An adenylated protein A8 mutant, its coding gene and applications

Abstract

The present invention discloses an adenylated protein A8 mutant, its coding gene, and its application as a key enzyme of the adenylation domain in a structural analog of daptomycin substituted with Gly<supgt;8< / supgt>. The adenylated protein A8 mutant is obtained by point mutation of the 277th amino acid of the adenylated protein A8 with the amino acid sequence shown in SEQ ID NO.1. By means of genetic engineering and enzyme engineering, the present invention studies the active site of the A domain, and determines the key amino acid residue (the 277th residue of A8) related to the substrate specificity of the A domain through constructing mutants, providing a new way for the structural expansion of daptomycin family compounds. Compared with the adenylated protein A8, the obtained mutant Y277P has an altered substrate specificity, with a significantly reduced activity towards L-Ala and a significantly increased activity towards L-Gly, providing a new way for obtaining a structural analog of daptomycin substituted with Gly<supgt;8< / supgt>.

Description

(1) Technical Field

[0001] The present invention relates to an adenylated protein A8 mutant, its encoding gene, and its application as a key enzyme of the adenylation domain in the catalytic synthesis of Gly 8 substituted daptomycin structural analogs. (2) Background Art

[0002] Daptomycin is a cyclic lipopeptide produced by Streptomyces roseosporus, which has good bactericidal effects on highly pathogenic drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP), and has high clinical application value. Different from the traditional mechanism of ribosome synthesizing proteins or polypeptides through the central dogma of transcription and translation, daptomycin is a class of polypeptides synthesized by non-ribosomal peptide synthetases (NRPSs). Non-ribosomal peptide synthetases are a complex enzyme system composed of multiple modules. Each module is responsible for one round of peptide bond extension, and there is a one-to-one correspondence between the module and the assembly order of amino acids. Among them, the most basic functional domains of the modular catalytic unit include a condensation domain, an adenylation domain, and a peptidyl carrier protein domain (Thiolation domain, T domain). The NRPS of daptomycin contains three subunits, DptA, DptBC, and DptD, and each subunit contains 5, 6, and 2 modules respectively. It is composed of 13 modules and a terminal thioesterase domain arranged linearly. Each module sequentially loads a specific amino acid onto the growing peptide chain. After the A domain recognizes and activates the corresponding amino acid, under the action of Mg2+, it forms aminoacyl-AMP using ATP; phosphopantetheinyl transferase captures phosphopantetheine from the coenzyme A molecule and transfers it to the Ser hydroxyl group of the T domain, making the T domain in an activated form. The aminoacyl-AMP is transported to the thiol group of the activated T domain to form an aminoacyl thioester; then it is transferred to the C domain, and the C domain catalyzes the condensation of this aminoacyl thioester with the upstream peptide chain; finally, under the catalysis of the thioesterase domain, an ester bond is formed between the carboxyl group of Kyn13 and the hydroxyl group of Thr4, and the cyclic peptide is released from the NRPS; there is a n-decanoyl group attached to the Trp residue, which is activated by DptE and DptF and undergoes condensation with Trp1 on the T domain of module one under the catalysis of the first CIII domain of DptA through the interaction of DptE, DptF, and DptA, ultimately forming daptomycin.

[0003] During the biosynthesis of daptomycin, the A domain selects the corresponding specific amino acid from the amino acid substrate pool to synthesize the corresponding aminoacyl-AMP. It has been reported that ten non-consecutively encoded key amino acid residues in the A domain determine its substrate recognition specificity. Therefore, determining the active site of the A domain and modifying it to obtain more novel daptomycin structural analogs with important biological activities has great application significance. (III) Summary of the Invention

[0004] The object of the present invention is to provide an adenylation protein A8 mutant capable of changing the substrate specificity of adenylation protein A8, its encoding gene, and its application as a key enzyme of the adenylation domain in the catalytic synthesis of daptomycin structural analogs.

[0005] The technical solution adopted by the present invention is as follows:

[0006] An adenylation protein A8 mutant is obtained by point mutation of the 308th amino acid of adenylation protein A8 with the amino acid sequence shown in SEQ ID NO.1. The present invention uses genetic engineering and enzyme engineering means to study the active site of the A domain, and determines the key amino acid residue (the 308th residue of A8) related to the substrate specificity of the A domain through constructing mutants, providing a new way for the expansion of the structures of daptomycin family compounds.

