CN108587991A

CN108587991A - A kind of bacterial strain of high yield cyclic peptide compound

Info

Publication number: CN108587991A
Application number: CN201810326829.9A
Authority: CN
Inventors: 李青连; 鞠建华; 秦湘静; 刘静
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2015-10-10
Filing date: 2015-10-10
Publication date: 2018-09-28
Anticipated expiration: 2035-10-10
Also published as: CN105154487B; CN108486028A; CN108486028B; WO2017059626A1; JP2018530335A; CN108587991B; JP6585839B2; CN105154487A

Abstract

The invention discloses aminopherases and isomerase to form the application in L allo Ile in catalysis.The aminopherase is aminopherase DsaD or aminopherase MfnO, the isomerase is isomerase DsaE or isomerase MfnH, the amino acid sequence of the aminopherase DsaD is as shown in SEQ ID NO.7, the amino acid sequence of the isomerase DsaE is as shown in SEQ ID NO.8, the amino acid sequence of the aminopherase MfnO is as shown in SEQ ID NO.5, and the amino acid sequence of the isomerase MfnH is as shown in SEQ ID NO.6.The invention discloses the enzymes being made of aminopherase and isomerase to forming the application in L alloisoleucines or L isoleucines in catalysis.To explain that the biosynthesis mechanism of L allo Ile has established important basis in zymetology level.The explanation of L allo Ile biosynthesis zymetology mechanism by the Enzymology method preparation L allo Ile to utilize green, and has important Practical significance to the diagnosing and treating of maple syrup urine disease.

Description

A kind of bacterial strain of high yield cyclic peptide compound

The application is the applying date：On October 10th, 2015, application number：201510651516.7 denomination of invention：Transamination Enzyme and isomerase form the divisional application of the patent application of the application in L-allo-Ile in catalysis.

Technical field：

The invention belongs to genetic engineerings and biocatalysis technology field, and in particular to one kind is by phosphopyridoxal pyridoxal phosphate The aminopherase (PLP-linked aminotransferase) and one that (pyridoxal5 '-phosphate, PLP) is relied on The enzyme pair of novel isomerase (isomerase) composition of kind forms L- alloisoleucines (L-allo-Ile) in common cooperation catalysis In application.

Background technology：

There are two asymmetric centers for isoleucine tool, therefore there is 4 kinds of stereoisomers：L-Isoleucine (L-Ile), D-Ile (D-Ile), L- alloisoleucines (L-allo-Ile) and D- alloisoleucines (D-allo-Ile) are corresponded to and are closed System is as shown in Figure 1.In addition to L-Ile, D-Ile, L-allo-Ile and D-allo-Ile belong to nonprotein amino acid, Presence in nature has relevant report.Wherein, L-allo-Ile is due to its being widely present in nature and important section Meaning is learned, and causes the special attention of scientists.L-allo-Ile was reported discovery for the first time in 1985, was ground in subsequent In studying carefully, it is found that it, in addition to being present in plant, is also used as structural unit and is present in a large amount of cyclic peptidal antibiotics, such as derive from Aureobasidin A, cordyheptapeptides and the aspergillicin E of fungi, and from actinomyces Globomycin, cypemycin, desotamides and marformycins (structure such as Fig. 2).It is interesting that L-allo-Ile It is also found to be present in human plasma, in the blood plasma of healthy population, the concentration of L-allo-Ile is very low, in almost can be with Detected concentration level；However, with autosomal recessive hereditary diseases --- maple syrup urine disease (maple syrup urine Disease in patient's blood plasma), L-allo-Ile but because patient metabolic deficiency and accumulated, concentration reach 5 μM with On, therefore, concentration levels of the L-allo-Ile in blood plasma has been used as one of the important means of diagnosis maple syrup urine disease.

The structure of L-allo-Ile and gal4 amino acid L-Ile are closely similar, and the difference of L-allo-Ile and L-Ile exist The conformation of methyl on β carbon atoms is different.Although L-allo-Ile is closely similar with L-Ile in structure, and certainly Be widely present in right boundary, but so far, life entity be how this nonprotein amino acid of biosynthesis L-allo-Ile, with And enzyme and enzyme reaction mechanism or a unsolved mystery in this process involved in.

Desotamides and marformycins is from respectively from the streptomycete Streptomyces in South Sea deep-sea source What scopuliridis SCSIO ZJ46 and Streptomyces drozdowiczii SCSIO 10141 were isolated and purified Two class cyclic peptide antibiotic, studies have shown that desotamides has preferable inhibitory activity to gram-positive bacterium, and Marformycins has good inhibiting effect to propionibacterium acnes (Propionibacterium acnes), is preferable The lead compound for Retinoids, Retin-A, Renova, Accutane.What is more important in this two classes cyclic peptide compounds structure, contains non-egg White matter amino acid building blocks L-allo-Ile.Currently, the biological synthesis gene cluster of desotamides and margormycins oneself Through being cloned, it is important that the above result of study for us in zymetology level explains that the biosynthesis mechanism of L-allo-Ile has been established Basis.The explanation of L-allo-Ile biosynthesis zymetology mechanism will prepare L-allo-Ile for the Enzymology method of utilization green, And there is important Practical significance to the diagnosing and treating of maple syrup urine disease.

Invention content：

The object of the present invention is to provide aminopherases and the enzyme of isomerase formation to forming L- in catalysis l-Isoleucine Alloisoleucine or catalysis L- alloisoleucines form the application in l-Isoleucine.

The enzyme that the aminopherase and isomerase of the present invention is formed in catalysis l-Isoleucine to forming L- alloisoleucines Or catalysis L- alloisoleucines formed l-Isoleucine in application, the aminopherase be aminopherase DsaD or Aminopherase MfnO, the isomerase are isomerase DsaE or isomerase MfnH, the ammonia of the aminopherase DsaD For base acid sequence as shown in SEQ ID NO.7, the amino acid sequence of the isomerase DsaE is described as shown in SEQ ID NO.8 Aminopherase MfnO amino acid sequence as shown in SEQ ID NO.5, the amino acid sequence of the isomerase MfnH is such as Shown in SEQ ID NO.6.

It is preferred that the nucleotide sequence such as SEQ ID NO.1 of the encoding gene mfnO genes of the aminopherase MfnO It is shown.

It is preferred that the nucleotide sequence of the encoding gene mfnH genes of the isomerase MfnH is as shown in SEQ ID NO.2.

It is preferred that the nucleotide sequence such as SEQ ID NO.3 of the encoding gene dsaD genes of the aminopherase DsaD It is shown.

It is preferred that the nucleotide sequence of the encoding gene dsaE genes of the isomerase DsaE is as shown in SEQ ID NO.4.

The invention mainly relates to the contents of three aspects：When using bioinformatic analysis method respectively from The amino for participating in L-allo-Ile biosynthesis is identified in the biological synthesis gene cluster of desotamides and marformycins Transferase/isomerase-DsaD/DsaE and MfnO/MfnH；Second is that by the method for internal gene knockout, to aminopherase/ Isomerase-DsaD/DsaE and MfnO/MfnH carries out internal deletion mutation, obtains chemical combination of the production containing L-Val structural units Superior strain △ mfnH and the Streptomyces coelicolorM1152/07-6H-DKO of object 7,9 and 11 (Fig. 2), Streptomyces coelicolor M1152/07-6H-EKO；Third, the present invention relates to the use of the aminopherase of identification/different Structure enzyme-DsaD/DsaE and MfnO/MfnH is converted into the application in L-allo-Ile in catalysis L-Ile, and feature is, L- Two enzymes synergistic effect that the generation of allo-Ile needs, individual aminopherase either isomerase cannot be catalyzed L- The generation of allo-Ile, and this catalytic process need not add any co-factor.

