TWI410493B - Application of Methanophilic Lysine 2,3 - Amino - transposase Gene in Drug Synthesis - Google Patents

Abstract

Methanoarchaeal lysine 2,3-aminomutase genes from marine Methanosarcina mazei N2M9705, halotolerant Methanocalculus chunghsingensis K1F9705b and halophilic Methanohalophilus portucalensis FDF1 are used in the synthesis of pharmacological active compounds. Due to the high salt, organic solvent and drought resistant nature for halotolerant and halophilic organisms, the lysine 2,3-aminomutase from halophilic methanogens are more stable than that of normal microorganism for use in &bgr; -lysine production. Furthermore, methods for inducing &bgr; -lysine production in methanoarchaea in a condition of an elevated salt concentration are provided. Therefore, cost and time of production of &bgr; -lysine for synthesis of &bgr; -lysine associated drugs is reduced.

Description

Application of methane archaea lysine 2,3-aminotransposase gene in drug synthesis

The present invention relates to the use of an aminomutase gene for drug synthesis, in particular to a lysine 2,3-aminomutase gene derived from an ancient methane archaea. For the application of drug synthesis.

In the prior art, when chemical synthesis methods are used to prepare pharmacologically active compounds, it is often necessary to purify β-amino acid and β-amino-α-hydroxy acid (β-amino-). --hydroxy acids). Agents with β-amino acid and β-amino-α-hydroxy acid as important components include: (1) bacterial β-lactams (β-lactams) antibiotics, such as penicillins (penicillins), cephalosporins Antibiotics such as cephalosporins, carbapenems, monobactams; (2) anticancer drugs such as taxol; (3) antibacterial agents such as double deoxygenation Dideoxykanamicin, etc.; (4) immunopotentiators, such as potent anti-HIV protease active compounds including kynostatins; (5) protein inhibitors such as bestatin; and (6) Peptide-type blood pressure lowering agents, such as microginin. In the synthesis of these pharmacologically active compounds, enantiomerically pure β-amino acid and β-amino-α-hydroxy acids are required and Quite important.

The relationship between β-lysine and drugs has been noted in the 1950s. Streptomyces produced several antibiotics such as viomycin, streptolin A, streptothricin, roseothricin and geomycin. After hydrolysis, β-lysine can be produced (Stadtman, T. 1973. Adv. Enzymol. Relat. Areas Molec. Biol . 38: 413-448). Mycomycin produced by the fungus of the genus Nocardia , and β-lysine is also one of its constituents. Although currently enantiomerically pure β-type amino acids, such as β-lysine, can be produced in large quantities by chemical synthesis, chemical synthesis of β-lysine requires time and expensive raw materials, and is obtained externally. The racemic mixture still requires further purification. Therefore, there is a need for a method and technique for efficiently purifying enantiomerically pure β-amino acids such as β-lysine to improve drug synthesis.

An enzyme that can be used to synthesize β-lysine, lysine 2,3-aminomutase (AblA), which was purified by Chirpich et al. from Clostridium subterminal strain SB4 in 1970. And has been shown to have the ability to convert L-alpha-lysine and produce L-beta-lysine. The action of this enzyme catalyzes the involvement of cofactors including [4Fe-4S] clusters ([4Fe-4S]cluster), S-adenosylmethionine, SAM And pyridoxal 5'-phosphate (PLP), the mechanism of action is: intracellular reduced protein reduces [4Fe-4S] ²⁺ to activated [4Fe-4S] ¹⁺ , activated state [ 4Fe-4S] ¹⁺ releases electrons to react with the SAM, which cleaves the SAM to produce methionine and 5'-deoxyadensyl radical. The PLP and the ε-amminium group on the lysine of the lysine 2,3-aminotransposase form an internal aldimine bond. In the presence of the receptor 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 produced by the action of [4Fe-4S] ¹⁺ and SAM reacts with the PLP-bound receptor α-lysine to produce 5'-deoxyadenosine and has free radicals (5'-deoxyadenosine) Intermediate of the radical, the intermediate product is formed by isomerization and hydrogen transfer from 5'-deoxyadenosine to form β-lysine (Frey, PA and GH Reed. 1993. Adv. Enzymol. Relat. Areas. Mol. Biol . 66:1-39; Chen, D. et al . 2006. Biochemistry . 45: 12647-12653).

The action of lysine 2,3-aminotransposase (AblA) to catalyze the production of β-lysine by α-lysine is known to require the participation of [4Fe-4S] clusters, SAM, and PLP. On the amino acid sequence, the binding position to the [4Fe-4S] cluster is to retain cysteine (cysteine), and the position of binding to the SAM has more glycine, and the position of bonding with PLP is Lysine (Chen, D. and PA Frey. 2001. Biochem . 40: 596-602). Studies have also shown that the activation of this enzyme requires the participation of zinc (Ruzicka et al . 2000. J. Bacteriol . 182: 469-476), suggesting that the three cysteine contained in the amino acid sequence near the C-terminus is The location of the zinc bond. From the crystal structure of the lysine 2,3-aminotransposase protein of Clostridium subterminale , the 2,3-aminotransferase of lysine is an asymmetric tetramer structure, which is first polymerized into two by the same monomer. A homodimer, which is further polymerized into a homotetramer by two dimers; wherein the structure of each monomer comprises three domains, which can be divided into: N-terminal structure domains (N- Terminal domain), including the 3rd to 53th residues (residues 3-53); the central globular domain, including 54th to 339th residues (residues 54-339); and the C-terminal structure The C-terminal domain consists of three parts, 340 to 412 residues (residues 340-412). The aforementioned N-terminal structure domain comprises seven short-chain helices, and a cluster is formed to be coated at the center of the structure, separated from the C-terminal structure domain. The aforementioned central globular structure forms a (β/α) ₈ tubular structure [(β/α) ₈ barrel] similar to triose phosphate isomerase (TIM), and the resulting cylindrical structure is lacking. Two β-strands (β-strands) form a (β/α) ₆ - crescent structure [(β/α) ₆ -crescent], in which the crescent channel surrounds the receptor α - Lysine, PLP and SeSAM/[4Fe-4S] complexes form an activation site. The receptor L-α-lysine forms a hydrogen bond with Asp-293, Asp-330 and Arg-134; the PLP is located at the N-terminus of the crescent channel, and Arg-112, Tyr-113, Arg-116 and water molecules form hydrogen bonds, forming a covalent bond with Lys-337; three of the [4Fe-4S] clusters will be associated with Cys-125, Cys-129, Cys -133 bond, the fourth iron atom will form a bond with the α-amino group of SeSAM; and SeSAM will form hydrogen with His-131, Thr-133, His-230, Asp-293 and Gln-258 Bonding; the aforementioned C-terminal structure domain contains three cysteine residues (ie, Cys-375, Cys-377, and Cys-380) bonded to zinc, and a new crescent The cysteine residue (Cys-268) in the crescent also interacts with zinc to stabilize the tetrameric structure (Lepore et al ., 2005. Proc. Natl. Acad. Sci . USA 102: 13819-13824).

