mutant strain in the low salt (376.5 mM NaCl) environment was the same as that of the wild strain. Lower (800 mM NaCl) significantly inhibited the growth environment, the salinity of cell damage at 1 M NaCl almost no growth environment, show high osmotic pressure difference JJ response to the extracellular environment of a high salt accumulation is compatible M. maripaludis Qualitative N ^ε -ethinyl-β-lysine as an osmotic pressure adaptation mechanism (Pfl Ger, 2003, as mentioned above).

Methanohalophilus portucalensis FDF1 is an irregular bacterium isolated from Marthrani et al. from the salt pool of saturated sodium chloride in Figutria da Foz, Portugal, and was officially named by Boone D, R in 1993 (Boone et al . , 1993). M. portucalensis FDF1 can use methanol, mono-, di- or trimethylamine as a carbon source for methanogenesis, but cannot utilize H ₂ +CO ₂ , formate, acetate, methyl sulfide Alcohol or dimethyl hydrazine is the growth substrate (Boone et al ., 1993). M. portucalensis FDF1 can grow in an environment with a salt concentration range of 1.0-3.0 M. The optimum growth salt concentration is 2.1 M. In high-salt environment, the intracellular and extra- osseous osmotic pressure cannot be balanced by changing the cell volume. Density is its main osmotic pressure regulation mechanism (Lai et al ., 1991, 1992). It was confirmed by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) experiments that M. portucalensis FDF1 was not present in the environment when glycine betaine was absent. Mainly accumulate potassium ions, α-glutamine, β-glutamine, betaine and N ^ε -ethinyl-β-lysine in the cell in response to the high osmotic pressure difference generated by the extracellular high salt environment. Solutes are used to cope with the high osmotic pressure of the outside world. These solute have a low turnover rate in the cell, are not involved in protein synthesis and cellular metabolism, and can accumulate in the cell for a long time, and their cumulative amount increases as the extracellular salt concentration increases, indicating that these solutes are M. portucalensis FDF1 plays a complementary role in the cell (Lai et al ., 1991; Robertson et al ., 1992, supra). M. portucalensis FDF1 has the ability to automate the synthesis of betaine, N ^ε -ethinyl-β-lysine and β-glutamine as a compatible substance. The biosynthesis of N ^ε -ethinyl-β-lysine is the production of α-lysine via the diaminopimelate pathway, which is then converted to lysine 2,3-aminomutase by lysine 2,3-aminomutase. --lysine, followed by the synthesis of N ^ε -acetyl-β-lysine by the action of β-lysine acetyltransferase.

Salt-tolerant methane archaea Methanocalculus chunghsingensis K1F9705b is an irregular cocci isolated from the marine aquaculture site near Wanggong in Changhua County, Taiwan (Lai et al., 2004), using H ₂ and CO ₂ as carbon sources. To carry out methanation. M. chunghsingensis K1F9705b can be grown in an environment with a salt concentration ranging from 0 to 2.0 M. The optimum growth salt concentration is 0.175 M. According to the analysis of nuclear magnetic resonance (NMR), M. chunghsingensis K1F9705b accumulates β-glutamine and N ^ε -acetamidine in the cell in response to the high osmotic pressure difference generated by the extracellular high salt environment. Basal-β-lysine, and its amount increases as the concentration of external salt increases. The intracellular turnover rate of these solutes is very low, indicating N ^ε -ethinyl-β-lysine and Beta-glutamine is the major osmotic compatible compatibilizer of this strain.

