CN118546896A - Multi-domain acetyl-CoA carboxylase and application thereof - Google Patents
Multi-domain acetyl-CoA carboxylase and application thereof Download PDFInfo
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- CN118546896A CN118546896A CN202410870990.8A CN202410870990A CN118546896A CN 118546896 A CN118546896 A CN 118546896A CN 202410870990 A CN202410870990 A CN 202410870990A CN 118546896 A CN118546896 A CN 118546896A
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- 108010016219 Acetyl-CoA carboxylase Proteins 0.000 title claims abstract description 28
- 102000000452 Acetyl-CoA carboxylase Human genes 0.000 title claims abstract description 28
- 108010018763 Biotin carboxylase Proteins 0.000 title claims abstract description 28
- 239000002773 nucleotide Substances 0.000 claims abstract description 19
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 19
- 239000013598 vector Substances 0.000 claims abstract description 16
- 229930001119 polyketide Natural products 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- 150000003881 polyketide derivatives Chemical class 0.000 claims abstract description 8
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 8
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 6
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 6
- 238000007876 drug discovery Methods 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 14
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
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- 229960000318 kanamycin Drugs 0.000 claims description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 9
- 229930182823 kanamycin A Natural products 0.000 claims description 9
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- 102000004169 proteins and genes Human genes 0.000 claims description 8
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- 238000003287 bathing Methods 0.000 claims description 6
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 3
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- 241000999525 Streptomyces venezuelae ATCC 10712 Species 0.000 claims description 2
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- 230000002255 enzymatic effect Effects 0.000 abstract description 5
- 241001655322 Streptomycetales Species 0.000 abstract description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
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- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 6
- 239000011574 phosphorus Substances 0.000 description 6
- 229910052698 phosphorus Inorganic materials 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 108010064926 acyl-CoA carboxylase Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
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- 229960002685 biotin Drugs 0.000 description 3
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- 239000003795 chemical substances by application Substances 0.000 description 3
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- 125000000830 polyketide group Chemical group 0.000 description 3
- ACTOXUHEUCPTEW-BWHGAVFKSA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4r,5s,6r)-5-[(2s,4r,5s,6s)-4,5-dihydroxy-4,6-dimethyloxan-2-yl]oxy-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-10-[(2s,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-o Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)[C@@H]1CC[C@H](N(C)C)[C@@H](C)O1 ACTOXUHEUCPTEW-BWHGAVFKSA-N 0.000 description 2
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 2
- 108010030713 Geranoyl-CoA carboxylase Proteins 0.000 description 2
- 108010085747 Methylmalonyl-CoA Decarboxylase Proteins 0.000 description 2
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- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 2
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- 239000004187 Spiramycin Substances 0.000 description 2
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- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 2
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- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 2
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- VTIKDEXOEJDMJP-UHFFFAOYSA-N Actinorhodine Natural products CC1OC(CC(=O)O)CC2=C1C(=O)c3c(O)c(cc(O)c3C2=O)c4cc(O)c5C(=O)C6=C(C(C)OC(CC(=O)O)C6)C(=O)c5c4O VTIKDEXOEJDMJP-UHFFFAOYSA-N 0.000 description 1
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- VTIKDEXOEJDMJP-WYUUTHIRSA-N actinorhodin Chemical compound C([C@@H](CC(O)=O)O[C@@H]1C)C(C(C2=C(O)C=3)=O)=C1C(=O)C2=C(O)C=3C(C(=C1C2=O)O)=CC(O)=C1C(=O)C1=C2[C@@H](C)O[C@H](CC(O)=O)C1 VTIKDEXOEJDMJP-WYUUTHIRSA-N 0.000 description 1
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- 239000011609 ammonium molybdate Substances 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a multidomain acetyl-CoA carboxylase which is JadJ-pET41a vector; the JadJ-pET41a vector has a JadJ nucleotide sequence shown in SEQ ID NO.1 from 12 th to 1810 th in the sequence table and an amino acid sequence shown in SEQ ID NO.2 from 1 st to 584 st in the sequence table. Meanwhile, the invention also discloses application of the carboxylase. The carboxylase has good enzymatic properties, and can be widely applied to drug discovery and high yield and synthesis of polyketide antibiotics, in particular to synthesis and high yield of streptomycete polyketide.