Frey and Ruzicka, US Patent No. 6,248,874, 2001, purify and sequence lysine 2,3-aminomutase from anaerobic Clostridium subterminal strain SB4. The gene sequence, and the gene was placed in the expression vector to express a large amount of protein on E. coli, and after induction by IPTG, it was confirmed that the recombinant Escherichia coli did have β-lysine production under anaerobic conditions. At the same time, the purified recombinant protein lysine 2,3-aminotransposase can convert α-lysine into β-lysine with enantiomeric purity of 87% in solution or in an immobilized state. Many strains of Clostridium belong to highly toxic pathogenic bacteria such as botulinum and tetanus.

In summary, the prior art requires a large amount of time and cost for chemical synthesis, and the synthesized racemic mixture (β-type amino acid) needs further purification, and is treated with Clostridium. When β-lysine is produced, many strains of Clostridium are highly toxic pathogens, so the β-type amino acid synthesized by them has the risk of being contaminated by pathogenic bacteria.

Summary of invention

Based on the development and application of β-amino acid-producing enzymes, there is a lack of a synthetic method for synthesizing a β-type amino acid-related synthetic drug using a non-pathogenic bacteria, which is simple and economical. In particular, a synthetic method for a synthetic drug related to β-lysine, since the salt-tolerant and halophilic microbial protein has the characteristics of salt tolerance, solvent resistance and low water activity, The 2,3-aminotransferase of lysine in salt-tolerant and halophilic methanogens should be more stable than general microbial enzymes, so this case will be derived from lysine which can grow methane archaea in harsh environments. The 2,3-aminotransposase enzyme and its gene are used in the field of pharmaceutical synthesis.

Thus, in a first aspect, the invention provides the use of an isolated nucleic acid molecule for a pharmaceutical synthesis, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) SEQ ID NO a nucleotide sequence of 3; (ii) a nucleotide sequence having a nucleotide sequence similarity to SEQ ID NO: 3 greater than 90%; (iii) an amino acid sequence encoding SEQ ID NO: a nucleotide sequence; and (iiii) a nucleotide sequence encoding an amino acid sequence substantially identical to SEQ ID NO: 4.

In a second aspect, the invention provides the use of an isolated nucleic acid molecule for drug synthesis, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) SEQ ID NO: 7 a nucleotide sequence; (ii) a nucleotide sequence having a nucleotide sequence similarity to SEQ ID NO: 7 greater than 90%; (iii) a nucleotide encoding the amino acid sequence of SEQ ID NO: a sequence; and (iiii) a nucleotide sequence encoding an amino acid sequence substantially identical to SEQ ID NO: 8.

In a third aspect, the invention provides the use of an isolated nucleic acid molecule for drug synthesis, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) SEQ ID NO: a nucleotide sequence; (ii) a nucleotide sequence having a nucleotide sequence similarity to SEQ ID NO: 9 greater than 90%; (iii) a nucleotide encoding the amino acid sequence of SEQ ID NO: a sequence; and (iiii) the coding is substantially identical to SEQ ID NO: 10. The nucleotide sequence of the amino acid sequence.

In a fourth aspect, the present invention provides a polypeptide fragment having lysine 2,3-aminotransposase activity encoded by an isolated nucleic acid molecule as described above for use in a pharmaceutical synthesis.

In a fifth aspect, the present invention provides a method for producing β-lysine in vitro , comprising: providing a nucleic acid according to the foregoing first, second and third aspects a lysine 2,3-aminotransposase encoded by a molecule; and the use of the lysine 2,3-aminotransposase to produce β-lysine in vitro using α-lysine as a substrate Acetylic acid.

Therefore, the present invention provides the use of the lysine 2,3-aminomutase gene of methane archaea for drug synthesis, and also provides an easy use of methane archaea belonging to non-pathogenic bacteria. And an economical β-lysine-related drug synthesis method, and the lysine 2,3-aminotransposase gene of the methane archaea screen selected by the present invention comprises MmablA, McablA and MpablA, It has been confirmed by research that it has great potential and advantages in the field of research and development and application of β-amino acid and β-amino acid producing enzyme.

Detailed description of the invention

In order to make the present invention more comprehensible, the terminology of the present invention is further described, and unless otherwise indicated, it is intended to be understood by those of ordinary skill in the art.

Absolutely anaerobic methane archaea (Archaea, Archaea) needs to grow in an anaerobic, low redox potential environment. Methane archaea can adapt to a wide range of salt concentration environments (0 to 4.5 M NaCl). In order to adapt to the pressure of salt concentration in the environment, methane archaea will accumulate potassium ions, α-glutamate, betaine (glycine). betaine), β- glutamic acid (β-glutamate), β- bran Amides (β-glutamine) and ^N ε - acetyl -β- yl lysine ^{(N ε -acetyl-β-lysine} ) and the like are compatible Compatible solutes against osmotic stress (Lai, M.-C. et al. 1991. J. Bacteriol . 173: 5352-5358), in which cumulative N ^ε -acetyl-β-lysine is present as compatible Only found in methane archaea. Sowers first discovered N ^ε -acetyl-β-lysine, which may play a compatible role in the Methanogenium cariaci and non-marine methane archaea Methanosarcina thermophila (Sowers, KR et al. 1990. Proc. Natl. Acad. Sci USA 87: 9083-9087). Lai confirmed in 1991 that all methane archaea would accumulate a specific β-amino acid N ^ε -acetyl-β-lysine as a compatible substance of salt stress (Lai, M.-C. et al. 1991, supra). It was subsequently confirmed that the N ^ε -acetyl-β-lysine (N ^ε -acetyl-β-lysine) biosynthetic pathway was derived from α-lysine via lysine 2,3-aminotransposase (lysine 2, The conversion of 3-aminomutase to β-lysine followed by the synthesis of N ^ε -acetyl-β-lysine via β-lysine acetyltransferase (Roberts, MF et al. 1992. J. Bacteriol . 174: 6688-6693). Pflüger confirmed that Methanosarcina mazei 's lysine 2,3-aminomutase and β-lysine acetyltransferase are biocompatible N _ε - The genes necessary for acetyl-β-lysine are also salt-induced (Pflüger, K. et al. 2003. Appl. Environ. Microbiol . 69: 6047-6055).