The lysine 2,3-aminotransposase (AblA) enzyme functions to catalyze the conversion of alpha-lysine to beta-lysine. This enzyme catalysis requires cofactors: [4Fe-4S]cluster, S-adenosylmethionine (SAM), and pyridoxal 5'-phosphate (PLP). The mechanism of action is that the intracellular reducing protein reduces [4Fe-4s] ²⁺ to the activated state of [4Fe-4S] ¹⁺ , and the activated [4Fe-4S] ¹⁺ releases electrons to react with the SAM to cleave the SAM. Produces methionine and 5'-deoxyadensyl free radicals. The PLP and the ε-ammonium group on the lysine amino acid of the AblA protein form an internal aldimine bond. In the presence of the substrate α-lysine, PLP undergoes a transaldimation reaction with the α-amino group of the α-lysine to form an external aldimine bond. The 5'-deoxyadensyl radical generated by the action of [4Fe-4S] ¹⁺ and SAM interacts with the PLP-bonded α-lysine to produce 5'-deoxyadenosine and an intermediate with free radicals. The isomerization reaction, as well as the transfer of hydrogen atoms from 5'-deoxyadenosine to form β-lysine (Frey and Reed, Adv. Enzymol. Relat. Areas. Mol. Biol. 66 : 1-39, 1993; Chen and Milne, Biochemistry. 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 The cluster binding position is the retention of cysteine, which has more glycine acid in the binding position with the SAM, and the position of the PLP linkage is lysine (Chen and Frey. Biochem . 40: 596-602, 2001). Studies have also indicated that the activation of this enzyme requires the participation of zinc (Ruzicka, 2000), presuming that the three cysteine amino acids contained at the position near the C-terminus of the amino acid sequence are zinc-binding sites. In the published methane archaic creature M. mazei G The location of these cofactor bindings was found in the lysine 2,3-aminotransposase sequence of 1.

The β-lysine acetyltransferase (AblB) enzyme functions to catalyze the production of N ^ε -ethinyl-β-lysine by β-lysine. The enzyme acetyltransferase belongs to the superfamily of GNAT (GCN-5-related N -acetyltransferase). This GNAT superfamily enzyme acts to catalyze the transfer of its acetamino group to its first grade. Primary amine. The role of the receptor can be divided into three types: (1) histone: involved in transcriptional activation, chromatin assembly and DNA repair process, acetyltransferase mainly acts on specific lysine; (2) aminiglycosides: ethionyl transfer Enzyme activity can cause antibiotic inhibition of pathogenic bacteria, which acts on antibiotics of aminiglycosides, resulting in decreased affinity of such antibiotics for targets; (3) arylalkylamines: currently only found in vertebrates, participation Catalyzing the process by which serotonin produces melatonin. The amino acid sequence of the acetaminotransferase can be divided into four conserved motifs according to the secondary structure, and the order of the amino acid is CDAB. The functions of the phenotypes B, C and D are mainly related to the structural stability of the protein. And phantom A is a region containing a long and highly retained region, which is involved in the binding of acetaminophen A, and contains a retained amino acid in this region as Q/RXXGXG/A (Dyda, 2000; Wolf, 1998). ). To date, there has been no study on the properties of beta-lysine acetyltransferase ( Abl B) enzymes, but in the published methane archaic organism M. mazei G In the β-lysine acetyltransferase sequence of 1 , the amino acid sequence found to be retained is RGKGHMK (Pfl Ger, 2003, as mentioned above).

In one aspect, the invention provides an encoding salt of methanogenic archaea Methanocalculus chunghsingensis K1F9705b compatible substance of ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) biosynthetic enzyme depends amine 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. 2 or the same Functional variants.

In a specific aspect, the isolated nucleic acid molecule encoding a lysine 2,3-aminotransposase comprises a nucleotide sequence selected from the group consisting of (i) SEQ ID NO: 1; and (ii) having the SEQ ID NO: A nucleotide sequence of a group consisting of nucleotide sequences having a nucleotide sequence similarity greater than 90%.

In another aspect, the invention provides an encoding salt of methanogenic archaea Methanocalculus chunghsingensis K1F9705b compatible mass of ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) biosynthetic enzyme beta] - an isolated nucleic acid molecule of ?-lysine acetyltransferase, wherein the beta-lysine acetyltransferase has the amino acid sequence of SEQ ID No. 4 or a functionally equivalent variant thereof .

In one embodiment, the isolated nucleic acid molecule encoding beta-lysine acetyltransferase comprises a nucleotide sequence selected from (i) SEQ ID NO: 3; and (ii) has SEQ ID NO : a nucleotide sequence of a group consisting of nucleotide sequences having a nucleotide sequence similarity greater than 90%.