Description
Technical Field
The invention relates to the technical field of novel enzymes, in particular to a multi-domain acetyl-CoA carboxylase and application thereof.
Background
Biotin-dependent carboxylase enzymes are widely distributed in nature and play an important role in cellular processes such as fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide synthesis, urea utilization and the like [Tong L. Structure and function of biotin-dependent carboxylases. Cell Mol Life Sci.2013 Mar;70(5):863-91.]. biotin-dependent carboxylase enzymes include acetyl-coa carboxylase (ACC), propionyl-coa carboxylase (PCC), 3-methylcrotonyl-coa carboxylase (MCC), geranyl-coa carboxylase (GCC), pyruvate Carboxylase (PC) and urea carboxylase （UC）[Tong L. Structure and function of biotin-dependentcarboxylases. Cell Mol Life Sci. 2013 Mar;70(5):863-91.],, which have defective mutations associated with serious metabolic diseases in humans. Furthermore, recent studies indicate that ACC is a drug discovery target for type two diabetes, obesity, cancer, microbial infection, and other diseases, ACC within the Gramineae's body is a target of action of three classes of commercial herbicides. However, the number of ACCs found in the prior studies is small, the ACCs are concentrated in eukaryotes, and almost all of the existing ACCs belong to protein complexes, and 2-4 enzymes are required for biotinylation and carboxyl transfer, respectively, which makes the study of ACC application difficult.
Thus, it is necessary to explore novel long chain multi-domain acyl-coa carboxylase enzymes.
Disclosure of Invention
The technical problem to be solved by the invention is to provide acidophilic multi-domain acetyl-CoA carboxylase.
Another technical problem to be solved by the present invention is to provide the use of the multidomain acetyl-CoA carboxylase.
In order to solve the problems, the multi-domain acetyl-CoA carboxylase of the invention is characterized in that: the carboxylase is JadJ-pET41a vector; the JadJ-pET41a vector has a JadJ nucleotide sequence shown in SEQ ID NO.1 from 12 th to 1810 th in the sequence table and an amino acid sequence shown in SEQ ID NO.2 from 1 st to 584 st in the sequence table.
A method of preparing a multidomain acetyl-coa carboxylase as described above comprising the steps of:
⑴ Constructing primers Seq1 and Seq2:
Seq1 is a nucleotide sequence shown in SEQ ID NO.3 from 1 st to 44 th positions in the sequence table;
Seq2 is a nucleotide sequence shown in SEQ ID NO.4 from 1 st to 50 th positions in the sequence table;
the underlined HindIII cleavage site for Seq1, and the underlined BamHI cleavage site for Seq 2;
⑵ Obtaining a target gene:
Carrying out PCR amplification by taking genomic DNA of a strain Streptomyces venezuelaeATCC 10712 as a template and sequences shown in Seq1 and Seq2 as primer pairs JadJ to obtain a segment JadJ nucleotide sequence, wherein the JadJ nucleotide sequence has coding genes from 1 st to 1831 st of SEQ ID NO.1 in a sequence table, and from 12 th to 1810 th of JadJ;
⑶ Construction JadJ of heterologous expression vectors:
double-enzyme digestion is carried out on the pET41a plasmid by using restriction enzymes HindIII and BamHI, and then a JadJ nucleotide sequence is connected with the plasmid, so that a JadJ-pET41a vector is obtained;
⑷ Plasmid transformation:
Adding JadJ-pET41a vector 10 μl into escherichia coli BL21 (DE 3) competence, ice-bathing for 30min, heat-shocking at 42 ℃ for 45s, ice-bathing for 5min, adding LB liquid culture medium, incubating for 1h, and culturing on culture medium containing kanamycin;
⑸ Heterologous expression and purification of proteins:
And (3) inducing protein expression of the successfully transformed escherichia coli BL21 (DE 3) in the step ⑷ by using IPTG at 18 ℃, collecting thalli after the expression is finished, re-suspending the thalli in a cell lysate, then ultrasonically crushing cells, centrifugally collecting supernatant, purifying by using nickel column affinity chromatography, and concentrating the enzyme solution by ultrafiltration to obtain JadJ with the purity of more than or equal to 95 percent.