According to the present invention, the methane archaea includes, but is not limited to, an archaea belonging to the family Methanobacteria , Methanococci , Methanomicrobia or Methanopyri .

In a preferred embodiment of the invention, the methane archaea is Methanosarcina sp., Methanocalculus sp., or Methanohalophilus sp. Specifically, according to methanogenic archaea Methanosarcina mazei, Methanocalculus chunghsingensis or Methanohalophilus portucalensis.

In addition to the specific sequences disclosed herein, a nucleic acid molecule also encompasses complementary sequences of the particular sequence, as well as conservative analogs, related naturally occurring structural variants, or synthetic unnatural. Analogs present, such as: homologous sequences of degenerative codon substitutions. In particular, one nucleotide residue of a particular sequence as disclosed herein may be substituted with another nucleotide residue without affecting the activity of the polypeptide encoded by the particular sequence or itself as a promoter.

In accordance with the present invention, the term "Similarity" is defined herein as the degree of association between two sequences, which may be determined by the same sequence and/or conservative ratios between sequences.

According to the invention, the phrase "substantially identical" means that among different species, variations in the amino acid sequence do not necessarily affect the activity of the protein they constitute, so that as long as the amino acid sequence has a certain degree of similarity Does not affect the activity of the protein. The above degree of similarity is preferably 70% or more; more preferably 80% or more; and still more preferably 90% or more.

The use of the isolated nucleic acid molecule of the present invention for a pharmaceutical synthesis, wherein the drug means a synthetic drug associated with β-lysine.

The aforementioned β-lysine-related synthetic drug means any synthetic drug consisting of β-lysine, including, but not limited to, bacterial β-lactam type (β-lactams) Antibiotics, such as penicillins, cephalosporins, carbapenems, monobactams and other antibiotics; anticancer drugs such as taxol; antibacterial agents Such as dideoxykanamicin, etc.; immunopotentiators such as potent anti-HIV protease active compounds such as kynostatins; protein inhibitors such as bestatin; and peptide lowering Medicaments such as microginin. A vector according to the present invention comprising a nucleic acid molecule encoding an enzyme having lysine 2,3-aminotransposase activity and a heterologous control sequence according to the present invention, wherein the nucleic acid molecule is operably associated with the The control sequences are connected.

According to the present invention, in order to express any of the aforementioned nucleic acid molecules comprising a lysine 2,3-aminotransposase gene, a nucleic acid molecule comprising any of the nucleic acids as described above may be operably linked to the control sequence. The recombinant vector of the heterologous control sequence is expressed in a suitable microbial system in a suitable culture medium to produce a lysine 2,3-aminotransposase.

The term "control sequence" as used herein, refers to a sequence that enables a nucleic acid sequence of a gene to be initiated or shut down, thereby affecting the expression of the nucleic acid sequence of the gene; for example, the control sequence can be a promoter, enhancer (enhancer) ), polyadenylation signal, terminator, and the like, which can make a protein or polypeptide The code sequence is expressed in a host cell.

The term "vector" refers to any recombinant vector which can be a recombinant expression system which can be constitutively or in any host cell either in vitro or in vivo . A selected nucleic acid sequence is inducibly expressed. The expression vector can be a linear or circular form, an episomal expression system, or a expression system incorporated into the genome of the host cell. The expression system may or may not have the ability to self-replicate, and may only initiate transient expressions in a host cell.

The term "heterologous" as used herein, refers to a gene other than the species itself, and the term "heterologous control sequence" as used herein means the lysine 2,3-aminotransposase used. The (lysine 2,3-aminomutase) gene is not derived from all control sequences of the same methanogen archaea; in a preferred embodiment of the invention, the "heterologous control sequence" is a control sequence for non-methane archaea.

According to the present invention, the aforementioned heterologous control sequence may be derived from Methanosarcina sp., Methanocalculus sp. or Methanohalophilus sp. Control sequences other than . Specifically, the aforementioned heterologous control sequence may be a control sequence such as the T7 promoter of pET21b of a large number of heterologous genes in Escherichia coli BL21 (DE3), which can be used by standard techniques. The desired nucleic acid sequence is inserted into a suitable vector having the desired control sequences. A specific vector is used to transport a gene message to a cell, and the vector is not particularly limited. Any of the usual vectors for use in recombinant protein expression systems can be employed.

The term "operably linked" as used herein, means that a first sequence is disposed in close proximity to a second sequence such that the first sequence can affect the second sequence or be in the second Under sequence control. For example, a promoter sequence operability is linked to a gene sequence and is typically at the 5' end of the gene sequence such that expression of the gene sequence is under the control of the promoter sequence. In addition, a regulatory sequence can be operably linked to a promoter sequence to enhance the ability of the promoter sequence to initiate transcription. In this case, the regulatory sequence is typically located at the 5' end of the promoter sequence.