In a further aspect, the present invention provides a compatible N ^ε -acetyl-β-lysine biosynthetic enzyme lysine 2,3-aminotransposase (lysine 2) encoding Methanohalophilus portucalensis FDF1. An isolated nucleic acid molecule of 3-aminomutase, wherein the lysine 2,3-aminotransposase has the amino acid sequence of SEQ ID No. 6 or an isofunctional variant thereof.

In one embodiment, the isolated nucleic acid molecule encoding a lysine 2,3-aminotransposase comprises a nucleotide sequence selected from the group consisting of (i) SEQ ID NO: 5; and (ii) having the SEQ ID NO: A nucleotide sequence of a group consisting of nucleotide sequences having a nucleotide sequence similarity greater than 90%.

In another aspect, the invention provides an encoding halophilic methanogenic archaea Methanohalophilus portucalensis FDF1 compatible substance of ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) biosynthetic enzyme beta] - an isolated nucleic acid molecule of ?-lysine acetyltransferase, wherein the beta-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 (i) SEQ ID NO: 7; and (ii) has SEQ ID NO : The nucleotide sequence of the group consisting of nucleotide sequences having a nucleotide sequence similarity greater than 90%.

Another aspect of the present invention provides a method for expressing a large amount of methane archaea lysine 2,3-aminotransposase using Escherichia coli, which is characterized in that a nucleic acid encoding a 2,3-aminotransferase of lysine is used. The molecule is selected in an appropriate expression vector, and the resulting recombinant plasmid is transformed into a large amount of expression in an E. coli host cell, and the recombinant lysine 2,3-aminotransposase is purified. In one particular aspect, the nucleic acid encoding lysine 2,3-amino translocase based molecules derived from the salt of methanogenic archaea Methanocalculus chunghsingensis K1F9705b genome. In another embodiment, the nucleic acid molecule encoding a lysine 2,3-aminotransposase is derived from the Methanohalophilus portucalensis FDF1 genome of the halophilic methanogen.

According to still another aspect of the present invention, a method for expressing a large amount of methane archaea β-lysine acetyltransferase using Escherichia coli, characterized in that a nucleic acid molecule encoding β-lysine acetyltransferase is The recombinant plasmid is transformed into an appropriate expression vector, and the resulting recombinant plasmid is transformed into a large amount of expression in an E. coli host cell, and the β-lysine acetyltransferase recombinant protein is purified. In one embodiment, the nucleic acid molecule encoding beta-lysine acetyltransferase is derived from the salt-tolerant Methane archaea Methanocalculus chunghsingensis K1F9705b genome. In another embodiment, the nucleic acid molecule encoding beta-lysine acetyltransferase is derived from the Methanohalophilus portucalensis FDF1 genome of the halophilic methanogen.

Another aspect of the present invention provides a method for producing a compatible N ^ε -ethinyl-β-lysine in vitro, characterized in that a methane archaea lysine 2 produced in large quantities by Escherichia coli is used. The 3-aminotransposase and the B-lysine acetyltransferase recombinant protein convert the L-α-lysine matrix into a compatible N ^ε -ethinyl-β-lysine.

According to still another aspect of the present invention, a method for converting L-α-lysine into an organic compatible N ^ε -ethinyl-β-lysine in vivo comprises encoding Mc AblA, Mc The nucleic acid molecule of AblB is expressed together in a host cell, and L-α-lysine is reacted in the host cell to synthesize N ^ε -ethinyl-β-lysine. In a specific aspect, L-α-lysine is converted into N ^ε -acetyl-β-lysine by in vivo to improve the salt and drought resistance of transgenic or transgenic organisms. High temperature resistance. In another embodiment, the host organism is selected from the group consisting of a microorganism, a plant, and an animal.

According to still another aspect of the present invention, a method for converting L-α-lysine into an organic compatible N ^ε -ethinyl-β-lysine in vivo comprises encoding Mp AblA and Mp The nucleic acid molecule of AblB is expressed together in a host cell, and L-α-lysine is reacted in the host cell to synthesize N ^ε -ethinyl-β-lysine. In a specific aspect, L-α-lysine is converted into N ^ε -acetyl-β-lysine by in vivo to improve the salt and drought resistance of transgenic or transgenic organisms. High temperature resistance. In another embodiment, the host organism is selected from the group consisting of a microorganism, a plant, and an animal.