[0007] The adenylation protein A8 gene is derived from Streptomyces roseospoous, its amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2.

[0008] The sequence of SEQ ID NO.1 is as follows:

[0009] ARVLTEWNDTGVPGVPETFLELFEAQVAARGDAPAVVYEGEVLSYRELDARAN

[0010] RLAGLLVGRGAGPEHFVGVALPRGLDLIVALLAVLKSGAAYVPLDPEYPAERLVHM

[0011] VTDAAPVVVVTSTDVRTLRTVPRVELDDEATRATLVAAPATGPDVKMSASHPAYVI

[0012] YTSGSTGRPKGVVISHGSLANFLAWAREDLGAERLRHVVLSTSLSFDVSVVELFAPLS

[0013] CGGTVEIVRNLLALVDRPGRWSASLVSGVPSAFAQLLEAGLDRADVGMIALAGEALS

[0014] ARDVRRVRAVLPGARVANFYGPTEATVYATAWYGDTPMDAAAPMGRPLRNTCVY

[0015] VLDDGLRVVPVGVVGELYVAGVGLARGYLGRVGLTAERFVACPFGARGERMYRTG

[0016] DLVRWRVDGTLEFVGRADDQVKVRGFRVELGEVEGAVAAHPDVVRAVVVVREDR

[0017] PGDHRLVAYVTGVDTGGLSSAVMRAVAERLPAYMVPSAVVVLDEIPLTPNGKVDRA

[0018] GLPVPVVSVAGFCAPSSPREEVLCGLFAEVLGVERV

[0019] Preferably, the amino acid sequence of the adenylated protein A8 mutant is as shown in SEQ ID NO.3.

[0020] The sequence of SEQ ID NO.3 is as follows (the mutated sites are underlined):

[0021] ARVLTEWNDTGVPGVPETFLELFEAQVAARGDAPAVVYEGEVLSYRELDARANRLAGLLVGRGAGPEHFVGVALPRGLDLIVALLAVLKSGAAYVPLDPEYPAERLVHMVTDAAPVVVVTSTDVRTLRTVPRVELDDEATRATLVAAPATGPDVKMSASHPAYVIYTSGSTGRPKGVVISHGSLANFLAWAREDLGAERLRHVVLSTSLSFDVSVVELFAPLSCGGTVEIVRNLLALVDRPGRWSASLVSGVPSAFAQLLEAGLDRADVGMIALAGEALSARDVRRVRAVLPGARVANFYGPTEATV PATAWYGDTPMDAAAPMGRPLRNTCVYVLDDGLRVVPVGVVGELYVAGVGLARGYLGRVGLTAERFVACPFGARGERMYRTGDLVRWRVDGTLEFVGRADDQVKVRGFRVELGEVEGAVAAHPDVVRAVVVVREDRPGDHRLVAYVTGVDTGGLSSAVMRAVAERLPAYMVPSAVVVLDEIPLTPNGKVDRAGLPVPVVSVAGFCAPSSPREEVLCGLFAEVLGVERV

[0022] Due to the particularity of the amino acid sequence, any fragment or variant of the polypeptide with the amino acid sequence shown in SEQ ID NO.3, such as its conservative variant, bioactive fragment or derivative, as long as the homology between the fragment or polypeptide variant of the polypeptide and the aforementioned amino acid sequence is more than 95%, belongs to the scope of protection of the present invention. The changes may include deletion, insertion or substitution of amino acids in the amino acid sequence; for conservative changes of the variant, the substituted amino acid has a similar structure or chemical property to the original amino acid, such as replacing isoleucine with leucine, and the variant may also have non-conservative changes, such as replacing glycine with tryptophan.

[0023] The present invention also relates to a gene encoding the adenylated protein A8 mutant.

[0024] Specifically, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.4 (this gene encodes the amino acid shown in SEQ ID NO.3).