The present invention compares L-allo-Ile and the difference in L-Ile structures by observation, and the difference of discovery is former in β carbon The conformation of methyl on son is different, wherein the carbon atom of the positions β of L-Ile is 3S types, and the carbon atom of the positions the β of L-allo-Ile For 3R types, therefore the present inventor speculates that L-allo-Ile may be converted by L-Ile and is formed that this conversion process can be aminopherase Transformation between the two is completed with two enzyme molecule cooperations of isomerase.It is L-Ile first under the action of aminopherase, sloughs Amino becomes carbonyl and fixes α carbon atoms planar, prevents it from rotating freely.Secondly complete under the action of isomerase At the overturning of methyl on β carbon atoms.The present invention is in desotamides and marformycins biological synthesis gene clusters points The DsaD and MfnO for not being noted as aminopherase have carried out bioinformatic analysis, and Multiple Sequence Alignment is shown, DsaD/MfnO With it has been reported that branched-chain amino acid transferase (branched-chain aminotransferase, BCATs) have it is very high Sequence homology, and there is signature motif " EXGXXNLFXnLXTXnLXGVXR " identical with IV- type aminotransferases, And with the catalysis lysine residue (Lysine) (such as Fig. 3) being covalently attached to PLP, prompt DsaD/MfnO have PLP- according to Bad aminotransferase activity.The present invention is also right respectively using the online resource HHpred for carrying out structural homology analysis to albumen The DsaE and MfnH that isomerase is noted as in the biological synthesis gene cluster of desotamides and marformycins are finished Structure is analyzed, and shows that it has with the albumen for belonging to 2 superfamily of the nuclear translocation factor (nuclear transport factor 2, NTF2) There is similar secondary structure folding mode, 2 superfamily of the nuclear translocation factor is containing there are many have different function and mutual amino acid The very low albumen of sequence similarity, including the △ that has reported⁵- 3- ketosteroid isomerases (delta⁵-3-ketosteroid Isomerse), △ can be catalyzed⁵- 3- ketone sterol isomerization generates △⁴- 3- ketosteroids, the present inventor speculate The MfnH in DsaE and marformycins gene clusters in desotamides gene clusters may be participated on the β carbon atoms of L-Ile Methyl carry out isomerization generate L-allo-Ile.In the biological synthesis gene cluster of marformycins and desotamides Aminopherase/the isomerase enzyme that may participate in L-allo-Ile synthesis is identified respectively to (such as Fig. 4), prompts L-allo-Ile Biosynthesis mechanism has conservative.

The present invention relates in marformycins wild type producing strains Streptomyces drozdowiczii SCSIO Knockout mutations (such as Figures 5 and 6) is carried out to the gene of aminopherase/isomerase MfnO/MfnH in 10141, generation is constructed and contains There are the superior strain △ mfnH of the compound 7 of L-Val structural units.Fermentation by HPLC to mutant strain △ mfnO and △ mfnH Product is analyzed, it is found that △ mfnH do not produce the compound 3 and 4 containing L-allo-Ile structural units completely, but generate and contain There is the compound 5 and 7 of L-Val structural units, and the yield of compound 7 improves about 100 times or so (such as compared with wild-type strain Fig. 7)；△ mfnO do not produce the compound 4 (such as Fig. 7) containing L-allo-Ile structural units completely yet, but remain to generate and contain The compound 5 and 7 of L-Val structural units.These data confirm thats MfnO/MfnH playing during synthesizing L-allo-Ile Essential effect causes precursor L-allo-Ile that cannot synthesize due to the deletion mutation of MfnO/MfnH, therefore, with L- There is allo-Ile the L-Val of similar structure can be integrated with the uncompetitive advantage of milli during synthesizing marformycin Into marformycins peptide backbones, to obtain the superior strain △ for generating the compound 7 containing L-Val structural units mfnH。

Therefore, second object of the present invention is to provide a kind of bacterial strain △ mfnH of high yield compound 7, which is characterized in that The bacterial strain △ mfnH are to carry out the mfnH genes of wild type Streptomyces drozdowiczii SCSIO 10141 It knocks out deletion mutation and obtains；

The structural formula of the compound 7 is as shown in Equation 1, wherein R₁=H, R₂=CH₃, R₃=OH；

The invention further relates to aminopherase/isomerase in desotamides gene clusters --- the base of DsaD/DsaE Because carrying out with frame knockout mutations (in-frame deletion), then in heterologous host Steptomyces coelicolor (such as Fig. 8 and 9) is expressed in M1152, constructs the superior strain for the compound 9 and 10 for generating the structural unit containing L-Val Streptomyces coelicolor M1152/07-6H-EKO and Streptomyces coelicolor M1152/07-6H- DKO.By HPLC to Streptomyces coelicolor M1152/07-6H-EKO and Streptomyces The tunning of coelicolor M1152/07-6H-DKO is analyzed, and finds dasE genes by same frame deletion mutation Streptomyces coelicolor M1152/07-6H-EKO do not produce the chemical combination containing L-allo-Ile structural units completely Object 8 and 10, but remain to produce the compound 9 and 11 containing L-Val structural units, and the yield of the two compounds with compare bacterium Strain is compared and is greatly improved (about 100 times of compound 9, about 140 times or so of compound 11) (such as Figure 10).DsaD genes are same Although the heterogenous expression bacterial strain Streptomyces coelicolorM1152/07-6H-DKO of frame deletion mutation remain to generate contains There is the compound 8 and 10 of L-allo-Ile structural units, but its yield substantially reduces, remain to generate contains L-Val structures The compound 9 and 11 of unit, and yield but greatly improves (about 80 times of compound 9；About 50 times of compound 11) (such as Figure 10).These Data similarly illustrate that DsaD/DsaE playing the role of during synthesizing L-allo-Ile is essential.

Therefore, third object of the present invention is to provide a kind of bacterial strain Streptomyces of high yield compound 9 and 11 Coelicolor M1152/07-6H-EKO or Streptomyces coelicolor M1152/07-6H-DKO, feature exist In the bacterial strain Streptomyces coelicolorM1152/07-6H-EKO are by DsaE genes with frame deletion mutation The biological synthesis gene cluster of desotamides is imported in bacterial strain Streptomyces coelicolor M1152 and is expressed And obtain, the Streptomyces coelicolorM1152/07-6H-DKO are by DsaD genes with frame deletion mutation The biological synthesis gene cluster of desotamides import in bacterial strain Streptomyces coelicolorM1152 and expressed And obtain；

The structure of the compound 9 and 11 is as shown in Equation 2, wherein compound 9：R₁=H, R₂=NH₂；Compound 11：R₁ =H, R₂=OH；

The invention further relates in E. coli (DE3) to aminopherase/isomerase-DsaD/DsaE and MfnO/MfnH is expressed, is purified (such as Figure 11), and the enzyme of acquisition under conditions of not adding any co-factor to can be catalyzed Substrate L-Ile converts to form L-allo-Ile.In the 50mM phosphate buffers of pH 8.0, any co-factor is not being added Under the conditions of, DsaD/DsaE or MfnO/MfnH cooperation catalysis substrate L-Ile are converted into L-allo-Ile, and conversion ratio reaches about 67% (such as Figure 12), but individually aminopherase DsaD/MfnO or individual isomerase DsaE/MfnH cannot be catalyzed L- Ile is converted into L-allo-Ile.DsaD/DsaE or MfnO/MfnH cooperation catalysis L-Ile is converted into L-allo-Ile categories In reversible reaction, when using L-allo-Ile as substrate, under the above same reaction conditions, product L-Ile can be obtained (as schemed 14).When using L-Ile as substrate, the equilibrium constant of the reversible reaction of DsaD/DsaE catalysis is 1.37 (such as Figure 15). Functionally mutual may be implemented in aminopherase and isomerase in Desotamides and marformycins biosynthesis pathways It mends, DsaD/MfnH and MfnO/DsaE can cooperate mutually converting (such as Figure 16) between catalysis L-Ile and L-allo-Ile.

The invention discloses the enzymes being made of aminopherase and isomerase to forming L- alloisoleucines (L- in catalysis Allo-Ile the application) or in l-Isoleucine.To explain that the biosynthesis mechanism of L-allo-Ile is established in zymetology level Important basis is determined.The explanation of L-allo-Ile biosynthesis zymetology mechanism, by the Enzymology method preparation L- to utilize green Allo-Ile, and there is important Practical significance to the diagnosing and treating of maple syrup urine disease.

The streptomycete Streptomyces scopuliridis SCSIO ZJ46 of the present invention are disclosed in document： Yongxiang Song,Qinglian Li,Xue Liu,Yuchan Chen,Yun Zhang,Aijun Sun,Weimin Zhang, Jingren Zhang, and Jianhua Ju, Cyclic Hexapeptides from the Deep South China Sea-Derived Streptomyces scopuliridis SCSIO ZJ46 Active Against Pathogenic Gram-Positive Bacteria.J.Nat.Prod.,2014,77(8),pp 1937–1941.The bacterial strain The applicant also holds, and ensures to provide to the public from 20 years.

10141 bacterial strains of Streptomyces drozdowiczii SCSIO of the present invention are disclosed in Xiao Zhou, Hongbo Huang,Jie Lia,Yongxiang Song,Renwang Jiang,Jing Liu,Si Zhang,Yan Hua, Jianhua Ju.New anti-infective cycloheptadepsipeptide congeners and absolute stereochemistry from the deep sea-derived Streptomyces drozdowiczii SCSIO 10141.Tetrahedron.Volume 70,Issue 42,21 October 2014,Pages 7795–7801.The bacterial strain sheet Applicant also holds, and ensures to provide to the public from 20 years.

Streptomyces coelicolor (Streptomyces coelicolor) M1152 of the present invention is disclosed in Master's thesis：Summer China seas are studied in Drimentines class compound biosynthesis in the Streptomyces sp.OUC6819 of beautiful mangrove source Foreign university .2013.Bacterial strain the applicant also holds, and ensures to provide to the public from 20 years.