Another aspect of the present invention to provide line A in methanogenic archaea compatible substance induces massive biosynthesis ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) of the method, characterized in that Methanocalculus chunghsingensis K1F9705b or Methanohalophilus portucalensis FDF1, salt-tolerant, is cultured in an anaerobic medium containing a final salt concentration of 1.5 to 3.0 M high salt (NaCl) to enhance the lysine of methane archaea Expression of 2,3-aminotransposase and β-lysine acetyltransferase gene.

Other features and advantages of the present invention will be apparent from the description of the appended claims and appended claims.

Methanocalculus chunghsingensis K1F9705b, which is used in the present invention, is readily available to those of ordinary skill in the art, and is deposited at the German Bacterial Center, under the accession number DSM 14539 and the American Methanogen Center, under the accession number OCM772. Methanohalophilus portucalensis FDF1 is readily available to those of ordinary skill in the art, and has been deposited with the German Stem Center (DSMZ) under the accession number DSM 7471T, the American Type Culture Center (ATCC) under the accession number BBA-912TM and the US methane. The center of the strain, the registration number is OCM59.

In this study, we designed the primers to use the published Methanococcoides burtonii and Methanococcus marpaludis gene-named lysine 2,3-aminomutase ( abl A) sequence to maintain the salt tolerance of Methanocalculus. chromosomal DNA chunghsingensis K1F9705b halophilic Methanohalophilus portucalensis FDF1 and performing a polymerase chain reaction (the PCR), sequence Mc abl a-0.8 kb and Mp abl a-0.8 kb fragment portions respectively, and then the two fragments were prepared as probes Southern hybridization analysis was carried out on the Methanocalculus chunghsingensis K1F9705b and Methanohalophilus portucalensis FDF1 genomes respectively, and the two methane archaeal compatible N ^ε -acetyl-β-lysine biosynthesis genes were obtained, respectively named Mcabl. A, Mcabl B and Mpabl A, Mpabl B genes.

In addition, the present invention has two methane archaea lysine 2,3-aminotransposase genes ( Mcabl A, Mpabl A), and β-lysine acetyltransferase genes ( Mcabl B, Mpabl B ), using the heterologous expression of E. coli, respectively, with a primer with a specific restriction enzyme cleavage site for polymerase chain reaction (PCR), after the selection and then using the same restriction enzymes, the gene fragments are sent to the pET system in E . coli BL21 (DE3) -RIL performance of a large number, so that the produced recombinant protein may be used for the in vitro synthesis is compatible substance ^N ε - acetyl -β- lysine group.

In addition, the present invention has used Methanocalculus chunghsingensis K1F9705b lysine 2,3-aminotransposase gene ( Mcabl A), and β-lysine acetyltransferase gene ( Mcabl B), using Escherichia coli heterologous In the performance, the Mcabl A gene fragment was constructed into the pET21b plastid, and the Mcabl B gene fragment was constructed into the pET28a plastid, and the constructed pET21b- Mcabl A and pET28a- Mcabl B plastids were simultaneously sent to E. coli BL21 (DE3). A large number of expressions in -RIL, so that the recombinant protein produced can be used to synthesize a compatible N ^ε -acetyl-β-lysine in vivo. Methanohalophilus portucalensis FDF1 lysine 2,3-aminotransposase gene ( Mpabl A), and β-lysine acetyltransferase gene ( Mpabl B), using E. coli heterologous co-expression, Mpabl The A gene fragment was constructed into the pET21b plastid, and the Mpabl B gene fragment was constructed into the pET28a plastid, and the constructed pET21b- Mpabl A and pET28a- Mpabl B plastids were simultaneously introduced into E. coli BL21(DE3)-RIL. The performance is such that the recombinant protein produced can be used to synthesize a compatible N ^ε -acetyl-β-lysine in vivo.

The invention will be described in detail with particular embodiments. These specific examples are provided by way of illustration of the invention and are not intended to limit the invention. The present invention is intended to embrace these and other modifications and variations within the scope and spirit of the invention.