[0025] The sequence of SEQ ID NO.4 is as follows (the underlined part is the mutation site):

[0026] gcccgtgtcctgacggagtggaatgacacgggcgtccccggtgtgccggaaacattcctggagttgttcgaggcgcaggtcgcggcccggggtgacgcgccggcggtcgtgtacgagggtgaggttctgtcgtaccgggaactcgacgcgcgggcgaaccgcctggccgggctgctggtggggcgcggtgcgggcccggagcatttcgtgggggtggcgctgccgcgtgggctggatctgatcgtggccctgctggccgtgctcaagtccggtgccgcgtacgttcccctggacccggagtacccggccgagcggctggtccacatggtcaccgacgccgcccccgtcgtggtcgtgacctccaccgacgtacgtactctgcggaccgttccccgggtcgagctggacgacgaggcgacccgcgccaccctggtcgcagcccccgccacagggcccgacgtgaagatgtccgcctcccaccccgcgtacgtgatctacacctccgggtccacgggccgccccaagggcgtcgtcatcagccacggcagcctggccaacttcctcgcctgggcgcgggaagacctgggtgccgagcggctccggcacgtcgtgttgtccacgtccctcagcttcgacgtctccgtggtcgaactcttcgccccgctgtcctgcggcggcaccgtcgagatcgtccggaatctgctggccctcgtcgaccgccccggccgatggtccgcgagcctggtcagcggcgtgccgtcggccttcgcgcagctgctggaagccggcctcgaccgggccgacgtgggcatgatcgccctggccggcgaggcgctgtccgctcgcgacgtgcgccgcgtccgcgctgtgctgcccggggcccgcgtggccaacttctacggcccgaccgaagccaccgtc cccgccacggcctggtacggcgacacccccatggacgccgcggcccccatgggccggcccctgcgcaacacgtgtgtgtatgtgctggacgacgggctgcgcgtggtgccggtcggtgtggtgggtgagctgtatgtggcgggtgtgggtctggcgcggggctatctcgggcgtgtgggtctgacggcggagcggtttgtggcgtgtccgttcggtgcgcggggtgagcgtatgtatcgcacgggggatttggtgcggtggcgggtggacggcacgcttgagtttgttggtcgtgcggatgatcaggtgaaggtccgtggtttccgtgtggagttgggtgaggtggagggtgctgttgcggcgcatcctgatgtggtgcgtgcggttgttgtggtgcgtgaggaccggccgggtgatcaccggttggttgcgtatgtcaccggtgttgacacgggtggactgtcctctgcggtgatgcgtgccgttgctgagcgtctgcctgcgtacatggtgccgtcggcggtggtggttctggatgagatcccgttgacgccgaacgggaaggtggaccgggcgggtcttccggtgccggtggtgtcggtggcggggttctgtgcgccgtcgtcgccgcgggaggaggtgttgtgtggtctgttcgcggaggtgctgggtgttgagcgggtg

[0027] Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.4, as long as it has more than 90% homology with the polynucleotide, falls within the scope of protection of the present invention. The variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. Such variants of the polynucleotide can be native allelic variants or non-native variants, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of encoding amino acids.

[0028] The present invention also relates to a recombinant bacterium containing the coding gene.

[0029] The present invention also relates to the use of the adenylated protein A8 mutant as a key enzyme in the adenylation domain in the catalytic synthesis of daptomycin structural analogs. For the biosynthetic pathway of daptomycin, see Figure 1 , and the mutants modified in vitro in the present invention are derived from the A domain in module 8.

[0030] The key of the present invention lies in the selection of mutation sites. On the premise of knowing the sequence of the adenylated protein A8 and its mutation sites, those of ordinary skill in the art can design mutant primers for site-directed mutagenesis according to the adenylated protein gene of SEQ ID NO.1, perform site-directed mutagenesis to construct mutants using the cloning vector carrying the adenylated protein A8 as a template, use plasmid pET-28a or a vector capable of expressing the enzyme as an expression vector, transform the recombinant plasmid into E. coli BL21(DE3) cells, and culture the verified positive monoclonal clones to obtain a recombinant strain containing the mutant of the present invention.