Description of the drawings：

Fig. 1 is l-Isoleucine (L-Ile), D-Ile (D-Ile), L- alloisoleucines (L-allo-Ile) and D- The chemical structural formula of alloisoleucine (D-allo-Ile).

Fig. 2 is marformycins and desotamides chemical structural formulas, wherein 1,2,3,4,5,6,7,8,9,10,11 Compound 1,2,3,4,5,6,7,8,9,10,11 is indicated respectively.

Fig. 3 be aminopherase DsaD/MfnO with it has been reported that branched-chain amino acid transferase Multiple sequence alignments.Arrow Head is signified to be participated in be conservative with the covalently bound lysine catalysis of PLP, the feature base of the referred to IV- aminopherases of box Sequence " EXGXXNLFXnLXTXnLXGVXR ".

Fig. 4 be maformycins and desotamides biological synthesis gene cluster in aminopherase and isomerase institute In position.Aminopherase DsaD/MfnO is marked with red；Isomerase DsaE/MfnH is marked with green.

Fig. 5 is to utilize PCR-targeting technologies in marformtcins producing strains to aminotransferase gene mfnO Carry out deletion mutation.(A) mutation process schematic diagram；(B) PCR identifications, W are carried out to mutant strain △ mfnO：With wild-type strain The genomic DNA of Streptomyces drozdowiczii SCSIO 10141 is template；M：With mutant strain △ mfnO genomes DNA is template, maker:NDA molecular weight standards.

Fig. 6 is to utilize PCR-targeting technologies in marformtcins producing strains to aminotransferase gene mfnH Carry out deletion mutation.(A) mutation process schematic diagram；(B) PCR identifications, W are carried out to mutant strain △ mfnH：With wild-type strain The genomic DNA of Streptomyces drozdowiczii SCSIO 10141 is template；M：With mutant strain △ mfnH genomes DNA is template, maker:NDA molecular weight standards.

Fig. 7 is the HPLC analyses of mutant strain △ mfnO and △ mfnH tunnings.

Fig. 8 is the same frame deletion mutation of dsaD genes and in heterologous host Streptomyces coelicolor Expression in M1152.(A) schematic diagram；(B) the PCR identifications of deletion mutation, WT：With control strain Streptomyces The genomic DNA of coelicolor M1152 is template；DKO：With heterogenous expression bacterial strain Streptomyces coelicolor M1152/07-6H-DKO genomic DNAs are template, M:DNA molecular amount standard items.

Fig. 9 is the same frame deletion mutation of dsaE genes and in heterologous host Streptomyces coelicolor Expression in M1152.(A) schematic diagram；(B) the PCR identifications of deletion mutation, WT：With control strain Streptomyces The genomic DNA of coelicolor M1152 is template；EKO：With heterogenous expression bacterial strain Streptomyces CoelicolorM1152/07-6H-EKO genomic DNAs are template, M:DNA molecular amount standard items.

Figure 10 is dsaD and dsaE genes by the heterogenous expression bacterial strain Streptomyces of same frame deletion mutation The tunning of coelicolor M1152/07-6H-DKO and Streptomyces coelicolor M1152/07-6H-EKO HPLC analysis.i：Control strain Streptomyces coelicolorM1152；ii：Biology containing desotamides closes At the heterogenous expression bacterial strain Streptomyces coelicolor M1152/07-6H of gene cluster；iii：DsaD is by same frame The heterogenous expression bacterial strain Streptomyces coelicolor M1152/07-6H-DKO of deletion mutation；iv：DsaE is same The heterogenous expression bacterial strain Streptomyces coelicolorM1152/07-6H-EKO of frame deletion mutation.

Figure 11 is that DsaD/DsaE and MfnO/MfnH are expressed in E. coli (DE3), utilized after purification SDS-PAGE is analyzed.

Figure 12 is that the recombination DsaD/DsaE and MfnO/MfnH of purifying are catalyzed L-Ile and are converted into L-allo- in vitro Ile。

Figure 13 is by preparing enzymatic preparation L-allo-Ile using DsaD/DsaE¹H NMR spectras.A：Enzymatic produces Object L-allo-Ile's¹H NMR spectras；B：L-allo-Ile standard items¹H NMR spectras

Figure 14 is that the recombination DsaD/DsaE and MfnO/MfnH of purifying are catalyzed L-allo-Ile and are converted into L- in vitro Ile。

Figure 15 is the measurement of the equilibrium constant of the reversible reaction of DsaD/DsaE catalysis.But when using L-Ile as substrate, balance Constant Keq can be calculated Keq=([l-allo-Ile]/[l-Ile])=(2.89/2.11)=1.37 according to formula.

Figure 16 is derived from aminopherase/isomerase enzyme of different biosynthesis pathways to DsaD/MfnH and MfnO/ DsaE can cooperate catalysis L-Ile and L-allo-Ile between mutually convert.

Specific implementation mode：

The following examples are further illustrations of the invention, rather than limiting the invention.

Embodiment 1

MfnO genes (its nucleotide sequence as shown in SEQ ID NO.1, the amino acid of the aminopherase MfnO of coding Sequence is as shown in SEQ ID NO.5) and mfnH genes (its nucleotide sequence as shown in SEQ ID NO.2, the isomerase of coding The amino acid sequence of MfnH is as shown in SEQ ID NO.6) in wild type producing strains Streptomyces drozdowiczii Deletion mutation in SCSIO10141

External knockout mutant strain is obtained using the method for PCR-targeting.According to the life of the marformycins of acquisition Object synthetic gene cluster sequence, the PCR-targeting systems of reference literature report, designs the knockout of a pair mfnO and mfnH genes Primer, primer sequence see mfnO and mfnH knockout primers in table 1.Referring next to the method construct of PCR-targeting Then the outer plasmid that knocks out is transferred in the donor bacterium of engagement transfer.It is as follows：(1) by the life containing marformycins Cosmid plasmids (the nucleotide of the biological synthesis gene cluster of plasmid cosmid 247E, marformycins of object synthetic gene cluster The GenBank accession number of sequence is：KP715145.1 it) is transferred in E. coli BW25113/pIJ790 and is contained The E.coli BW25113/pIJ790 bacterial strains of purpose plasmid are expressed with L-arabinose induction λ/red recombination systems of 10mM, And it is prepared into turning competent cell for electricity for use.(2) restriction endonuclease EcoR I and Hind III digestion plasmid pIJ778 are used, are returned The DNA fragmentation that wherein about 1.4kb contains transfer origin (oriT) and spectinomycin (spectinomycin) resistant gene is received, with This is amplified as pcr template, with primer mfnOdelF/mfnOdelR and mfnHdelF/mfnHdelR by PCR respectively The PCR product of 1.4kb, the PCR reaction systems of 50 μ L：5 μ L of high-fidelity DNA polymerase 3U, 10 × Buffer, 2.5 μ L of dNTPs0.5mmol/L, DMSO, primer each 0.5 μm of ol/L, DNA profiling about 1ng, add water to mend to 50 μ L.PCR reacts item Part is：94 DEG C of 5min of pre-degeneration；Amplification cycles are 94 DEG C of denaturation 45s, and 58 DEG C of annealing 45s, 72 DEG C of extension 90s, 30 recycle；Most 72 DEG C of extension 10min afterwards.It is for use that purifying is separately recovered in the PCR product of 1.4kb.(3) PCR product electricity is transferred to (1) step respectively Recombination is brought it about in the competent cell E.coli BW25113/pIJ790 prepared in rapid, LB screening flat boards is coated on and (contains 100 μ g/mL ampicillins, 50 μ g/mL kanamycins, 50 μ g/mL spectinomycins) on, 37 DEG C are incubated overnight.It is chosen from tablet Go out positive monoclonal, extract plasmid, recombinant plasmid is named as delmfnO and delmfnH, the mfnO in delmfnO and delmfnH It is transferred origin and spectinomycin resistance gene substitution respectively with the Partial Fragment of mfnO genes.(4) recombination built is dashed forward Become plasmid delmfnO and delmfnH to be transformed into respectively in E.coli ET12567/pUZ8002, obtains recombinant bacterial strain E.coli ET12567/pUZ8002/delmfnO and E.coli ET12567/pUZ8002/delmfnH, the donor bacterium as engagement transfer.