Example

Example 1. Isolation and cloning of methanogenic archaea salt M. Chunghsingensis K1F9705b halophilic methanogenic archaea and M. Abl portucalensis FDF1 of gene A and abl B A

The method for extracting methane Pacific 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 the cells were collected by a high-speed centrifuge (Sorvall RC5C, DuPont Co.) at 8000 rpm and centrifuged at 4 ° C for 15 minutes. The obtained pellet was centrifuged and suspended in 2 ml of lysis solution (10 mM Tris, pH 8.0; 1 mM EDTA pH 8.0; 2% SDS; 100 μg/ml proteinase K) and disrupted. The bacteria pieces were dispersed, and then allowed to stand on ice for 10 minutes, centrifuged at 14000 rpm in a centrifuge (Sigma, 2K15) at 4 ° C for 10 minutes, and the supernatant was recovered, and an equal volume of Phenol/chloroform/isoamyl alcohol was stored at 4 ° C for storage. (25:24:1), gently shake it evenly, centrifuge with a centrifuge (Sigma, 2K15) at 14000 rpm, 4 ° C for 10 minutes, and recover the supernatant to repeat the above extraction. The supernatant after centrifugation was carefully taken out and added to RNase (50 μg/ml) for one hour at 37 °C. Continue to extract twice with Phenol/chloroform/isoamyl alcohol, remove the supernatant and add half volume of chloroform/isoamyl alcohol (24:1), shake gently and centrifuge (Sigma, 2K15) 14000 rpm, 4 °C Centrifuge for 10 minutes, remove the supernatant and repeat the chloroform/isoamyl alcohol extraction twice until there is no protein residue in the interface layer. The sodium acetate concentration of the supernatant fraction was adjusted to 0.3 M with 3 M sodium acetate to precipitate DNA, 0.6-0.7 volumes of isopropanol was added and mixed uniformly, and centrifuged at 14000 rpm, 4 ° C in a centrifuge (Sigma, 2K15) 10 Minutes, carefully remove the supernatant, and add a suitable amount of 70% alcohol to wash the DNA sample, centrifuge (Sigma, 2K15) 14000 rpm, 4 ° C for 10 minutes, remove the supernatant, vacuum decompression concentrator (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 OD _260/280 ratio and the DNA content was calculated.

In this study, we designed ablA bm-F 5'-GAAGATCCTCTTTCCGAAGAT-3', ablAbm-R 5'- using the highly retained region of the published lysine 2,3-aminomutase ( abl A) sequence named Methanococcoides burtonii and Methanococcus marpaludis. GGTGATAACACCTTCATAATT-3 ', in our laboratory salt tolerance purified chromosomal DNA chunghsingensis K1F9705b M. and M. portucalensis FDF1 halophilic performing a polymerase chain reaction (PCR) respectively sequence Mc abl a-0.8 kb portion and Mp Abl A-0.8 kb fragment, and then the two fragments were separately made into probes, and Southern hybridization analysis was performed on M. chunghsingensis K1F9705b and M. portucalensis FDF1 genomes, respectively. M. chunghsingensis K1F9705b and M. portucalensis FDF1 chromosomal DNA were treated with restriction enzyme Nsi I, respectively. Electrophoresis analysis was performed. The colloid was shaken with 0.25 N HCl soak for 20 minutes, then with denaturation buffer (0.5 N NaOH, 1.5 M NaCl). Soak the gel and shake for 20 minutes. Finally, soak the gel with a neutralization buffer (1.5 M NaCl, 1 M Tris) and shake for 20 minutes. Followed by vacuum blotting of nucleic acids blotted to Hybond ^TM -N ⁺ Nylon membrane (Amersham) to short-wave UV nucleic acids immobilized nucleic acids immobilized on the membrane will. The probe prepared by the DIG DNA Labeling and Detection Kit was used for hybridization and immunodetection. As a result, the biosynthetic profiles of two methane archaeal compatible N ^ε -acetyl-β-lysine were obtained, which were named Mcabl A-1320 bp (listed in SEQ ID NO: 1) and Mcabl B-858 bp ( The sequences are listed in SEQ ID NO: 3), and Mpabl A-1314 bp (sequences are listed in SEQ ID NO: 5) and Mpabl B-831 bp (sequences are listed in SEQ ID NO: 7, 8), The relative positions of the genes are shown in Figure 1.