The LB liquid medium in the step ⑷ is obtained by adding 10g of tryptone, 5g of yeast extract and 5g of sodium chloride into 1L of deionized water, uniformly mixing, adjusting pH to be neutral, and steam sterilizing at a high pressure of 5psi for 21 min.
The culture medium containing kanamycin in the step ⑷ is obtained by adding 50mg of kanamycin into 1L of deionized water and uniformly mixing.
The use of a multidomain acetyl-coa carboxylase as described above, characterized in that: the carboxylase is used for drug discovery or synthesis of polyketide antibiotics.
Compared with the prior art, the invention has the following advantages:
1. the invention takes JadJ in the Jettanomycin synthetic gene cluster as a research object, carries out heterologous expression after cloning, and explores the enzymatic properties of the Jettanomycin. As a result, it was found that the enzyme was an acidophilic multidomain acetyl-CoA carboxylase.
2. The carboxylase has good enzymatic properties, and can be widely applied to drug discovery and high yield and synthesis of polyketide antibiotics, in particular to synthesis and high yield of streptomycete polyketide.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 is a plasmid map of JadJ-pET41a of the invention.
FIG. 2 shows SDS-PAGE detection according to the invention JadJ.
FIG. 3 shows the optimum pH of JadJ of the present invention.
FIG. 4 shows the optimum temperature of JadJ of the present invention.
FIG. 5 is a graph showing the enzymatic kinetics of the present invention JadJ.
FIG. 6 is a graph showing comparison of the yields of actinorubicin, daunorubicin, spiramycin and monensin before and after the introduction of acetyl-CoA carboxylase JadJ of the present invention.
Detailed Description
A multidomain acetyl-coa carboxylase that is a JadJ-pET41a vector and a JadJ-pET41a vector. JadJ-pET41a vector is a vector which has JadJ nucleotide sequence shown in SEQ ID NO.1 from 12 th to 1810 th in a sequence table and has JadJ amino acid sequence shown in SEQ ID NO.2 from 1 st to 584 th in the sequence table.
The preparation method of the multidomain acetyl-CoA carboxylase comprises the following steps:
⑴ Constructing primers Seq1 and Seq2:
Seq1 is a nucleotide sequence shown in SEQ ID NO.3 from 1 st to 44 th positions in the sequence table;
Seq2 is a nucleotide sequence shown in SEQ ID NO.4 from 1 st to 50 th positions in the sequence table;
The underlined HindIII cleavage site for Seq1 and the underlined BamHI cleavage site for Seq 2.
⑵ Obtaining a target gene:
PCR amplification was performed on JadJ gene using genomic DNA of strain Streptomyces venezuelaeATCC and 10712 as templates and sequences shown in Seq1 and Seq2 as primer pairs. The PCR system is as follows:
2X Vazyme LAMP MASTER Mix 12.5. Mu.L, primer Seq1 1. Mu.L, primer Seq 21. Mu.L, DNA template 2. Mu.L, DMSO 1. Mu.L, and water was added to make up to 25. Mu.L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 65℃for 30 seconds, elongation at 72℃for 60 seconds, 32 cycles; finally, the extension is carried out at 72 ℃ for 10 minutes.
2X Vazyme LAMP MASTER Mix 12.5. Mu.L, primer Seq 31. Mu.L, primer Seq4 1. Mu.L, DNA template 2. Mu.L, DMSO 1. Mu.L, and water was added to make up to 25. Mu.L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 65℃for 30 seconds, elongation at 72℃for 60 seconds, 32 cycles; finally, the extension was carried out at 72℃for 10 minutes.
Amplifying a fragment JadJ nucleotide sequence, wherein the JadJ nucleotide sequence has coding genes from 1 st to 1831 st of SEQ ID NO.1 in a sequence table, and from 12 th to 1810 th of the coding genes are JadJ;
⑶ Construction JadJ of heterologous expression vectors:
the pET41a plasmid was double digested with restriction enzymes HindIII and BamHI, and then the JadJ nucleotide sequence was ligated thereto to give a JadJ-pET41a vector, as shown in FIG. 1.