According to the present invention, the aforementioned microorganism suitable for expressing the 2,3-aminotransposase of lysine may be all bacteria or archaea; more specifically, the aforementioned bacteria are Escherichia coli; the aforementioned archaea is absolutely anaerobic. Methane archaea, including but not limited to: Archaea belonging to the family Methanobacteria , Methanococci , Methanomicrobia or Methanopyri , including: (Methanobacteria) methane mesh bacilli (Methanobacteriales) of Methanobacterium Section (Methanobacteriaceae) methane genus of bacteria (Methanobacterium sp.), methane Brevibacterium (Methanobrevibacter sp.), species of methane bacteria spherical (Methanosphaera sp.), methane thermal Bacillus bacteria, thermophilic bacteria and methane Section (Methanothermaceae) of thermal methane bacteria belonging to the genus (Methanothermobacter sp.) (Methanothermus sp .); Methanococcus Gang (Methanococci) of Methanococcus mesh (Methanococcales) of methane bacteria warm Section ( Methanocaldococcaceae ) Methanocaldococcus sp., Methanotorris sp. And Methanococcus Section (Methanococcaceae) of methane Lactococcus bacteria, thermal methane bacteria belonging to the genus (Methanococcus sp.) (Methanothermococcus sp .); And methane micro Oomycetes (Methanomicrobia) of methane MICROORGANISM mesh (Methanomirobiales) of methane grain bacteria Section (Methanocorpusculaceae) methane bacteria belonging to the genus particles (Methanocorpusculum sp.), and methane bacteria micro branch (Methanomicrobiaceae) methane bacteria genus bladder (Methanoculleus sp.), species of methane bacteria bubble (Methanofollis sp.), methanogens Methanogenium sp., Methanolacinia sp., Methanomic robium sp., Methanoplanus sp., Methanospirillaceae Methanospirillum sp., and not yet classified Methanocalculus sp., Methanolinea sp.; and Methanosarcinales ( Methanosaetaceae ) Methanosaeta sp., Methanothrix sp., and Methanomes Osarcinaceae ) Halomethanococcus sp., Methanirnicrococcus sp., Methanococcoides sp., Methanohalobium sp., Methane halophile Methanohalophilus sp., Methanolobus sp., Methanomethylovorans sp., Methanosalsum sp., M. mazei (. Methanosarcina sp), and a Pyrococcus Section (Methermicoccaceae) methyl Pyrococcus genus bacteria (Methermicoccus sp.); and methane fire Oomycetes (Methanopyri) of methane fire bacteria mesh (Methanopyrales) of methane fire aceae (Methanopyraceae) Methanopyrus sp.

As described above, when the microorganism to be used is Escherichia coli, the medium to which it is applied is a general standard medium used in the technical field such as LB medium or the like; and when the microorganism used is a methane-like substance such as the aforementioned In the case of bacteria, the appropriate medium is an anaerobic medium, and specific examples thereof are HP and MM/W medium. In a preferred embodiment, the anaerobic medium is further supplemented with a vitamin and a trace elment solution.

In a preferred embodiment of the present invention, the aforementioned methane archaea is Methanosarcina sp., Methanocalculus sp. or Methanohalophilus sp. .).

In a preferred embodiment of the invention, the salt medium comprises an anaerobic medium having a sodium salt concentration ranging from about 0.1 M to 4.5 M. In a preferred embodiment of the invention, the salt medium comprises an anaerobic medium of sodium chloride having a concentration ranging from about 1.0 M to 3.0 M.

According to the present invention, in order to express the aforementioned lysine 2,3-aminotransposase, The β-lysine can be produced by the methane archaea in an anaerobic environment by means of salt induction, which is an oxygen-free closed environment. In a preferred embodiment, the anaerobic environment is a closed environment filled with nitrogen, carbon dioxide, hydrogen, an inert gas, or the like.

In summary, the present invention utilizes molecular biotechnology to provide lysine 2,3-aminomutase from three strains of methane archaea grown in different salt concentrations and using different methane substrates. The use in a pharmaceutical synthesis has the following characteristics: The present invention uses a non-pathogenic fungus of methane archaea, which can be applied to a technique for producing methane by fermentation as an energy source. Since lysine 2,3-aminomutase has the ability to convert L-lysine to L-β-lysine, Thus the lysine 2,3-aminotransposase gene can be used to prepare L-beta-lysine to reduce and avoid the cumbersome steps of the chemical synthesis process. The invention utilizes molecular biotechnology to purify and sequence three lysine 2,3-aminotransposase genes of anaerobic methane archaea grown in different salinities; and construct methane archaea lysine 2,3 A carrier in which the aminotransposase gene is abundantly expressed in the bacterium. The present invention confirms that the lysine 2,3-aminomutase gene of the methane archaea is induced by an increase in salt concentration. Therefore, it can be produced under salt induction by microbial fermentation, and the recombinant protein can be mass-produced intracellularly in vivo to transform L-α-lysine and produce β-lysine. Alternatively, the recombinant protein can be purified first and then in vitro ( in vitro ) to produce β-lysine. In this way, β-lysine can reduce the cost and shorten the time, and no need to The product was further purified.

Since the lysine 2,3-aminomutase of the methane archaea acts together with β-lysine acetyltransferase, L-α- Lysine is converted to a compatible N ^ε -acetyl-β-lysine to maintain the intracellular and extracellular pressure balance of methanogens in high salt and high osmotic pressure environments and to protect intracellular proteins; therefore, gene performance increases with salinity And increase. Salt-induced performance can be achieved, and costs can be reduced in in vivo production. Based on the above, the salt-tolerant, halophilic protein proteinase used in the present invention has the characteristics of salt tolerance, solvent resistance and low water activity, and thus the present invention is superior to the general microbial protein enzymes such as Frey and Ruzicka in the US patent case. The Clostridium protein gene used in No. 6,248,874 should be more stable when producing β-lysine in a test tube in a culture independent method.

The present invention utilizes the complete genotype sequence of the completed methane archaea Methansarcina mazei Gö1 compatible N ^ε -acetyl-β-lysine biosynthetic enzyme lysine 2,3-aminomntase (lysine 2,3-aminomntase , abl A) sequence design primer ablA mF-2 5'-GTGAAATCCAGGATATTTGACTGT-3' (SEQ ID NO: 1), ablA mR-2 5'-TCAGAAACGCTGTTTCTCTTCGAG-3' (SEQ ID NO: 2), to the applicant's experiment The chromosomal DNA of the purified marine type M. maz e l N2M9705 is used as a template for polymerase chain reaction (PCR) to obtain a Mmabl A-1260 bp sequence, as shown in SEQ ID NO: 3, which is encoded by The amino acid sequence is shown in SEQ ID NO: 4. In addition, the region of the highly expressed region of the lysine 2,3-aminotransposase ( abl A) sequence named after the published Methanococcoides burtonii and Methanococcus marpaludis genome was designed as ablA bm-F5'-GAAGATCCTCTTTCCGAAGAT-3' (SEQ ID) NO: 5), ablAbm-R5'-GGTGATAACACCTTCATAATT-3' (SEQ ID NO: 6), using the chromosomal DNA of the salt-tolerant M. chunghsingensis K1F9705b and the halophilic M. Portucalensis FDF1 purified by the applicant's laboratory as a template A partial sequence of Mc abl A-0.8 kb and Mp abl A-0.8 kb fragments were obtained by polymerase chain reaction (PCR), and the two fragments were respectively made into probes for M. chunghsingensis K1F9705b and M. portucalensis FDF1 genes, respectively . The Southern hybridization analysis was performed to obtain: Mcabl A-1320 bp, as shown in SEQ ID NO: 7, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 8. And the Mpabl A-1314 bp sequence, as set forth in SEQ ID NO: 9, and the amino acid sequence encoded thereby is set forth in SEQ ID NO: 10.