The amino acid sequences of the 2,3-aminotransposases of the two strains of methane archaea and Clostridium subterminale were analyzed, and the 2,3-aminotransfer of lysine of the two strains of methane archaea obtained by the present invention was found. The amino acid sequence of the enzyme gene is the same as the lysine 2,3-aminotransposase amino acid sequence of Clostridium subterminale , which has (1). The positional region bound to the [4Fe-4s] cluster contains a retained amine. The cysteine (CXXXCXXC); (2). The binding position to the SAM is DAPG/HGGGKIPV; (3). The binding position to the PLP is the lysine (K) in the SAM binding region; in addition, (4). The C-terminal position contains three cysteines, presumably in the position to bind to zinc (see Figure 2). The results of sequence analysis revealed that the abl A gene obtained from the methane archaea of the present invention is a lysine 2,3-aminotransposase gene.

The characteristics of the β-lysine acetyltransferase enzyme can be divided into four conserved motifs according to the structure, and the order is CDAB, wherein the phantom A is a segment containing a longer and highly retained one. The region, which is involved in the binding of acetyl coenzyme A, and contains a retained amino acid in this region as Q/RXXGXG/A, and found that the two strains of methane archaea obtained by β-lysine transfer The amino acid sequence of the enzyme gene, like the GNAT superfamily enzyme, has a similar retention sequence of R/QG/KK/LGH/LM/SK/G (Fig. 2). Sequence analysis showed that the abl B gene we obtained from the methane archaea was the β-lysine acetyltransferase gene. And we can convert these methane archaea into β-lysine by the action of α-lysine via lysine 2,3-aminotransferase, followed by β-lysine acetyltransferase. biosynthesis ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) (Roberts 1992), the two genes can be confirmed this product does depend alanine 2,3-aminomutase and beta] translocase - Activity of lysine acetyltransferase.

Systematic evolution analysis of the lysine 2,3-aminotransposase and the amino acid sequence of β-lysine acetyltransferase of the two methane archaea, resulting in Mc AblA, M pAblA and other methane archaea The amino acid sequence similarity of AblA is 47-80%; the amino acid sequence similarity between Mc AblB and M pAblB and other methane archaea AblB is 24-52%, indicating the compatibility of halophilic and salt-tolerant methanogens. quality ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) biosynthesis genes has considerable differences. These two strains of methane archaea have lower protein pI values and more negatively charged amino acids, which can bond with hydroxide ions to maintain their surface as a hydration layer and maintain the hydrophobicity of their surface. The formation of a group is reduced in a high-salt environment; proteins of halophilic and salt-tolerant organisms can be hydrated with salts, so that proteins of halophilic and salt-tolerant organisms can withstand low-water activities such as organic solvents. The lysine 2,3-aminotransposase and β-lysine acetyltransferase of methane archaea have the characteristics of salt tolerance and high solvent resistance, and are more suitable for industrial and extracellular production applications.

Example 2. Abl A and abl B genes of heterologous expression of methane archaea in E. coli host will be introduced with restriction enzyme cleavage sites ( Nhe I, Not I and Xho I), and the template source is obtained by Southern blotting method. The fragment was ligated with the plastid pGEM-7zf, and the obtained plastid was screened, and the plastid carrying the gene of lysine 2,3-aminotransposase and β-lysine acetyltransferase was confirmed by sequencing. . After the polymerase chain reaction, the restriction enzyme cleavage sites can be increased at both ends ( Nhe I- Mcabl A- Not I, Nhe I- Mcabl B- Not I, Nhe I- Mpabl A- Xho I and Nhe I- Mpabl The product of B- Xho I). Colonize it to After the -T Easy vector, it was transformed into competent cell Escherichia coli JM101, and plated on an LA plate containing IPTG/X-gal and Ampicillin for screening. After screening through blue and white, white colonies were picked and plastids were extracted, followed by cleavage with restriction enzymes ( Nhe I, Not I and Xho I), and fragments having restriction enzyme cleavage sites were screened and recovered. Then, the expression vector pET21b cleavage by the same restriction enzyme was ligated at 16 ° C for 16 hours, and then transformed into competent cell E. coli JM101, respectively, and the drug resistance gene Amp ^{r was} used as a screening marker. After further confirmation by restriction enzyme cleavage, the confirmed plastids (pET21b- M cablA, pET21b- Mp ab1A, pET21b- M cab1B and pET21b- Mp ablB) were separately transformed into competent cell E. coli BL21 (DE2). In the -RIL, protein expression was induced by IPTG, and the expression of the recombinant gene protein was analyzed by protein electrophoresis. From the results of Fig. 3, Mc AblA, Mp AblA, Mc AblB and Mp AblB showed a large amount of recombinant protein expression in Escherichia coli under the induction of IPTG. Moreover, these recombinant proteins expressed by E. coli are present as soluble proteins.