⑷ Plasmid transformation:
Adding JadJ-pET41a vector 10 μl into escherichia coli BL21 (DE 3) competence, ice-bathing for 30min, heat-shocking at 42 ℃ for 45s, ice-bathing for 5min, adding LB liquid culture medium, incubating for 1h, and culturing on culture medium containing kanamycin.
Wherein: the LB liquid medium is obtained by adding 10g of tryptone, 5g of yeast extract and 5g of sodium chloride into 1L of deionized water, uniformly mixing, adjusting pH to be neutral, and steam sterilizing at high pressure of 5psi for 21 min.
The culture medium containing kanamycin is obtained by adding 50mg kanamycin into 1L deionized water and uniformly mixing.
⑸ Heterologous expression and purification of proteins:
The successfully transformed E.coli BL21 (DE 3) in step ⑷ is induced to express protein by IPTG at 18 ℃, after the expression is completed, thalli are collected and resuspended in cell lysate, cells are crushed by ultrasound, supernatant is collected by centrifugation, and purified by using nickel column affinity chromatography, and the purified enzyme solution is concentrated by ultrafiltration to obtain JadJ with the purity of more than or equal to 95 percent, as shown in figure 2.
The acetyl-CoA carboxylase obtained by the invention is characterized by enzymatic properties:
① Determination of optimum pH:
The reaction system, 100. Mu.L, comprises 3mM ATP,15mM NaHCO 3, 10mM enzyme, 20mM Hepes and 0.5mM acetyl-CoA, and the enzymatic reaction is carried out at pH 3-10. Boiling water bath after the enzymatic reaction is completed, centrifuging 10000g to obtain supernatant, adding phosphorus fixing agent, reacting for 30min at room temperature, and detecting absorbance at 650 nm. The amount of inorganic phosphorus produced per mg protein per hour was 1. Mu.M, which was one ACC activity unit.
Phosphorus fixative formula (mass ratio g/g): 3M H 2SO4: 2.5% ammonium molybdate: 10% anti-chemosanguinic acid: h 2 o=1:1:1:2.
The enzyme activity is shown in figure 3, and JadJ has an optimal pH of 5-6, is neutral meta-acid, and can be applied to high yield of polyketides in most streptomyces.
② Determination of optimum temperature:
The reaction system of 100. Mu.L comprises 3mM ATP,15mM NaHCO 3, 10mM enzyme, 20mM Hepes and 0.5mM acetyl-CoA, and the enzymatic reaction is carried out at a temperature of 15-65 ℃. Boiling water bath after the enzymatic reaction is completed, centrifuging 10000g to obtain supernatant, adding phosphorus fixing agent, reacting for 30min at room temperature, and detecting absorbance at 650 nm. The amount of inorganic phosphorus produced per mg protein per hour was 1. Mu.M, which was one ACC activity unit.
The enzyme activity is shown in figure 4, the optimal temperature of JadJ is 25-35 ℃, and the method can be applied to high yield of polyketides of most strains.
③ Enzyme kinetic assay:
The reaction system comprises 1mM ATP,5mM NaHCO 3, 50. Mu.M enzyme, 20mM Hepes and 0-0.2mM acetyl-CoA in 100. Mu.L. Boiling water bath after the enzymatic reaction is completed, centrifuging 10000g to obtain supernatant, adding a phosphorus fixing agent, reacting for 30min at room temperature, detecting absorbance at 650nm, and fitting Km according to the result by using a Milker equation.
As a result, FIG. 5 shows that JadJ has a Km of 0.8 mM and a Vmax of 2.02 mol/min.
Use of a multidomain acetyl-coa carboxylase for drug discovery or synthesis of polyketide antibiotics.
For the long-chain multi-domain acyl-CoA carboxylase sequences of the invention, the comparison in the database shows that 99% of similar sequences are from actinomycetes (mainly Streptomycetaceae) and are mostly non-researched sequences, the sequences are greatly different from the sequences of the acyl-CoA carboxylase known to be involved in fatty acid synthesis, and Streptomyces are the main sources of various antibiotics at present, so that the long-chain multi-domain acyl-CoA carboxylase can have preferential participation in the production of polyketides in Streptomyces.