It is known that the action of lysine 2,3-aminotransposase requires the participation of cofactor [4Fe-4S]cluster, SAM and PLP, and requires the activation of zinc, so the applicant further analyzes and compares three strains of methane archaea with Clostridium. Subterminale lysine 2,3-aminotransposase amino acid sequence, and found that the amino acid sequence of the lysine 2,3-aminotransposase gene of the three strains of methane archaea obtained by the present invention The amino acid sequence of the lysine 2,3-aminotransposase of Clostridium subterminale is similar, and is similar in that: (1) the positional region bound to the [4Fe-4S] cluster contains the retained amino acid cysteamine. Acid (cysteine) (such as CXXXCXXC); (2) the binding site with SAM is DAPG/HGG-GKIPV; (3) the binding site with PLP is the lysine (K) in the SAM binding region; in addition, (4) Three cysteine acids are included at the position near the C-terminus, presumably in the position to bind to zinc (as shown in Annex I). It was confirmed by sequence analysis that the gene obtained by the applicant from the methane archaea was indeed the lysine 2,3-aminotransposase gene.

According to the present invention, the amino acid sequence of the lysine 2,3-aminotransposase of the three strains of methane archaea is homology modeling, and the lysine of the C. subterminale having the crystal structure is solved . The stereostructure of the 2, 3-aminomutase amino acid sequence was predicted. The results are shown in Annex 2. The predicted three-dimensional structure is very similar to the lysine 2, 3-aminomutase structure of C. subterminale . Docked models were used to analyze the relationship between lysine 2,3-aminotransposase and cofactor binding. The results are shown in Annex III. The binding position of the cofactors is highly retained in the protein structure. From the structural analysis showing the high degree of retention of the functional region, it is speculated that this gene product has the activity of the lysine 2,3-aminotransposase. These methane archaea can be converted to β-lysine by α-lysine via lysine 2,3-aminotransferase, followed by β-lysine. Lysine acetyltransferase produces N ^ε -acetyl-β-lysine (Roberts, MF et al. 1992. J. Bacterial . 174: 6688-6693), and it can be confirmed that this gene product does have lysine 2,3-amino translocation Enzyme activity.

The present invention further analyzes the amino acid sequence similarity between C. subterminale and the lysine 2,3-aminotransposase of the three strains of methane archaea by a system evolution of 53% to 57%. The protein of methane archaea has a low pI value and a large amount of negatively charged amino acid, which can bond with hydroxide ions to maintain its surface as a hydration layer and maintain its surface hydrophobicity. In the salt environment, the formation group is reduced; the protein of the halophilic and salt-tolerant organisms can be hydrated with the salt, so that the protein of the halophilic and salt-tolerant organism can resist the low-water activity environment such as an organic solvent. The lysine 2,3-aminotransposase showing methane archaea has the characteristics of salt tolerance and high solvent resistance compared with C. subterminale , and is more suitable for industrial and extracellular production applications.

The function of the known lysine 2,3-aminotransposase of methane archaea is to convert α-lysine to β-lysine and then to synthesize compatible N ^ε via β-lysine acetyltransferase. -acetyl-β-lysine to help methane archaea adapt to the environment of salt stress, increase intracellular solute concentration and protect intracellular proteins from salt and osmotic pressure, and methane archaeal compatible N ^ε -acetyl-β The amount of -lysine biosynthesis increases as the extracellular salt concentration increases (Sower 1990; Lai, M.-C. et al . 1991. J. Bacteriol . 173: 5352-5358). The ink stain analysis was carried out by using the lysine 2, 3-aminomutase and the β-lysine acetyltransferase gene abl AB, which are compatible with the halophilic methanogens, N ^ε -acetyl-β-lysine, and the results are as shown in the first figure A. In the B, C and C regions, M. portucalensis FDF1 will increase the expression of intracellular N ^ε -acetyl-β-lysine biosynthesis genes as the concentration of external salt increases. It can be inferred that the expression of the lysine 2,3-aminotransposase gene of the methane archaea increases with the increase of the salt concentration, so that the salt-induced expression can be used to increase the expression of the gene. At the same time, the methane produced by the methane archaea can be used as a lower cost of biomass energy; and the methane archaea is not a pathogen, compared to the safer problematic anaerobic used by Frey and Ruzicka in U.S. Patent No. 6,248,874. Clostridium , a methane archaea for in vivo production of beta-lysine, has no safety concerns.

In the present invention, the applicant has used three methionine 2,3-aminotransposase genes ( MmablA , McablA , MpablA ) of the methane archaea to express heterologously in E. coli to add a specific restriction enzyme cleavage site. The primers were subjected to polymerase chain reaction (PCR), and after selection, the same restriction enzymes were used, and the gene fragments were separately sent to the pET system and expressed in E. coli BL21 (DE3)-RIL. Mm AblA, Mc AblA, and Mp AblA exhibited a large number of recombinant proteins in E. coli induced by IPTG (as shown in the second figure). These recombinant proteins are all presented as soluble proteins. In the US Patent No. 6,248,874, Frey and Ruzicka have demonstrated that the Clostridium subterminal strain SB4 has a large amount of lysine 2,3-aminotransposase gene E. coli. After induction by IPTG, it is confirmed that it is reconstituted in the absence of oxygen. Escherichia coli does have β-lysine production; at the same time, its purified recombinant protein lysine 2,3-aminotransposase can convert α-lysine into enantiomeric purity in solution or in immobilized state. Up to 87% beta-lysine. Compared with the lysine 2,3-aminotransposase gene product of Clostridium, the gene and protein of the lysine 2,3-aminotransposase provided by the present invention for the environment Salinity, osmotic pressure and solvent resistance are better, and it is more suitable for industrial production.