[0031] Specifically, the steps for constructing mutants are as follows:

[0032] (1) Clone the gene encoding the adenylated protein A8 obtained from the daptomycin biosynthetic gene cluster of Streptomyces roseosporus into an expression vector to construct an A8 recombinant expression plasmid;

[0033] (2) Using site-directed mutagenesis technology, design complementary primers containing the codon bases of the mutant amino acids, perform inverse PCR using the recombinant plasmid as a template to obtain a target vector fragment containing site-directed mutagenesis;

[0034] (3) Digest the PCR reaction solution containing the target vector fragment with site-directed mutagenesis with Dpn I endonuclease to remove the original plasmid that has not been successfully mutated, and recover the fragment by 1% agarose gel electrophoresis;

[0035] (4) Transform the recovered target vector fragment containing site-directed mutagenesis into competent Escherichia coli DH5α, culture on a plate, pick the correct monoclonal, and extract the plasmid with the correct mutation;

[0036] (5) Transform the correct recombinant plasmid and five molecular chaperone plasmids into Escherichia coli BL21(DE3) respectively for induced expression.

[0037] The method further includes purifying the adenylated protein using a 1 mL Ni-NTA nickel column, a desalting column, and an ultrafiltration tube.

[0038] Pick the recombinant strain obtained in step (5) of the said method and inoculate it into a 5 mL LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol. Incubate it overnight in a shaker at 37 °C and 220 rpm. Take 1.5 mL of the LB liquid culture and inoculate it into 150 mL of TB liquid medium. Incubate at 37 °C until the OD 600 is 0.6 - 0.8. Add IPTG with a final concentration of 0.4 mM and induce it overnight at 16 °C. After incubation, centrifuge at 12,000 rpm and 4 °C to collect the bacterial cells, weigh them, add a cell disruption solution with a volume 5 times the weight, resuspend, disrupt the cells in a cryogenic high-pressure cell disruptor at 4 °C, and centrifuge at 12,000 rpm for 10 min at 4 °C to collect the supernatant, which is the crude enzyme solution.

[0039] The beneficial effects of the present invention are mainly reflected in that: compared with the adenylated protein A8, the mutant Y308P obtained in the present invention has an altered substrate specificity, with a significantly reduced activity towards L-Ala and a significantly increased activity towards L-Gly, providing a new way to obtain Gly 8 -substituted daptomycin structural analogs. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 shows the biosynthetic pathway of daptomycin.

[0041] Figure 2 shows the chemical structural formulas of daptomycin and Gly 8 -substituted structural analogs, and the amino acids within the boxes are alanine and glycine respectively.

[0042] Figure 3 shows the protein expressions of adenylated protein A8 and mutants G251T, A273L, Y308PY308P, G251T / A273L; lane 1 is the protein Marker, lane 2 is the supernatant of A8, lane 3 is the supernatant of A8 - G251T, lane 4 is the supernatant of A8 - A273L, lane 5 is the supernatant of A8 - G251T / A273L, lane 6 is the supernatant of A8 - Y308P.

[0043] Figure 4 shows the reaction principle of the FeCl3 detection method.

[0044] Figure 5 shows the comparison of the activities of adenylated protein A8 towards different substrates.

[0045] Figure 6 shows the comparison of the activities of adenylated protein mutant G251T towards different substrates.

[0046] Figure 7 Activity comparison of adenylated protein mutant A273L towards different substrates

[0047] Figure 8 Activity comparison of adenylated protein mutants G251T / A273L towards different substrates

[0048] Figure 9 Activity comparison of adenylated protein mutant Y308P towards different substrates (V) Specific implementation manners

[0049] To deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments. These embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0050] Example 1: Construction of wild-type adenylated protein A8 expression plasmid

[0051] (1) Using the Streptomyces roseosporus genome preserved in the laboratory as a template, upstream and downstream primers F / R with recombination homologous fragments to pET-28a(+) were designed for PCR amplification. The PCR products were detected by 1% agarose gel electrophoresis,

[0052] and the target fragments were recovered using a small-scale DNA purification kit for standby.

[0053] F: TGCCGCGCGGCAGCCATATGGCCCGTGTCCTGACGGAGTG

[0054] R: CAAGCTTGTCGACGGAGCTCCTACACCCGCTCAACACC

[0055] (2) The expression plasmid pET-28a(+) was extracted and double-digested with Nde I and Hind III, and then recovered for standby.

[0056] (3) The above-mentioned recovered vector and fragments were recombined using the SoSoo kit and transformed into Escherichia coli DH5α competent cells to obtain a cloned strain containing the recombinant expression plasmid pET-28a+A8.