10141 bacterial strains of wild type streptomycete Streptomyces drozdowiczii SCSIO are in ISP2 culture medium (wheats Bud extract 4g, yeast extract 4g, glucose 4g, sea salt 30g, agar powder 20g add water to 1L, pH 7.2) it crosses in tablet Culture 3-5 days, in the TSB culture mediums that the spore grown is collected in aseptic cotton carrier, vortex oscillation makes spore disperse.Filtering point From mycelium and spore, spore suspension is in the TSB culture mediums of 5mL, 50 DEG C of heat shock 10min, and it is small then to sprout 2-4 in 28 DEG C When, the recipient bacterium as engagement transfer.As donor bacterium E.coli ET12567/pUZ8002/delmfnO and E.coli ET12567/pUZ8002/delmfnH contains 50 μ g/mL kanamycins in 50mL respectively, and 25 μ g/mL chloramphenicol and 50 μ g/mL are grand In the LB liquid medium of mycin when growing to OD600 values and being about 0.6 for 37 DEG C, thalline were collected by centrifugation (4000rpm, 10min), Thalline is cleaned with LB 3 times, is suspended in 300 μ L LB culture mediums, the donor bacterium as engagement transfer.Take 400 μ L of above-mentioned recipient bacterium It is uniformly mixed with 100 μ L of donor bacterium, is coated on M-ISP4 solid mediums (soluble starch 10g, ferment without any antibiotic Female extract 0.5g, peptone 1g, NaCl 1g, MgSO₄·7H₂O 1g, (NH₄)₂SO₄2g, K₂HPO₄1g, CaCO₃2g, sea Salt 30g, 100 μ L of trace element, adds water to 1L, pH 7.2) on, after drying, 18-20h is cultivated in 28 DEG C.Then tablet is taken Go out, tablet, final concentration of 100 μ g/mL spectinomycins and 50 μ g/mL methoxybenzyl aminopyrimidines covered with the water containing antibiotic, It after drying, is placed in 28 DEG C of incubators, culture is observed after 3-4 days.

After growing petite on engagement transfer tablet, it is transferred to containing 100 μ g/mL spectinomycins with sterile toothpick On the ISP2 tablets of 50 μ g/mL methoxybenzyl aminopyrimidines, after 28 DEG C are cultivated 2-3 days, the genomic DNA of each mutant strain is extracted, It is detected by PCR using detection primer (primer sequence sees the detection primer sequence of mfnO and mfnH in table 2) and obtains the positive Clone obtains aminopherase mfnO gene knockout double crossing over mutant strain △ mfnO and isomerase mfnH gene knockout double crosses Change mutant strain △ mfnH.

Table 1：Build the knockout Primer and sequence needed for mfnO and mfnH gene mutation strains

Table 2：Build the detection primer title and sequence needed for mfnO and mfnH gene mutation strains

Embodiment 2

DsaD genes (its nucleotide sequence as shown in SEQ ID NO.3, the amino acid of the aminopherase DsaD of coding Sequence is as shown in SEQ ID NO.7) and dsaE genes (its nucleotide sequence as shown in SEQ ID NO.4, the isomerase of coding The amino acid sequence of DsaE is as shown in SEQ ID NO.8) same frame deletion mutation and in heterologous host Streptomyces Expression in coelicolor M1152

It is to carry out dsaD and dsaE genes with frame deletion mutation first.Specific operation process is as follows：(1) reference literature report The PCR-targeting systems in road, first by restriction endonuclease EcoR I and Hind III digestion plasmid pIJ773, recycling is wherein about 1.4kb contains the DNA fragmentation of transfer origin (oriT) and apramycin (apramycin) resistant gene, is struck as PCR amplification Except the DNA fragmentation needed for dsaD and dsaE genes.(2) it according to the sequence of dsaD and dsaE genes, separately designs a pair of knock out and draws Object, this primer feature are have 39 nucleotide and target deletion mutation DNA homolog (being shown in Table 3, shown in capitalization), there is 19 Either 20 nucleotide and resistance gene fragment left end or right end are homologous (being shown in Table 3, shown in lowercase), in addition, at 39 The sites restriction endonuclease SpeI (being shown in Table the sites 3, SpeI to be marked with underscore) are added between nucleotide and 19/20 nucleotide.Profit With this primer, the DNA of transfer origin (oriT) and apramycin (apramycin) resistant gene is contained with the 1.4kb of recycling Segment carries out the PCR product that PCR amplification obtains 1.4kb, the PCR reaction systems of 50 μ L as template：High-fidelity DNA polymerase 5 2.5 μ L of μ L, dNTPs 0.5mmol/L, DMSO of 3U, 10 × Buffer, primer each 0.5 μm of ol/L, DNA profiling about 1ng add Water is mended to 50 μ L.PCR reaction conditions are：94 DEG C of 5min of pre-degeneration；Amplification cycles are 94 DEG C of denaturation 45s, 58 DEG C of 45s that anneal, 72 DEG C extend 90s, 30 cycle；Last 72 DEG C of extensions 10min.It is for use that purifying is separately recovered in the PCR product of 1.4kb.(3) it connects down Select the clay 07-6H of SuperCos1 plasmid origins as the starting clay carried out to dsaD and dsaE with frame deletion mutation, This clay contains the biological synthesis gene cluster (nucleotide sequence of the biological synthesis gene cluster of desotamides of desotamides GenBank accession number be：KP769807.1).Clay 07-6H is transferred in E. coli BW25113/pIJ790 E.coli BW25113/pIJ790/07-6H are obtained, are expressed with L-arabinose induction λ/red recombination systems of 10mM, and will It prepares that as electricity to turn competent cell for use.(4) the 1.4kb PCR product electricity obtained in (2) step is transferred to (3) step respectively Recombination is brought it about in the competent cell E.coli BW25113/pIJ790/07-6H prepared in rapid, it is flat to be coated on LB screenings On plate (containing 100 μ g/mL ampicillins, 50 μ g/mL kanamycins, 50 μ g/mL apramycins), 37 DEG C are incubated overnight.From Choose positive monoclonal on tablet, extract plasmid, recombinant cosmid is named as 07-6H-DKO and 07-6H-EKO, 07-6H-DKO and The Partial Fragment of dsaD and dsaE genes in 07-6H-EKO is transferred origin and the substitution of apramycin resistant gene respectively. (5) digestion is carried out to recombinant cosmid 07-6H-DKO and 07-6H-EKO using SpeI, through phenol：Chloroform after ethanol precipitation, is used T4 ligases are attached, and transformed competence colibacillus cell E.coli DH5 are coated on containing 100 μ g/mL ampicillins, 50 μ g/ On the LB tablets of mL kanamycins, it is incubated overnight in 37 DEG C.PCR identifications, picking are carried out to clone using detection primer (being shown in Table 4) The clay lost transfer origin and apramycin resistance gene DNA segment and connected certainly, is named as 07-6H-DKO-IF and 07- 6H-EKO-IF。

Followed by by dsaD and dsaE genes by the clay 07-6H-DKO-IF and 07-6H- of same frame deletion mutation EKO-IF imported into heterologous host Streptomyces coelicolor M1152 and is expressed.It is importing Before Streptomyces coelicolor M1152, first have to carry out clay 07-6H-DKO-IF and 07-6H-EKO-IF Transformation, to be appropriate for heterogenous expression.The strategy of transformation is λ/red recombination systems using Escherichia coli by clay 07-6H- The kalamycin resistance gene from plasmid SuperCos1 on DKO-IF and 07-6H-EKO-IF is respectively with containing A Pula Mycin resistant gene aac (3) IV, engagement transfer origin original paper oriT, the NDA pieces of integrase gene and int ψ C31 integration sites Section is replaced.The DNA fragmentation of this aac (3) IV-oriT-int ψ C3 derives from the plasmid pSET152AB of this laboratory structure, utilizes After BamH I/EcoR I complete degestion plasmids pSET152AB, recycling 5.5kb or so segment, this segment contain aac (3) IV- OriT-int ψ C3 DNA fragmentations and the 1.0kb DNA fragmentation homologous with the kanamycin gene both sides that are replaced).By 07- 6H-DKO-IF and 07-6H-EKO-IF is transferred in E.coli BW25113/pIJ790, obtains E.coli BW25113/ PIJ790/07-6H-DKO-IF and E.coli BW25113/pIJ790/07-6H-EKO-IF.The 5.5kb that about 100mg is recycled DNA fragmentation is added separately to E.coli BW25113/pIJ790/07-6H-DKO-IF and E.coli BW25113/pIJ790/ In 07-6H-EKO-IF competent cells, it is transferred in electric shock cup, 1.4kv voltages carry out electrotransformation.It is rapidly added after the completion of electric shock The LB culture mediums of the 0.5mL of precooling, 37 DEG C of recoveries be coated on after 1 hour containing 100 μ g/mL ampicillins and 50 μ g/mL Ah The LB tablets of platenomycin.After son to be transformed is grown after 12 hours, positive weight is verified using PCR by detection primer (table 4) Group plasmid, positive recombinant plasmid are named as 07-6H-DKO-AB and 07-6H-EKO-AB.The recombinant plasmid electricity built is gone to In E.coli ET12567/pUZ8002, E.coli ET12567/pUZ8002/07-6H-DKO-AB and E.coli are obtained Donor bacterium of the ET12567/pUZ8002/07-6H-EKO-AB as engagement transfer.