The invention is further illustrated by the following examples which are not intended to limit the invention. A person skilled in the art can make some modifications and modifications without departing from the scope of the invention.

Example 1: Methane archaea culture medium composition and anaerobic inoculation

Methanosarcina mazei N2M9705 is widely available in the technical field of the art, and has been deposited in the American Methanogen Center, the registration number is OCM668; and the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute, The numbers are BCRC16179. Methanocalculus chunghsingensis K1F9705b is readily available to those of ordinary skill in the art, and is deposited at the American Methanogen Center, under the accession number OCM772; and the German Bacterial Center, with accession number DSM 14539. Methanohalophilus portucalensis FDF1 is readily available to those of ordinary skill in the art, and has been deposited with the American Methanogen Center, under the accession number OCM59.

Methane archaea culture is operated by an anaerobic system. This system is referenced by Balch et al . (Balch, WE et al . 1979. Microbiological Reviews . 43: 260-296) with Hungate technology (Hungate, RE, 1969, Method in Microbiology ., In JR Norris and DW Ribbons (ed.) Academic Press Inc. , New York, NY, vol 3B: p117-132) Modified oxygen removal system. Methanohalophilus portucalensis FDF1 was cultured in 12% NaCl anaerobic medium HP medium (120 g NaCl, 3.0 g MgCl ₂ ‧6H ₂ O, 2.0 g KCl, 0.1 g CaCl ₂ ‧2H ₂ O, 0.4 g K ₂ HPO ₄ , 1.0 g NH ₄ Cl); 10 mL vitamin solution at a concentration of 2.0 mg biotin per liter, 10 mg pyridoxine hydrochloride, 2.0 mg folic acid, 5.0 mg riboflavin (riboflavin), 5.0 mg thiamine, 5.0 mg nicotinic acid, 5.0 mg pantothenic acid, 0.1 mg vitamin B ₁₂ , 5.0 mg thioctic acid, 5.0 mg p-aminobenzene Formic acid (p-aminobenzonic acid), stored at -20 ° C (Wolin et al ., 1963); and added 10 mL trace element solution (0.01 g H ₂ SeO ₃ per liter, 0.10 g MnCl ₂ ‧ 4H ₂ O, 0.01 g FeSO ₄ ‧7H ₂ O, 0.15 g CoCl ₂ ‧6H ₂ O, 0.10 g ZnCl ₂ , 0.01 g H ₃ BO ₃ , 0.01 g NaMoO ₄ ‧2H ₂ O, 0.02 g CuCl ₂ ‧2H ₂ O, 0.02 g NiSO ₄ ‧6H ₂ O, 0.04 g AlCl ₃ ‧6H ₂ O, 0.05 g disodium EDTA dehydrate, 30 mg Na ₂ WO ₄ ‧6H ₂ O) (Ferguson, TJ and RA Ma h. 1983. Appl. Environ. Microbiol . 45: 265-274), 0.5 g cysteine HCl and 1 mg resazurin (resazurin is a redox indicator, purplish red when aerobic, colorless and transparent when anaerobic). Change the nitrogen gas to the mixed gas (N ₂ :CO ₂ =4:1), and add 4.0 g of NaHCO _{3 to} adjust the pH value of the culture solution [NaHCO _{3 is} dissolved in the hydrolyzed bicarbonate ion (HCO ₃ ^- )). The pH of the culture solution (about pH 7.2) can be maintained after equilibrating with water-soluble CO ₂ . After the culture solution was quantified to 1000 mL, the mixture was continuously filled with a mixed gas (N ₂ : CO ₂ = 4:1) into a round bottom flask containing 12% NaCl HP medium, and the indicator resazurin was changed from purple to colorless. The bottle is sealed with a gas-tight rubber stopper and sealed with a lead wire.

The methane archaea M. chunghsingensis K1F9705b and M. mazei N2M9705 were separately cultured in 1% NaCl anaerobic medium MM/W medium (10 g NaCl, 1.0 g MgCl ₂ ‧6H ₂ O, 0.5 g KCl, 0.1 g CaCl ₂ ‧2H ₂ O, 0.4 g K ₂ HPO ₄ , 1.0 g NH ₄ Cl). Warm the round bottom bottle to cool down. Add 10 mL of vitamin solution, 10 mL of trace element solution, 0.25 g cysteine HCl and 0.1 mg resazurin until the temperature drops to about 45 °C. The nitrogen gas that was introduced was changed to a mixed gas (N ₂ :CO ₂ = 4:1), and 4.0 g of NaHCO _{3 was} added to adjust the pH of the culture solution. After the culture solution was quantified to 1000 mL, the mixture was continuously filled with a mixed gas (N ₂ : CO ₂ = 4:1) into a round bottom flask containing 1% NaCl MM/W medium, and the indicator resazurin was changed from lavender to After colorless, use a gas-tight silicone stopper to stopper the bottle and seal the bottle with a lead wire.

Open the Hungate station (Hungate station), set up the venting needle and pipeline, open the nitrogen and insert the sterile syringe into the venting needle, pump nitrogen back and forth at least 30 times to completely remove the oxygen; take the carbon from the syringe after the nitrogen pumping Source (0.2 mL 1 M TMA/10 mL medium, 0.5 mL 1M methanol/10 mL medium, 1.0 mL 1M sodium formate/10 mL medium, 0.2 mL 1M sodium acetate/10 mL medium), reducing agent (0.1 mL 2.5% Na) ₂ S‧9H ₂ O/10 mL medium) and strain (bacteria: culture solution = 1:20), quickly added to the liquid culture solution in sequence (Yang Dao Ren, 1995, halophilic methane archaea compatible Studies on the synthesis of enzymes related to glycine betaine and its influencing factors, National Institute of Plant Science, National Chung Hsing University).