[0057] Example 2: Prediction of key amino acid residues by sequence alignment

[0058] To predict the key residues related to the substrate binding pocket of the adenylated protein, the amino acid sequences of five proteins (GrsA, SlgN1, CmiS6, IdnL1, and DltA) whose protein structures and active sites have been determined by X-ray diffraction were downloaded from NCBI. Their PBD numbers are 1AMU, 4GR5, 5JJP, 5JJQ, and 3DHV respectively. These amino acid sequences were aligned with the amino acid sequence of A8 in Example 1 by multiple sequence alignment, and the key residues that may be related to the substrate specificity of the A domain of this module were preliminarily screened as shown in Table 1.

[0059] Table 1: Results of predicting active sites by multiple sequence alignment

[0060]

[0061] Example 3: Construction of an expression plasmid for an adenylated protein mutant

[0062] (1) Using the pET-28a+A8 expression plasmid constructed in Example 1 as a template, site-directed mutagenesis primers G251T-F / R, A273L-F / R, G251T / A273L-F / R, and Y308P-F / R were designed to perform inverse PCR respectively.

[0063] The specific site-directed mutagenesis primers are as follows (the underlined part is the mutation site):

[0064] G251T-F:ACTGTGCCGTCGGCCTTCGCGCAGCTGCTGGAAGCCGGCC

[0065] G251T-R:GCGAAGGCCGACGGCAC AGT GCTGACCAGGCTCGC GGACC

[0066] A273L-F: CTC CTGGCCGGCGAGGCGCTGTCCGCTCGCGACGTGCGCC

[0067] A273L-R:AGCGCCTCGCCGGCCAG GAG GATCATGCCCACGTCGGCCC

[0068] G251T / A273L-F: CTC CTGGCCGGCGAGGCGCTGTCCGCTCGCGACGTGCGCC

[0069] G251T / A273L-R:AGCGCCTCGCCGGCCAG GAG GATCATGCCCACGTCGGCCC

[0070] Y308P-F: CCC GCCACGGCCTGGTACGGCGACACCCCCATGGACGCCG

[0071] Y308P-R:CCGTACCAGGCCGTGGCG GGG ACGGTGGCTTCGGTCGGGC

[0072] (2) The PCR products were detected by 1% agarose gel electrophoresis. After detecting the target product, Dpn I enzyme was added to digest the template, and the target fragment was recovered using a small-scale DNA purification kit for standby.

[0073] (3) The recovered mutant PCR fragments were transformed into Escherichia coli DH5α competent cells to obtain the expression plasmids pET-28a+G251T, pET-28a+A273L, pET-28a+G251T / A273L, pET-28a+Y308P of the mutant G251T encoding gene (SEQ ID NO.5), A273L encoding gene (SEQ ID NO.6), G251T / G251T / A273L (encoding gene SEQ ID NO.7), Y308P (SEQ ID NO.3, encoding gene SEQ ID NO.4).

[0074] Example 4: Expression and purification of adenylylated protein A8 and mutant proteins

[0075] (1) The constructed plasmids pET-28a+A8, pET-28a+G251T, pET-28a+A273L, pET-28a+G251T / A273L, pET-28a+Y308P (together with the molecular chaperone plasmid) and the pET-28a empty plasmid were transformed into BL21(DE3) competent cells to construct expression strains. Single colonies were picked from the plate and inoculated into an LB medium test tube with a kanamycin concentration of 50 μg / ml and a chloramphenicol concentration of 25 μg / ml, and cultured overnight with constant shaking at 37°C and 220 rpm. Then, they were inoculated into a TB liquid medium with a kanamycin concentration of 50 μg / ml and a chloramphenicol concentration of 25 μg / ml at an inoculation amount of 1%. Cultured at 37°C until the OD 600 reached 0.6 - 0.8, and 0.4 mM IPTG was added to induce protein expression overnight at 16°C.

[0076] (2) After overnight induction at 16 °C and 180 rpm, the cells were collected by centrifugation at 12,000 rpm for 10 min at 4 °C and weighed. Five volumes of cell disruption buffer were added based on the weight of the cells, and the cells were resuspended. The cells were disrupted using a cryogenic high-pressure cell disrupter at 4 °C, and the supernatant was collected by centrifugation at 12,000 rpm for 10 min at 4 °C. This was the crude enzyme solution.