Connect down is by E.coli ET12567/pUZ8002/07-6H-DKO-AB and E.coli ET12567/pUZ8002/ 07-6H-EKO-AB and Streptomyces coelicolor M1152 carry out engagement transfer.By bacterial strain E.coli ET12567/ The LB liquid medium that pUZ8002/07-6H-DKO-AB and 07-6H-EKO-AB is inoculated in 3mL respectively (contains 100 μ g/ ML ampicillins, 50 μ g/mL apramycins, 25 μ g/mL chloramphenicol and 50 μ g/mL kanamycins) in, 37 DEG C of cultures 12 are small Shi Hou, it is 0.6 to take the switching of 40 μ L bacterium solutions to be cultivated in 4mL same mediums to OD, and thalline were collected by centrifugation, with without any antibiosis The LB liquid medium of element is washed 2 times, washes away antibiotic, centrifugal concentrating thalline is spare.At the same time, 10% glycerine is collected S.coelicolor M1152 spores, after being filtered, 3600rpm centrifuges 8min, abandons supernatant, appropriate LB culture mediums are added Suspension spore is placed in heat shock 10 minutes in 50 DEG C of water-baths.It will conversion bacterial strain E.coli ET12567/pUZ8002/07-6H-DKO- AB and 07-6H-EKO-AB is respectively with S.coelicolorM1152 spores according to volume ratio 2:1 ratio mixes, and is coated on MS+ MgCl₂On (final concentration of 10mM) solid plate.After 20~24 hours, tablet is taken out, is covered with the water containing antibiotic flat Plate, final concentration of 100 μ g/mL apramycins and 50 μ g/mL methoxybenzyl aminopyrimidines, after drying, are placed in 28 DEG C of incubators, Culture is observed after 3-4 days.After growing petite on engagement transfer tablet, it is transferred to containing 100 μ g/mL with sterile toothpick (soy meal 20g, mannitol 20g, agar powder 20g add water to the MS culture mediums of apramycin and 50 μ g/mL methoxybenzyl aminopyrimidines To 1L, pH 7.2) on tablet, after 28 DEG C of cultures 2-3 days, the genomic DNA of each mutant strain is extracted, (is drawn using detection primer Object sequence sees table 4) acquisition positive colony is detected by PCR, it is it is prominent by same frame missing that dsaD and dsaE genes are obtained respectively The desotamides biological synthesis gene cluster heterogenous expression bacterial strain Streptomyces coelicolor M1152/07-6H- of change DKO and Streptomyces coelicolor M1152/07-6H-EKO.

Table 3：Build the knockout Primer and sequence needed for dsaD and dsaE gene mutation strains

Table 4：Build the detection primer title and sequence needed for dsaD and dsaE gene mutation strains

Embodiment 3

The biofermentation of marformycins and detection：

By 10141 wild mushrooms of marine streptomyces Streptomyces drozdowiczii SCSIO or mutant strain △ mfnO In ISP2 culture mediums, (malt extract 4g, yeast extract 4g, glucose 4g, sea salt 30g, agar powder 20g add with △ mfnH Water is to 1L, pH 7.2) on tablet after activation production spore, the spore of equivalent is inoculated into the 50mL m- of 250mL triangular flasks respectively AM2ab fermentation mediums (soy meal 10g, starch 5g, peptone 2g, glucose 20g, yeast extract 2g, K₂HPO₄0.5g, MgSO₄·7H₂O 0.5g, calcium carbonate 2g, sea salt 30g add water to 1L, pH 7.0) in, bacterium hair is shaken in 28 DEG C, under the conditions of 200rpm Ferment.The butanone that 100mL is added after cultivating the 7th day is extracted, and ultrasonic 30min smudge cells are then allowed to stand layering.Wait for that butanone extracts After taking liquid separated from the water, draws supernatant extract liquor and be evaporated butanone with Rotary Evaporators, residue is dissolved in methanol and forms sample Product carry out high performance liquid chromatography (HPLC) and detect.

Testing conditions are：Alltima C18 (250 × 4.6mm, 5 μm) reversed-phase column, mobile phase A are mutually 15% acetonitrile, are contained 0.03% acetic acid, Mobile phase B are mutually 85% acetonitrile；Flow velocity is 1mL/min, and Detection wavelength is 215nm and 275nm.

HPLC injection procedures：0-20min, 0%-100%B phase；20-25min, 100%B phase；25.01-30min, 100%-0%B phases.

The results are shown in Figure 7,10141 generationizations of wild type producing strains Streptomyces drozdowiczii SCSIO Object 3,4 and 5 is closed, △ mfnH do not produce the compound 3 and 4 containing L-allo-Ile structural units completely, but generate production and contain L- The compound 5 and 7 of Val structural units, and the yield of compound 7 improves about 100 times or so (as schemed compared with wild-type strain 7)；△ mfnO do not produce the compound 4 containing L-allo-Ile structural units completely yet, but still generate and contain L-Val structure lists The compound 5 and 7 (such as Fig. 7) of member.

Embodiment 4

The biofermentation of desotamides and detection：

Desotamides wild type producing strains Streptomyces scopuliridis SCSIO ZJ46 are coated on Activation production spore on ISP4 culture medium flat plates, and by heterogenous expression control strain Streptomyces coelicolor M1152, DsaD and dsaE deletion mutations heterogenous expression bacterial strain Streptomyces coelicolor M1152/07-6H-DKO and Streptomyces coelicolor M1152/07-6H-EKO are coated on MS culture mediums (soy meal 20g, mannitol 20g, fine jade Cosmetics 20g adds water to 1L, pH 7.2) activation production spore on tablet.The spore of equivalent is inoculated into 250mL triangular flasks respectively 50mL m-AM2ab fermentation mediums (soy meal 10g, starch 5g, peptone 2g, glucose 20g, yeast extract 2g, K₂HPO₄ 0.5g, MgSO₄·7H₂O 0.5g, calcium carbonate 2g, sea salt 30g add water to 1L, pH 7.0) in, in 28 DEG C, under the conditions of 200rpm Shake bacterium fermentation.The butanone that 100mL is added after cultivating the 7th day is extracted, and ultrasonic 30min smudge cells are then allowed to stand layering.It waits for After butanone extract liquor is separated from the water, draws upper layer butanone extract liquor and be evaporated butanone with Rotary Evaporators, residue is dissolved in Methanol forms sample, carries out high performance liquid chromatography (HPLC) and detects.

HPLC injection procedures：0-20min, 0%-100%B phase；20-25min, 100%B phase；25.01-30min, 100%-0%B phases；

The results are shown in Figure 10, and dasE genes are by the Streptomyces coelicolor of same frame deletion mutation M1152/07-6H-EKO does not produce the compound 8 and 10 containing L-allo-Ile structural units completely, but remains to production and contain L- The compound 9 and 11 of Val structural units, and the yield of the two compounds greatly improves (compound 9 compared with control strain About 100 times, about 140 times or so of compound 11) (such as Figure 10).DsaD genes are by the heterogenous expression bacterial strain of same frame deletion mutation Although Streptomyces coelicolor M1152/07-6H-DKO remain to generate the change containing L-allo-Ile structural units Object 8 and 10 is closed, but its yield substantially reduces；In addition, it remains to the compound 9 and 11 containing L-Val structural units generated, and Yield but greatly improves (about 80 times of compound 9；About 50 times of compound 11).

Embodiment 5

Aminopherase DsaD (its amino acid sequence is as shown in SEQ ID NO.7), isomerase DsaE (its amino acid sequence As shown in SEQ ID NO.8), aminopherase MfnO (its amino acid sequence is as shown in SEQ ID NO.5) and isomerase MfnH The expression and purifying of (shown in its amino acid sequence such as SEQ ID NO.6) in E.coli BL21 (DE3)：

By dsaD (its nucleotide sequence is as shown in SEQ ID NO.3), dsaE (its nucleotide sequence such as SEQ ID NO.4 It is shown), mfnO (its nucleotide sequence is as shown in SEQ ID NO.1), mfnH (its nucleotide sequence is as shown in SEQ ID NO.2) Gene is conventionally cloned between the sites NdeI and EcoRI of carrier pET28a (+) to obtain pET28a (+)/dsaD, Then pET28a (+)/dsaE, pET28a (+)/mfnO, pET28a (+)/mfnH are converted after being sequenced correctly to E.coli BL21 (DE3) is to express.Being linked into specification by 1% inoculum concentration after obtained conversion bacterial strain picking monoclonal is incubated overnight is 200mL LB culture liquid (each bacterial strain is inoculated with 1L LB culture mediums altogether) containing 50 μ g/mL kanamycins in the triangular flask of 1L, When 37 DEG C of shaking table 200rpm/min are cultivated and are about 0.6 to OD600, the isopropyl-of final concentration of 0.1mM is added into culture solution β-D- Thiogalactopyranosides (IPTG) continue induced expression 12-15h in 25 DEG C.Thalline were collected by centrifugation, uses After 50mLbinding buffer (50mM sodium phosphates, 500mM NaCl, 10mM imidazoles, pH 8.0) washing thalline 2 times, it is resuspended in In 30mL bindingbuffer, ultrasonic disruption is carried out to discharge albumen, then high speed freezing low-temperature centrifugation removes insoluble portion Point.Soluble upper fraction is loaded on nickel column HisTrap HT column (1mL, GE Healthcare), waits for that filtrate is complete After portion has been filtered, rinsed with 10mL wash buffer 1 (50mM sodium phosphates, 500mM NaCl, 50mM imidazoles, pH 8.0), then It is rinsed with 2mL wash buffer 2 (50mM sodium phosphates, 500mM NaCl, 90mM imidazoles, pH 8.0), then uses 5mL Elution buffer (50mM sodium phosphates, 500mM NaCl, 250mM imidazoles, pH 8.0) are eluted.Use super filter tube (Millipore, 10mL, 3kD) is concentrated into 2.5mL, after PD-10 desalting columns (GE Healthcare) desalination, is stored in and contains The sodium phosphate l buffer solutions (50mM, pH 8.0) of 10% glycerine, determination of protein concentration is measured using Bradford methods, point Dress be stored in -80 DEG C it is spare, thus respectively obtain the aminopherase DsaD of purifying, isomerase DsaE, aminopherase MfnO With isomerase MfnH.Protein electrophoresis figure after purification is as shown in figure 11.