Example 3. Abl A and abl B genes of methane archaea in a heterologous source of E. coli host

Obtained in Example 2 -T- Nhe I-Mc abl B- Not I plastid, cleavage with restriction enzymes ( Nhe I and Not I), screening for fragments with restriction enzyme cleavage sites. Then, it was ligated with the expression vector pET28a cleaved by the same restriction enzyme at 16 ° C for 16 hours, and then transformed into competent cell Escherichia coli JM101, and the drug resistance gene Kan ^{r was} used as a screening marker. Using restriction enzyme cleavage step closer confirmation, confirmed the plastid (pET21b- M cablA and pET28a- Mc ablB) simultaneously to the competent cells of Escherichia coli Transformation BL21 (DE2) -RIL in and induced with IPTG Mc AblA The Mc AblB protein co-expressed and analyzed the expression of recombinant protein by protein electrophoresis.

Obtained in Example 2 -T- Nhe I- Mpabl B- Xho I plastid, cleavage with restriction enzymes ( Nhe I and Xho I), screening for fragments with restriction enzyme cleavage sites. Then, it was ligated with the expression vector pET28a cleaved by the same restriction enzyme at 16 ° C for 16 hours, and then transformed into competent cell Escherichia coli JM101, and the drug resistance gene Kan ^{r was} used as a screening marker. After further confirmation by restriction enzyme cleavage, the confirmed plastids (pET21b- Mp ablA and pET28a- Mp ablB) were simultaneously transformed into competent E. coli BL21(DE2)-RIL, and Mp AblA was induced by IPTG. Mp AblB protein co-expressed and analyzed the expression of recombinant gene protein by protein electrophoresis. From the results shown in Fig. 4, Mc AblA, Mc AblB, Mp AblA and Mp AblB have a large amount of recombinant proteins co-expressed in E. coli under the induction of IPTG.

Example 4. Northern blot analysis of blots methanogenic archaea compatible substance ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) biosynthesis genes affected by salt stress

The halophilic methanogen M. portucalensis FDF1 was separately cultured in an anaerobic medium containing different salinities (1.2, 1.65, 2.1, 2.5 and 2.9 M NaCl), and cultured until the middle log phase to collect the cells by centrifugation. Or 2. M. portucalensis FDF1 was cultured in 1.2 M NaCl to the middle log phase. Anaerobic operation was carried out, and anaerobic medium with high salt (2.1, 3.0, 3.8 and 4.6 M NaCl) was added to make the final salt concentration 1.65. The culture was continued with 2.1, 2.5 and 2.9 M NaCl, and the cells were collected by centrifugation at different time points. Subsequently of Rare RNA kit (Jinheung Biosciences) whole cell RNA was extracted, analyzed by electrophoresis using TBE colloid, electroblotter system (Galileo Bioscience) RNA was blotted to Hybond ^TM -N ⁺ Nylon membranes using semi-dry, and to The short-wave UV holder immobilized the nucleic acid on the mold and analyzed the northern ink stain with the compatible N ^ε -acetyl-β-lysine biosynthesis gene ( Mpabl A) as a probe. The membrane was scanned into a pattern using a scanner IamgeScanner ^TM II (Amersham Co.), and the signal size on the membrane was analyzed by TINA software (Version 2.09e, raytest Isotopenmeβger Te). The relative transcript amount 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 Figure 5 show that the halophilic methane archaea M. portucalensis FDF1 will increase the expression of intracellular N ^ε -acetyl-β-lysine biosynthesis gene with the increase of external salt concentration. It can be inferred that the expression of lysine 2,3-aminomutase and β-lysine acetyltransferase genes of methane archaea will follow the salt concentration increases, and therefore may be utilized to improve the performance of salt-induced expression levels of the gene, to improve ^N ε - lysine accumulation acetylsalicylic -β- ^{(N ε -acetyl-β-lysine} ) of.