[0077] (3) Protein expression was verified by SDS-PAGE. As Figure 3 shown, the results indicated that the target protein was expressed solubly.

[0078] Example 4: Determination of the adenylation activity of A8 and each mutant protein towards the substrate by the FeCl3 detection method

[0079] Principle of the FeCl3 detection method: The A domain recognizes and activates the substrate amino acid, which will be adenylated to form aminoacyl-AMP. When hydroxylamine is present, aminoacyl-AMP will react with hydroxylamine to form hydroxamic acid, and hydroxamic acid will react with Fe 3+ to form a red or purple-red complex, which can be detected at an absorption wavelength between 480 nm and 540 nm. As Figure 4 shown.

[0080] The reagents used in the FeCl3 detection method include 2× adenylation detection buffer (100 mM Tris-HCl, pH 8.0), 100 mM MgSO4 solution, 4 M hydroxylamine solution, 7 M NaOH solution, 100 mM ATP, 100 mM amino acid solution, 8% trichloroacetic acid solution, 3.4% FeCl3 solution. The specific reaction system is shown in Table 2. Among them, the 2 M hydroxylamine solution needs to be prepared freshly. Use a pipette to take 400 μL of the 4 M solution, 225 μL of the 7 M NaOH, and 175 μL of deionized water, mix well and place on ice.

[0081] Table 2: Specific reaction system for detecting the adenylation activity of the A domain by the FeCl3 method

[0082]

[0083] Taking the selective determination of 20 natural amino acids by a protein as an example, the system mother liquor can be prepared by expanding 21 times on the basis of a 100 μL system, and all the required reagents except amino acids are added to the mother liquor. Take 0.2 mL PCR tubes labeled from tube 1 to tube 20 and add 5 μL of different 100 mM amino acid solutions. Tube 21 is added with 5 μL of deionized water as a control. 95 μL of the system mother liquor is added to each PCR tube, and the reaction is carried out in a 37 °C constant temperature incubator. After 24 h of reaction, 50 μL of 8% trichloroacetic acid solution is added to each reaction system to quench the reaction, and then 50 μL of 3.4% FeCl3 solution is added. Pipette and mix well, and develop color for 5 min. The mixture is centrifuged at 12000 rpm at room temperature for 10 min, and 100 μL of the supernatant is carefully aspirated into a transparent 96-well plate, and detected using an enzyme-linked immunosorbent assay at a wavelength of 490 nm. The absorbance value of the experimental group is subtracted from the absorbance value of the control group with water as the substrate as the selectivity of the protein for each amino acid.

[0084] The control group of PET-28A plasmid transformed into E. coli BL21(DE3), adenylylated protein A8 and mutants G251T, A273L, G251T / A273L, and Y308P protein were respectively used for substrate specificity detection of L-alanine, L-glycine, L-serine, and L-2-aminobutyric acid by the FeCl3 detection method. The results showed that the supernatant of the control group transformed with the empty PET-28A plasmid into BL21 had no activity for the substrates, and the interference of other miscellaneous proteins except the target protein in the supernatant on the activity detection could be excluded. Compared with the wild-type A8 protein, the mutant A8 - Y308P had a sharp decrease in the adenylylation activity for L-alanine, but a sharp increase in the adenylylation activity for L-glycine, which provided a reference for obtaining Gly 8 substituted daptomycin structural analogs. At the same time, it was shown that the amino acid at position 308 of the A8 protein was related to the substrate-specific recognition. Modifying the amino acid residues at this site could change the substrate specificity of the A8 protein. Mutational studies at this site could provide a new way for the structural expansion of daptomycin family compounds.

[0085] The above-described embodiments are only preferred embodiments cited for full illustration, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention, and the protection scope of the present invention is subject to the claims.

Claims

1. An adenylylated protein A8 mutant, characterized in that The amino acid sequence of the adenylated protein A8 mutant is shown in SEQ ID NO.

3.

2. A gene encoding the adenylylated protein A8 mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

4. A recombinant bacterium containing the gene according to claim 2.

5. Use of the adenylylated protein A8 mutant according to claim 1 as a key enzyme of the adenylylation domain in the catalytic synthesis of a Gly 8 substituted daptomycin structural analog.

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