Embodiment 6

The external enzymatic reactions of DsaD/DsaE and MfnO/MfnH and detection：

(1) using L-Ile as substrate aminopherase DsaD/ isomerases DsaE and aminopherase MfnO/ isomeries are detected when The enzymatic activity of enzyme MfnH：In 100 μ L sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of amino are added Transferase DsaD either 5 μM of isomerases DsaE or MfnH of MfnO, react 4 hours under the conditions of being placed in 30 DEG C.

(2) using L-allo-Ile as substrate aminopherase DsaD/ isomerases DsaE and aminopherase MfnO/ are detected when The enzymatic activity of isomerase：In 100 μ L sodium phosphate buffers (50mM, pH 8.0), it is added the substrate L-allo-Ile of 1mM, 5 μM Aminopherase DsaD either 5 μM of isomerases DsaE or MfnH of MfnO, react 4 hours under the conditions of being placed in 30 DEG C.

After reaction, the methanol of 200 μ L is added to terminate reaction, be vortexed concussion, in being stored at room temperature after twenty minutes, 1, 2000 × g is centrifuged 20 minutes, takes supernatant to be evaporated with Rotary Evaporators, residue is dissolved in 40 μ L 2mM CuSO₄Solution takes 25 μ L Chiral HPLC is carried out, to detect enzymatic reaction situation.Chiral HPLC condition is：Use MCI GEL CRS10W Column (Mitsubishi, 50 × 4.6mm, 3 μm) chiral analysis column；Flowing is 2mM CuSO₄Solution；Flow velocity is 1mL/min, Detection time is 30 minutes, Detection wavelength 254nm.

Embodiment 7

One, it is catalyzed using L-Ile as substrate

(1) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added DsaD, 5 μM of isomerase DsaE react 4 hours under the conditions of being placed in 30 DEG C.After reaction, it is anti-to terminate that the methanol of 20mL is added It answers, be vortexed concussion, and in being stored at room temperature after twenty minutes, 1,2000 × g is centrifuged 20 minutes, takes supernatant to be evaporated, residue is dissolved in 2mM CuSO₄Solution, is purified using chiral HPLC, and condition is：Using MCI GEL CRS10W column (Mitsubishi, 50 × 4.6mm, 3 μm) chiral analysis column；Mobile phase is 2mM CuSO₄Solution；Flow velocity is 1mL/min, and detection time is 30 minutes, Detection wavelength is 254nm.After the fraction that residence time is 13 minutes is picked out merging, ethylenediamine tetra-acetic acid (ethylene is added Diamine tetraacetic acid, EDTA) to final concentration of 2mM, and the pH value of the above mixed liquor is adjusted to 4.0.With etc. The mixed liquor (7 of the normal heptane of volume and two-(2- ethylhexyls phosphonic acids):3) 4.0 solution of the above pH is stripped twice, is received Collection merges organic phase.The organic phase for collecting merging above is stripped twice with 5% isometric HCl solution, collects and merges water Phase.The aqueous of merging will be collected to be evaporated, residue, which is further purified with forward direction silicagel column, (utilizes n-butanol-glacial acetic acid-water (4:1:5) Gradient elution is carried out), pure enzymatic reaction product L-allo-Ile is obtained (in the V in Figure 12 A, Figure 12 A and 12B I and ii is respectively the standard items of L-Ile and L-allo-Ile).Its¹H NMR spectras are as shown in figure 13.

Enzymatic reaction product l-allo-Ile:HR-ESI-MS[M+H]⁺=132.1038 (calc.for C₆H₁₃NO₂, 132.1019)；¹H NMR(500MHz,D₂), O δ 0.86 (3H, t, J=7.5Hz), 0.83 (3H, d, J=7.0Hz), 1.19~ 1.40(2H,m),1.96(1H,m),3.62(1H,m).[α]_D ²⁵+23.4°(c 0.0575,aq HCl,pH 2.5).CD[θ]₂₀₂+ 165.7°(c 0.0575,aq.HCl,pH 2.5).

L-allo-Ile standard items:¹H NMR(500MHz,D₂), O δ 0.86 (3H, t, J=8.0Hz), 0.84 (3H, d, J= 7.0Hz), 1.20~1.40 (2H, m), 1.98 (1H, m), 3.64 (1H, m) [α]_D ²⁵+23.2°(c 0.1,aq HCl,pH 2.5).CD[θ]₂₀₂+333.3°(c 0.1,aq.HCl,pH 2.5).

(2) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added DsaD reacts 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), does not obtain enzymatic reaction production Object L-allo-Ile (iii in Figure 12 A).

(3) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of isomerases are added DsaE reacts 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), does not obtain enzymatic reaction production Object L-allo-Ile (iV in Figure 12 A).

(4) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM is added, is added without any enzyme, It is reacted 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), does not obtain enzymatic reaction product L- Allo-Ile (Vi in Figure 12 A).

(5) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added MfnO, 5 μM of isomerase MfnH react 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), is obtained Obtain pure enzymatic reaction product L-allo-Ile (V in Figure 12 B).

(6) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added MfnO reacts 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), does not obtain enzymatic reaction production Object L-allo-Ile (iii in Figure 12 B).

(7) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of isomerases are added MfnH reacts 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), does not obtain enzymatic reaction production Object L-allo-Ile (iV in Figure 12 B).

(8) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM is added, is added without any enzyme, It is reacted 4 hours under the conditions of being placed in 30 DEG C.Analysis and Structural Identification are carried out according to step (1), does not obtain enzymatic reaction product L- Allo-Ile (Vi in Figure 12 B).

Two, using L-allo-Ile as substrate

(1) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase DsaD, 5 μM of isomerase DsaE, react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-Ile (figures I and ii is respectively the standard items of L-Ile and L-allo-Ile in V in 14A, Figure 14 A and 14B).

(2) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase DsaD reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (iii in Figure 14 A).

(3) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of isomeries are added Enzyme DsaE reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (iV in Figure 14 A).

(4) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM is added, is added without and appoints What enzyme, reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (Vi in Figure 14 A).

(5) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase MfnO, 5 μM of isomerase MfnH, react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-Ile (figures V in 14B).

(6) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase MfnO reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (iii in Figure 14 B).

(7) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of isomeries are added Enzyme MfnH reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (iV in Figure 14 B).

(8) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM is added, is added without and appoints What enzyme, reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (Vi in Figure 14 B).

Three,

(1) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added DsaD, 5 μM of isomerase DsaE react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-allo-Ile (figures I and ii is respectively the standard items of L-Ile and L-allo-Ile in iii in 16A, Figure 16 A and 16B).

(2) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added DsaD, 5 μM of isomerase MfnH react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-allo-Ile (figures IV in 16A).

(3) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added MfnO, 5 μM of isomerase MfnH react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-allo-Ile (figures V in 16A).

(4) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM, 5 μM of aminopherases are added MfnO, 5 μM of isomerase DsaE react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-allo-Ile (figures Vi in 16A).

(5) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile of 1mM is added, is added without any enzyme, It is reacted 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-allo-Ile (Vii in Figure 16 A).

(6) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase DsaD, 5 μM of isomerase DsaE, react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-Ile (figures Iii in 16B).

(7) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase DsaD, 5 μM of isomerase MfnH, react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-Ile (figures IV in 16B).

(8) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase MfnO, 5 μM of isomerase MfnH, react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-Ile (figures V in 16B).

(9) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM, 5 μM of amino are added Transferase MfnO, 5 μM of isomerase DsaE, react 4 hours under the conditions of being placed in 30 DEG C, obtain pure enzymatic reaction product L-Ile (figures Vi in 16B).

(10) in 10mL sodium phosphate buffers (50mM, pH 8.0), the substrate L-allo-Ile of 1mM is added, is added without Any enzyme reacts 4 hours under the conditions of being placed in 30 DEG C, does not obtain enzymatic reaction product L-Ile (Vii in Figure 16 B).