Examples
Acetyl-CoA carboxylase JadJ was introduced into Streptomyces coelicolor, streptomyces genitaliensis and Streptomyces cinnamomum strains, respectively, and detected using high resolution LC-MS: the yields of actinorhodin, daunorubicin, spiramycin and monensin were all increased to varying degrees, as shown in figure 6.
Claims (5)
1. A multidomain acetyl-coa carboxylase characterized by: the carboxylase is JadJ-pET41a vector; the JadJ-pET41a vector has a JadJ nucleotide sequence shown in SEQ ID NO.1 from 12 th to 1810 th in the sequence table and an amino acid sequence shown in SEQ ID NO.2 from 1 st to 584 st in the sequence table.
2. A method of preparing a multidomain acetyl-coa carboxylase according to claim 1, comprising the steps of:
⑴ Constructing primers Seq1 and Seq2:
Seq1 is a nucleotide sequence shown in SEQ ID NO.3 from 1 st to 44 th positions in the sequence table;
Seq2 is a nucleotide sequence shown in SEQ ID NO.4 from 1 st to 50 th positions in the sequence table;
the underlined HindIII cleavage site for Seq1, and the underlined BamHI cleavage site for Seq 2;
⑵ Obtaining a target gene:
Carrying out PCR amplification by taking genomic DNA of a strain Streptomyces venezuelae ATCC 10712 as a template and sequences shown in Seq1 and Seq2 as primer pairs JadJ to obtain a segment JadJ nucleotide sequence, wherein the JadJ nucleotide sequence has coding genes from 1 st to 1831 st of SEQ ID NO.1 in a sequence table, and from 12 th to 1810 th of JadJ;
⑶ Construction JadJ of heterologous expression vectors:
double-enzyme digestion is carried out on the pET41a plasmid by using restriction enzymes HindIII and BamHI, and then a JadJ nucleotide sequence is connected with the plasmid, so that a JadJ-pET41a vector is obtained;
⑷ Plasmid transformation:
Adding JadJ-pET41a vector 10 μl into escherichia coli BL21 (DE 3) competence, ice-bathing for 30min, heat-shocking at 42 ℃ for 45s, ice-bathing for 5min, adding LB liquid culture medium, incubating for 1h, and culturing on culture medium containing kanamycin;
⑸ Heterologous expression and purification of proteins:
And (3) inducing protein expression of the successfully transformed escherichia coli BL21 (DE 3) in the step ⑷ by using IPTG at 18 ℃, collecting thalli after the expression is finished, re-suspending the thalli in a cell lysate, then ultrasonically crushing cells, centrifugally collecting supernatant, purifying by using nickel column affinity chromatography, and concentrating the enzyme solution by ultrafiltration to obtain JadJ with the purity of more than or equal to 95 percent.
3. A method of preparing a multidomain acetyl-coa carboxylase according to claim 2, wherein: the LB liquid medium in the step ⑷ is obtained by adding 10g of tryptone, 5g of yeast extract and 5g of sodium chloride into 1L of deionized water, uniformly mixing, adjusting pH to be neutral, and steam sterilizing at a high pressure of 5psi for 21 min.
4. A method of preparing a multidomain acetyl-coa carboxylase according to claim 2, wherein: the culture medium containing kanamycin in the step ⑷ is obtained by adding 50mg of kanamycin into 1L of deionized water and uniformly mixing.
5. Use of a multidomain acetyl-coa carboxylase according to claim 1, wherein: the carboxylase is used for drug discovery or synthesis of polyketide antibiotics.
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| CN107805640A (en) * | 2017-10-13 | 2018-03-16 | 中国农业科学院植物保护研究所 | A kind of method for improving secondary metabolism of Streptomyces Product yields |
| CN118126920A (en) * | 2024-03-25 | 2024-06-04 | 中国科学院西北生态环境资源研究院 | High-yield aureomycin engineering strain based on malonyl-CoA regeneration pathway |
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| CN107805640A (en) * | 2017-10-13 | 2018-03-16 | 中国农业科学院植物保护研究所 | A kind of method for improving secondary metabolism of Streptomyces Product yields |
| CN118126920A (en) * | 2024-03-25 | 2024-06-04 | 中国科学院西北生态环境资源研究院 | High-yield aureomycin engineering strain based on malonyl-CoA regeneration pathway |
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