Other specific aspects

All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.

From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.

FIG 1 is a M. chunghsingensis K1F9705b and M. portucalensis FDF1 in ablA with ablB genomic sequence and the regulatory region of the gene.

Comparative analysis of acetylsalicylic -β- lysine ^{(N ε -acetyl-β-lysine} ) amino acid sequence of the synthetic raw enzyme of Figure 2 - is a methanogenic archaea N ^ε. (A) Sequence alignment of the 2,3-aminotransposase amino acid sequence of lysine by Clostridium subterminale (LAM), which is performed by Clustal W program (SDSC Biology) Workbench), wherein (a): a ligand for an iron atom; (b): a lysine residue of a SAM binding domain and a PLP-binding site; (c): a zinc binding site. (B) Sequence alignment analysis of the retention motif A of the primary sequence of the GCN-5-related N-acetyltransferase of the methicillin and the S. cerevisiae Hat1.

Figure 3 lists M. chunghsingensis K1F9705b and M. portucalensis FDF1 AblB performance of AblA and E. coli BL (DE3) -RIL of. The E. coli transformed strains that reached the mid-log phase were treated with IPTG for 4 (I1), 8 (I2), and 22 (I3) hours, respectively, to induce AblA and AblB expression, and were separated by 12.5% SDS-PAGE. M: Rainbow mark. The arrows indicate the induced AblA (blue) and AblB (red) proteins.

Figure 4 lists M. chunghsingensis K1F9705b and M. portucalensis FDF1 of AblA and AblB in E. coli BL (DE3) -RIL jointly performance. E. coli transformed plants arriving in the log phase were treated with IPTG for 2 (I1), 4 (I2), and 8 (I3) hours, respectively, to induce AblA and AblB to be co-expressed and separated by 12.5% SDS-PAGE. M: Rainbow mark. The arrows indicate the induced AblA (blue) and AblB (red) proteins.

Figure 5 shows the degree of transcription of AblAB grown by M. portucalensis under the specified salt concentration medium, and analyzed by northern blotting. (a) results of heterozygous AblAB- 2.1 probes; (b) one-quarter of total RNA isolated by denaturing agar gel, stained with EtBr and directly detected by UV light; (c) with TINA software Calculate the relative amount of transcription.

<110>Zhongxing University

<120>Compatible N ^ε -acetyl-β-lysine synthesis gene of methane archaea and its application

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<170> PatentIn Version 3.4

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<213> Methanohalophilus portucalensis FDF1

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Claims (5)

An encoding halophilic methanogenic archaea Methanohalophilus portucalensis FDF1 compatible substance of ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) biosynthetic enzyme lysine 2,3-aminomutase transposition 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. An isolated nucleic acid molecule encoding a lysine 2,3-aminotransposase according to claim 1 of the scope of the patent, which comprises the nucleotide sequence of SEQ ID NO: 5. An encoding halophilic methanogenic archaea Methanohalophilus portucalensis FDF1 compatible substance of ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) lysine biosynthesis enzyme β- acetyl transferase (β-lysine acetyltransferase) an isolated nucleic acid molecule wherein the β-lysine acetyltransferase has the amino acid sequence of SEQ ID No. 8. An isolated nucleic acid molecule encoding a beta-lysine acetyltransferase according to claim 3, which comprises the nucleotide sequence of SEQ ID NO: 7. One kind of substance in vivo compatibility producing ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) of the method, characterized in that patent utilize a range of halophilic 2 and 4 in accordance with The lysine 2,3-aminotransposase and β-lysine acetyltransferase nucleic acid molecules of Methanohalophilus portucalensis FDF1 are selected in an appropriate expression vector and the resulting recombinant plasmid is transformed into Recombinant proteins are expressed in large amounts in E. coli host cells to convert the L-alpha-lysine matrix into a compatible N ^ε -ethinyl-β-lysine.