Embodiment 8

The reversible reaction equilibrium constant of DsaD/DsaE catalysis measures：

In 50 μ L sodium phosphate buffers (50mM, pH 8.0), the substrate L-Ile or L-allo-Ile of 2.5mM is added, 0.1mM PLP, 5 μM of aminopherases DsaD, 5 μM of isomerase DsaE react 4 hours under the conditions of being placed in 30 DEG C.After reaction, The methanol of 200 μ L is added to terminate reaction, be vortexed concussion, and in being stored at room temperature after twenty minutes, 1,2000 × g is centrifuged 20 minutes, is taken Supernatant is evaporated with Rotary Evaporators, and residue is dissolved in 40 μ L 2mM CuSO₄Solution takes 25 μ L to carry out chiral HPLC, with inspection Survey enzymatic reaction situation.Chiral HPLC condition is：Using MCI GEL CRS10W column (Mitsubishi, 50 × 4.6mm, 3 μm) chiral analysis column；Flowing is 2mM CuSO₄Solution；Flow velocity is 1mL/min, and detection time is 30 minutes, detection Wavelength is 254nm.The final concentration of substrate and product is obtained by carrying out integral and calculating to corresponding HPLC peak values.The equilibrium constant (Keq) with descending according to this, calculation formula calculates and obtains Keq=([production concentration]/[concentration of substrate]).

When measuring using L-Ile as substrate, the equilibrium constant of the reversible reaction of DsaD/DsaE catalysis is 1.37 (such as Figure 15).

Sequence table

<110>Chinese Academy of Science Nanhai Ocean Research Institute

<120>A kind of bacterial strain of high yield cyclic peptide compound

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1122

<212> DNA

<213>Actinomyces SCSIO 10141 (Streptomyces drozdowiczii SCSIO 10141)

<400> 1

atgaccacga cctcatccgc agcaccggac attgtcctgc gccccggtac ctcggcgtct 60

tcggccgacc ggcgtcgaga ggcgctcgcc ggagcggcct tcggcgaggt gttcaccgat 120

cacatggtca ccgcacgatg gaccgccacc gacggctggc acgacgctgc actggaacca 180

ttcgctccat tggagttgag cccggccgct gtcggactgc actacgggca gagcgtgttc 240

gaaggattca aggcctacca tcggacaccg gagcgggctg ccatcttccg tccgtcggcc 300

cacgcccgcc ggttcgcgga ctcagcccgg cgcatggccc ttcccgaggt tccggtcgac 360

ttgttcgtcg gcgccgccga agccctggtc cgccaggaca aggactggat ccccgatggc 420

gacgaccgga gcctgtacct cagaccggtg ctcttcgctt ccgaggccca tctcgccctg 480

cggcccgccc gtgcatgcct gttcgtccta ctcgccttcc ccacgggcaa cttcttcgat 540

gcgggcgacc gcgcggtgac ggtggcggtc gccgatgagt acgtgcgcgc cgctccgggc 600

ggcacggggg cggccaagtg cggtggcaac tacgcgagca cctatctcgc ccaggagacg 660

gccgcccgga agggcgctga ccaggtggtc tggctggacg cagtggagcg gcgctgggtg 720

gaggaactgg gtggcatgaa cctctttttc gtgtacggca cgggagacca gaccacactc 780

acaaccccgc cactgactgg caccatcctg cccggcgtca cccgcgacac cctcctcacc 840

ctcgccggtg gccttggact acaagtcact gaggccccca taacggtcag ccggtggcgt 900

gaggagtgcg ccgcaggccg gatcacggag gtattcgcgt gcggcactgc ggcccgaatc 960

acgtccgtcg gccgtgtcct cagcgcggac gggccctgga cggtcggaga cggacggcca 1020

ggacccgtcg cgggccggct ctcggcggcg ctagctgccg tacaccgcgg cgaggcagcc 1080

gattcgtatg gctggtgcca tctggtgaac cacgaggcct ga 1122

<210> 2

<211> 351

<212> DNA

<213>Actinomyces SCSIO 10141 (Streptomyces drozdowiczii SCSIO 10141)

<400> 2

atggggcgct ccgagaccat ccgtcgctac tacgaactag tggacgcggc ggattacgag 60

gccatgttcc gtatattctg cgacgacctg atatacgagc gggccggaac cgaacccatc 120

gagggaatcg tggagttccg tcacttctat ctcgccgacc gcaagatcag gtcgggacgg 180

cactctctgg acgtgctcat cgagaatggc gactgggtcg ccgccagagg agtcttcacc 240

ggacaactcc gcacagggga agccgtgacc acccggtggg ccgacttcca ccagttccgg 300

ggagagaaga tctggcgtcg gtacacctat ttcgcggatc agtcggtgta g 351

<210> 3

<211> 1134

<212> DNA

<213>Actinomyces SCSIO ZJ46 (Streptomyces scopuliridis SCSIO ZJ46)

<400> 3

gtgcatatcg tgaccacacc cgtagcccga ccactgacgg cgcaggagcg cacggagcgc 60

tgcgccgccc ccgccttcgg caccgcgttc accgagcaca tggtctccgc ccgatggaac 120

cccgaacagg gctggcatga cgccgagttg gtgccctacg gtccgctgct gctggacccc 180

gccacggtcg gtctgcacta tggccaggtc gtcttcgagg gactcaaggc gttccgttcg 240

cacaccggcg aggtcgcggt cttccggccg gacgcgcacg ccgaacggat gcgcgcctcg 300

gcccgccgcc tcatgatgcc cgagccgccc gaggaactgt tcctcgcggc ggtggacgcc 360

ctggtcgccc aggaccagga gtggataccc gacgaccccg gcatgagtct gtatctgcgc 420

cccatcctct tcgcgagcga gcggactctc gctctgcgtc ccgcccgtga ataccgcttc 480

ctgctggtgg cgttcatcac cgagggctac ttcggccctg cccagcgccc ggtacgggtg 540

tgggtcaccg acgagtactc ccgggccgcc gccggcggca ccggagccgc caagtgcgcg 600

ggcaactacg cgggaagcct gctcgcccag gaggaggccc agcgcaaggg gtgcgaccaa 660

gtcgtctggc tcgacccggt ggagcgcaac tgggtcgagg agatgggagg catgaatctc 720

ttcttcgtgt acgaagccgg tggctccgcc cgactggtca ccccgccgct gacgggcagc 780

ctgctgcccg gcgtcacgcg ggacgcgctg ctgcgactgg cccccaccct cggtgtgccg 840

gtgagtgagg cacccctgag cctggaacag tggcgggcgg actgcgcctc cggcgcgatc 900

accgaggtct tcgcctgcgg caccgccgcc cggatcagtc ccgtcaacga ggtcagcacc 960

aaggacggct cctggaccat cggcgcgggc gcccctgccg aaggcggcgt cgcggccggc 1020

gaggtcaccg gcagactctc cgccgcgctg ttcggcatcc agcgcggcga actgcccgac 1080

tcccactcct ggatgcggcc ggtgtccccg gccagacagt cggcgatcac atga 1134

<210> 4

<211> 375

<212> DNA

<213>Actinomyces SCSIO ZJ46 (Streptomyces scopuliridis SCSIO ZJ46)

<400> 4

atgaccgaga gctctcccac cgaggtcaat gaggcccggg tgcgtgagta ctaccggttg 60

gtggacgcgg acgacgtcct cggactcgtc tccctcttcg cggaggacgc cgtctaccgg 120

cggccgggat acgaacccat gcgcggtcac accggtctga ccgccttcta caccggcgag 180

cgcgtgatcg agagcggtcg gcacaccgtc gccacggtcg tcgcgcgagg cgatcaggtc 240

gcggtcaacg gagtcttcga gggcgtcctc aaggacggcc gccaagtccg cctggaattc 300

gccgacttct ttctgctcaa cggcgagcgg cggttcagtc ggcgtgacac gtacttcttc 360

gccccactgg tgtga 375

<210> 5

<211> 373

<212> PRT

<213>Actinomyces SCSIO 10141 (Streptomyces drozdowiczii SCSIO 10141)