Example 2: Extraction of methane archaea chromosomal DNA

This method was modified by the method of extracting the chromosomal DNA of methane archaea from Jarrell (1992). The 250 mL bacterial 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 pellet obtained after centrifugation was suspended in 2 mL of a lysis solution (10 mM Tris, pH 8.0; 1 mM EDTA pH 8.0; 2% SDS; 100 μg/mL proteinase K) and the cells were 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 phenol/chloroform/isoprene stored at an equal volume of 4 ° C was added. Phenol/chloroform/isoamyl alcohol (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. once. 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 a phenol/chloroform/isoamyl alcohol mixed solution. After taking out the supernatant, add half a volume of chloroform/isoamyl alcohol (24:1), gently shake it evenly. Centrifuge (Sigma, 2K15) at 14000 rpm, centrifuge at 10 ° C for 10 minutes, remove the supernatant and repeat the extraction of the chloroform / isoamyl alcohol mixture solution 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, and 0.6-0.7 volume of isopropanol was added and mixed uniformly to centrifuge (Sigma, 2K15). Centrifuge at 14000 rpm for 10 minutes at 4 °C, carefully remove the supernatant, and wash the DNA sample with an appropriate amount of 70% alcohol. Centrifuge (Sigma, 2K15) at 14000 rpm for 10 minutes at 4 °C to remove the supernatant. A vacuum concentrator (Savant Speed Vac System, Savant Co.) drains the alcohol, re-dissolves the DNA sample in sterile water, and examines the purity of the extracted DNA and calculates the DNA content by nucleic acid electrophoresis and measurement of the OD ₂₆₀ /OD ₂₈₀ ratio. . The OD ₂₆₀ /OD ₂₈₀ ratio of DNA and RNA should be at least 1.8 or more. If the value is too low, the protein content in the nucleic acid sample is too high. If the ratio of OD ₂₆₀ /OD ₂₈₀ is higher than 2.0, it may be caused by phenol residue. Under this standard, when the OD value of the nucleic acid at a wavelength of 260 nm is 1, the concentration of the double-stranded DNA is 50 μg/mL, the concentration of the single-stranded DNA and RNA is 40 μg/mL, and the single-stranded oligonucleotide (single oligonncleotide) The concentration is 20 μg/mL. The concentration of the nucleic acid can be calculated according to this principle (Sambrook et al ., 1989).

Example 3: Screening of the lysine 2,3-aminomutase gene of methane archaea by the Southern blotting method

Procedures for probe preparation Refer to Roche's DIG DNA Labeling and Detection Kit operating manual to prepare probes using Roche's DIG DNA Labeling and Detection Kit (Roche Diagnostics Corp.). 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, and then denaturation buffer (0.5 N). The gel was soaked with NaOH, 1.5 M NaCl) and shaken for 20 minutes. Finally, the colloid was soaked in a neutralization buffer (1.5 M NaCl, 1 M Tris) and shaken 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. The signaled nucleic acid fragments are then recovered and stored in a vector for storage.

Example 4: Analysis of methane archaeal compatible N by northern blotting method ^ε -acetyl-β-lysine biosynthesis gene is affected by salt stress

M. portucalensis FDF1 was separately cultured in an anaerobic medium containing different salinities (1.2 M, 1.65 M, 2.1 M, 2.5 M, 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, and anaerobic operation was carried out to add the high salt (2.1 M, 3.0 M, 3.8 M and 4.6 M NaCl) anaerobic medium to the final salt concentration. The culture was continued for 1.65 M, 2.1 M, 2.5 M, 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. Using a scanner IamgeScanner ^TM II (Amersham Co.) to a film scanning image file, and the software TINA software to analyze the signal magnitude film (Version 2.09e, raytest Isotopenmeβgeräte) analysis. 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.

Example 5: Large amount of heterologous protein

After designing a primer with a restriction enzyme cleavage site, the template was obtained by splicing the plastid obtained by the Southern blotting method and the plastid pGEM-7zf, and confirming that the plastid carries lysine 2,3 via sequencing. - the gene for the aminotransferase. After the polymerase chain reaction, the product containing the restriction enzyme cleavage site at both ends can be amplified, and then cloned into pGEM ^® -T Easy vector and transformed into competent cell Escherichia coli JM101, which is coated with IPTG/X-gal and Screening was performed on the LA plate of penicillin. After screening through blue and white, white colonies were picked and plastids were extracted, followed by cleavage with restriction enzymes, and fragments with restriction enzyme cleavage sites were screened for recovery. Then, it was ligated with the expression vector pET21b 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 Amp ^{r was} used as a screening marker. After further confirmation by restriction enzyme cleavage, the confirmed plastids were transformed into competent E. coli BL21 (DE2)-RIL, and protein expression was induced by IPTG, and the expression of recombinant protein was analyzed by protein electrophoresis. The method of protein purification is described in the "A handbook for high-level expression and purification of 6xHis-tagged proteins" operating manual by Amersham Biosciences.

It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In fact, the various modifications that are apparent to those skilled in the art are also contemplated by the scope of the invention.

The first panel shows the transcription levels of M. portucalensis ablAB grown in the specified salt concentration culture medium by Northern blotting , wherein the panel A shows 36 μg of total RNA (total RNA). After denaturing agarose gel electrophoresis, the result was transferred to nylon membrane (nylon AB ) for hybridization with ablAB -2.1 as probe, while panel B showed one quarter of total RNA. After another denaturing agarose gel electrophoresis analysis, the results were directly detected by EtBr staining. Panel C shows the relative transcript level calculated by the TINA software analysis of the M. portucalensis ablAB gene expression for each of the specified salt concentration cultures.

The second panel shows an electrophoretic analysis of the AblA protein in E. coli BL21(DE3)-RIL, E. coli BL21(DE3)-RIL(pET21b) and E.coli BL21(DE3)-RIL(pET21b- abl A The cells were cultured in LB medium and grown to the middle log phase. IPTG was added to the culture medium, and the cells were cultured for 4 hours to induce AblA expression, and analyzed by 12.5% SDS-PAGE, wherein the diameter P (Lane P) E.coli BL21(DE3)-RIL(pET21b), diameter A (Lane A) is E.coli BL21(DE3)-RIL(pET21b-ablA), and diameter M (Lane M) is rainbow marker. The path AI (Lane AI) is IPTG-induced E. coli BL21(DE3)-RIL (pET21b-ablA).

The third figure shows the sequence alignment of lysine 2,3-aminomutase derived from methane archaea and C. subterminale (LAM), which is a sequence alignment analysis. It is obtained by Clustal W program (SDSC Biology Workbench), in which (a) shows the ligands of iron atoms, (b) shows the SAM binding domain and the PLP binding site (PLP-binding site) ) lysine residue, and (c) shows zinc ion binding sites.