<400> 5

Met Thr Thr Thr Ser Ser Ala Ala Pro Asp Ile Val Leu Arg Pro Gly

1 5 10 15

Thr Ser Ala Ser Ser Ala Asp Arg Arg Arg Glu Ala Leu Ala Gly Ala

20 25 30

Ala Phe Gly Glu Val Phe Thr Asp His Met Val Thr Ala Arg Trp Thr

35 40 45

Ala Thr Asp Gly Trp His Asp Ala Ala Leu Glu Pro Phe Ala Pro Leu

50 55 60

Glu Leu Ser Pro Ala Ala Val Gly Leu His Tyr Gly Gln Ser Val Phe

65 70 75 80

Glu Gly Phe Lys Ala Tyr His Arg Thr Pro Glu Arg Ala Ala Ile Phe

85 90 95

Arg Pro Ser Ala His Ala Arg Arg Phe Ala Asp Ser Ala Arg Arg Met

100 105 110

Ala Leu Pro Glu Val Pro Val Asp Leu Phe Val Gly Ala Ala Glu Ala

115 120 125

Leu Val Arg Gln Asp Lys Asp Trp Ile Pro Asp Gly Asp Asp Arg Ser

130 135 140

Leu Tyr Leu Arg Pro Val Leu Phe Ala Ser Glu Ala His Leu Ala Leu

145 150 155 160

Arg Pro Ala Arg Ala Cys Leu Phe Val Leu Leu Ala Phe Pro Thr Gly

165 170 175

Asn Phe Phe Asp Ala Gly Asp Arg Ala Val Thr Val Ala Val Ala Asp

180 185 190

Glu Tyr Val Arg Ala Ala Pro Gly Gly Thr Gly Ala Ala Lys Cys Gly

195 200 205

Gly Asn Tyr Ala Ser Thr Tyr Leu Ala Gln Glu Thr Ala Ala Arg Lys

210 215 220

Gly Ala Asp Gln Val Val Trp Leu Asp Ala Val Glu Arg Arg Trp Val

225 230 235 240

Glu Glu Leu Gly Gly Met Asn Leu Phe Phe Val Tyr Gly Thr Gly Asp

245 250 255

Gln Thr Thr Leu Thr Thr Pro Pro Leu Thr Gly Thr Ile Leu Pro Gly

260 265 270

Val Thr Arg Asp Thr Leu Leu Thr Leu Ala Gly Gly Leu Gly Leu Gln

275 280 285

Val Thr Glu Ala Pro Ile Thr Val Ser Arg Trp Arg Glu Glu Cys Ala

290 295 300

Ala Gly Arg Ile Thr Glu Val Phe Ala Cys Gly Thr Ala Ala Arg Ile

305 310 315 320

Thr Ser Val Gly Arg Val Leu Ser Ala Asp Gly Pro Trp Thr Val Gly

325 330 335

Asp Gly Arg Pro Gly Pro Val Ala Gly Arg Leu Ser Ala Ala Leu Ala

340 345 350

Ala Val His Arg Gly Glu Ala Ala Asp Ser Tyr Gly Trp Cys His Leu

355 360 365

Val Asn His Glu Ala

370

<210> 6

<211> 116

<212> PRT

<213>Actinomyces SCSIO 10141 (Streptomyces drozdowiczii SCSIO 10141)

<400> 6

Met Gly Arg Ser Glu Thr Ile Arg Arg Tyr Tyr Glu Leu Val Asp Ala

1 5 10 15

Ala Asp Tyr Glu Ala Met Phe Arg Ile Phe Cys Asp Asp Leu Ile Tyr

20 25 30

Glu Arg Ala Gly Thr Glu Pro Ile Glu Gly Ile Val Glu Phe Arg His

35 40 45

Phe Tyr Leu Ala Asp Arg Lys Ile Arg Ser Gly Arg His Ser Leu Asp

50 55 60

Val Leu Ile Glu Asn Gly Asp Trp Val Ala Ala Arg Gly Val Phe Thr

65 70 75 80

Gly Gln Leu Arg Thr Gly Glu Ala Val Thr Thr Arg Trp Ala Asp Phe

85 90 95

His Gln Phe Arg Gly Glu Lys Ile Trp Arg Arg Tyr Thr Tyr Phe Ala

100 105 110

Asp Gln Ser Val

115

<210> 7

<211> 377

<212> PRT

<213>Actinomyces SCSIO ZJ46 (Streptomyces scopuliridis SCSIO ZJ46)

<400> 7

Val His Ile Val Thr Thr Pro Val Ala Arg Pro Leu Thr Ala Gln Glu

1 5 10 15

Arg Thr Glu Arg Cys Ala Ala Pro Ala Phe Gly Thr Ala Phe Thr Glu

20 25 30

His Met Val Ser Ala Arg Trp Asn Pro Glu Gln Gly Trp His Asp Ala

35 40 45

Glu Leu Val Pro Tyr Gly Pro Leu Leu Leu Asp Pro Ala Thr Val Gly

50 55 60

Leu His Tyr Gly Gln Val Val Phe Glu Gly Leu Lys Ala Phe Arg Ser

65 70 75 80

His Thr Gly Glu Val Ala Val Phe Arg Pro Asp Ala His Ala Glu Arg

85 90 95

Met Arg Ala Ser Ala Arg Arg Leu Met Met Pro Glu Pro Pro Glu Glu

100 105 110

Leu Phe Leu Ala Ala Val Asp Ala Leu Val Ala Gln Asp Gln Glu Trp

115 120 125

Ile Pro Asp Asp Pro Gly Met Ser Leu Tyr Leu Arg Pro Ile Leu Phe

130 135 140

Ala Ser Glu Arg Thr Leu Ala Leu Arg Pro Ala Arg Glu Tyr Arg Phe

145 150 155 160

Leu Leu Val Ala Phe Ile Thr Glu Gly Tyr Phe Gly Pro Ala Gln Arg

165 170 175

Pro Val Arg Val Trp Val Thr Asp Glu Tyr Ser Arg Ala Ala Ala Gly

180 185 190

Gly Thr Gly Ala Ala Lys Cys Ala Gly Asn Tyr Ala Gly Ser Leu Leu

195 200 205

Ala Gln Glu Glu Ala Gln Arg Lys Gly Cys Asp Gln Val Val Trp Leu

210 215 220

Asp Pro Val Glu Arg Asn Trp Val Glu Glu Met Gly Gly Met Asn Leu

225 230 235 240

Phe Phe Val Tyr Glu Ala Gly Gly Ser Ala Arg Leu Val Thr Pro Pro

245 250 255

Leu Thr Gly Ser Leu Leu Pro Gly Val Thr Arg Asp Ala Leu Leu Arg

260 265 270

Leu Ala Pro Thr Leu Gly Val Pro Val Ser Glu Ala Pro Leu Ser Leu

275 280 285

Glu Gln Trp Arg Ala Asp Cys Ala Ser Gly Ala Ile Thr Glu Val Phe

290 295 300

Ala Cys Gly Thr Ala Ala Arg Ile Ser Pro Val Asn Glu Val Ser Thr

305 310 315 320

Lys Asp Gly Ser Trp Thr Ile Gly Ala Gly Ala Pro Ala Glu Gly Gly

325 330 335

Val Ala Ala Gly Glu Val Thr Gly Arg Leu Ser Ala Ala Leu Phe Gly

340 345 350

Ile Gln Arg Gly Glu Leu Pro Asp Ser His Ser Trp Met Arg Pro Val

355 360 365

Ser Pro Ala Arg Gln Ser Ala Ile Thr

370 375

<210> 8

<211> 124

<212> PRT

<213>Actinomyces SCSIO ZJ46 (Streptomyces scopuliridis SCSIO ZJ46)

<400> 8

Met Thr Glu Ser Ser Pro Thr Glu Val Asn Glu Ala Arg Val Arg Glu

1 5 10 15

Tyr Tyr Arg Leu Val Asp Ala Asp Asp Val Leu Gly Leu Val Ser Leu

20 25 30

Phe Ala Glu Asp Ala Val Tyr Arg Arg Pro Gly Tyr Glu Pro Met Arg

35 40 45

Gly His Thr Gly Leu Thr Ala Phe Tyr Thr Gly Glu Arg Val Ile Glu

50 55 60

Ser Gly Arg His Thr Val Ala Thr Val Val Ala Arg Gly Asp Gln Val

65 70 75 80

Ala Val Asn Gly Val Phe Glu Gly Val Leu Lys Asp Gly Arg Gln Val

85 90 95

Arg Leu Glu Phe Ala Asp Phe Phe Leu Leu Asn Gly Glu Arg Arg Phe

100 105 110

Ser Arg Arg Asp Thr Tyr Phe Phe Ala Pro Leu Val

115 120

Claims

1. a kind of bacterial strain Streptomyces coelicolor M1152/07-6H-EKO of high yield compound 9 and 11 or Streptomyces coelicolor M1152/07-6H-DKO, which is characterized in that the bacterial strain Streptomyces Coelicolor M1152/07-6H-EKO are the biosynthesis gene with the desotamides of frame deletion mutation by DsaE genes Cluster imports in bacterial strain Streptomyces coelicolor M1152 and is expressed and obtained, described Streptomyces coelicolor M1152/07-6H-DKO are the desotamides with frame deletion mutation by DsaD genes Biological synthesis gene cluster import in bacterial strain Streptomyces coelicolorM1152 and expressed and obtained；

The structure of the compound 9 and 11 is as shown in Equation 2, wherein compound 9：R₁=H, R₂=NH₂；Compound 11：R₁=H, R₂=OH；