The fourth panel shows the space-filled protein structure of the lysine 2,3-aminotransposase derived from C.subterminale , M.mazei N2M9705, M.chunghsingensis K1F9705b, and M. portucalensis FDF1. Comparison).

The fifth panel shows a structural comparison of the binding of lysine 2,3-aminotransposase derived from C.subterminale , M.mazei N2M9705, M.chunghsingensis K1F9705b, and M. portucalensis FDF1 to cofactors.

[A brief description of the attachment]

Annex I: Sequence alignment of lysine 2,3-aminomutase derived from methane archaea and C. subterminale (LAM), the alignment analysis is borrowed Obtained by Clustal W program (SDSC Biology Workbench), wherein (a) shows the ligands of iron atoms, (b) shows the SAM binding domain and the PLP-binding site. Lysine residue, and (c) shows zinc ion binding sites.

Annex 2: Space filling protein structure comparison from C. subterminale , M. mazei N2M9705, M. chunghsingensis K1F9705b and M. portucalensis FDF1 lysine 2,3-aminotransposase .

Annex III; structural comparison of the binding of lysine 2,3-aminotransposase from C. subterminale , M. mazei N2M9705, M. chunghsingensis K1F9705b and M. portucalensis FDF1 to cofactors.

<110>National Zhongxing University <120> Application of methane archaea lysine 2,3-aminotransposase gene in drug synthesis <130><160>10 <170>PatentIn version 3.4 <210>1 <211> 24 <212>DNA <213>Artificial <220><223>Synthetic DNA<400>1 <210>2 <211>24 <212>DNA <213>Artificial <220><223>Synthetic DNA<400>2 <210>3 <211>1260 <212>DNA <213> Methanosarcina mazei N2M9705 abl A gene encodes a lysine 2,3-aminomutase <400>3 <210>4 <211>419 <212>PRT <213> Methanosarcina mazei N2M9705 lysine 2,3-aminotransposase <400>4 <210>5 <211>21 <212>DNA <213>Artificial <220><223>Synthetic DNA <400>5 <210>6 <211>21 <212>DNA <213>Artificial <220><223>Synthetic DNA<400>6 <210>7 <211>1320 <212>DNA <213> Methanocalculus chunghsifgensis K1F9705b abl A gene encodes a lysine 2,3-aminotransposase <400>7 <210>8 <211>439 <212>PRT <213> Methanocalculus chunghsingensis K1F9705b lysine 2,3-aminotransposase <400>8 <210>9 <211>1314 <212>DNA <213> Methanohalophilus portucalensis FDF1 ablA gene encodes a lysine 2,3-aminotransposase <400>9 <210>10 <211>437 <212>PRT <213> Methanohalophilus portucalensis FDF1 lysine 2,3-aminotransposase <400>10

Claims (11)

Use of an isolated nucleic acid molecule for drug synthesis, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 7; (ii) the coding The nucleotide sequence of the amino acid sequence of SEQ ID NO: 8; wherein the drug is a synthetic drug associated with β-lysine. The use according to claim 1, wherein the β-lysine-related synthetic drug is selected from the group consisting of β-lactams (β-lactams). Antibiotics, anticancer drugs, antibacterial agents, immunopotentiators, protein inhibitors, and peptide-type blood pressure lowering agents. The application according to claim 1, wherein the β-lysine-related synthetic drug is selected from the group consisting of penicillins and cephalosporins. (cephalosporins), carbapenems, and monobactams antibiotics, taxol, dideoxykanamicin, bestatin, zynostatin ( Kynostatins), microginin and derivatives thereof. An use of an isolated nucleic acid molecule for drug synthesis, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 9; (ii) the coding The nucleotide sequence of the amino acid sequence of SEQ ID NO: 10; wherein the drug is a synthetic drug associated with β-lysine. The application of claim 4, wherein the β-lysine-related synthetic drug is selected from the group consisting of: Beta-lactam antibiotics, anticancer drugs, antibacterial agents, immunopotentiators, protein inhibitors, and peptide-type blood pressure lowering agents. The application according to claim 4, wherein the β-lysine-related synthetic drug is selected from the group consisting of penicillins and cephalosporins. (cephalosporins), carbapenems, and monobactams antibiotics, taxol, dideoxykanamicin, bestatin, zynostatin ( Kynostatins), microginin and derivatives thereof. A polypeptide fragment having lysine 2,3-aminotransposase activity for use in a pharmaceutical synthesis, wherein the polypeptide fragment having lysine 2,3-aminotransposase activity is encoded by a nucleic acid molecule The nucleic acid molecule is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 7; (ii) the nucleoside encoding the amino acid sequence of SEQ ID NO: An acid sequence; wherein the drug is a synthetic drug associated with β-lysine. A polypeptide fragment having lysine 2,3-aminotransposase activity for use in a pharmaceutical synthesis, wherein the polypeptide fragment having lysine 2,3-aminotransposase activity is encoded by a nucleic acid molecule The nucleic acid molecule is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 9; (ii) the nucleoside encoding the amino acid sequence of SEQ ID NO: 10. An acid sequence; wherein the drug is a synthetic drug associated with β-lysine. A method for producing β-lysine in vitro , comprising: providing a nucleic acid molecule with an operably linked heterologous control sequence to form a recombinant vector, Wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 7; (ii) a nucleotide encoding the amino acid sequence of SEQ ID NO: a sequence; placing the recombinant vector in a suitable microbial system and culturing the microbial system in an appropriate medium and inducing expression to produce a lysine 2,3-aminotransposase; and utilizing the lysine 2, 3- Aminotransposase produces β-lysine based on α-lysine in vitro . A method for producing β-lysine in vitro , comprising: providing a nucleic acid molecule with an operably linked heterologous control sequence to form a recombinant vector, Wherein the nucleic acid molecule comprises a sequence selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 9; (ii) a nucleotide encoding the amino acid sequence of SEQ ID NO: a sequence; placing the recombinant vector in a suitable microbial system and culturing the microbial system in an appropriate medium and inducing expression to produce a lysine 2,3-aminotransposase; and utilizing the lysine 2, The 3-aminotransposase produces β-lysine based on α-lysine in vitro. A method for synthesizing a medicament comprising: producing β-lysine using α-lysine as a substrate using M. chunghsingensis K1F9705b or M. portucalensis FDF1; and synthesizing β-lysine using the produced β-lysine Related synthetic drugs.