CN108486028B

CN108486028B - Strain delta mfnH for high yield of compound containing L-Val structural unit

Info

Publication number: CN108486028B
Application number: CN201810326214.6A
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: 2021-06-01
Anticipated expiration: 2035-10-10
Also published as: CN108587991A; WO2017059626A1; CN105154487B; CN108486028A; CN105154487A; JP2018530335A; JP6585839B2; CN108587991B

Abstract

The invention discloses application of aminotransferase and isomerase in catalyzing and forming L-allo-Ile. The aminotransferase is aminotransferase DsaD or aminotransferase MfnO, the isomerase is isomerase DsaE or isomerase MfnH, the amino acid sequence of the aminotransferase DsaD is shown in SEQ ID NO.7, the amino acid sequence of the isomerase DsaE is shown in SEQ ID NO.8, the amino acid sequence of the aminotransferase MfnO is shown in SEQ ID NO.5, and the amino acid sequence of the isomerase MfnH is shown in SEQ ID NO. 6. The invention discloses an application of an enzyme pair consisting of aminotransferase and isomerase in catalyzing and forming L-alloisoleucine or L-isoleucine. Thus, an important foundation is laid for explaining the biosynthesis mechanism of L-allo-Ile on the enzymology level. The explanation of the enzymatic mechanism of L-allo-Ile biosynthesis is to prepare L-allo-Ile by a green enzymatic method and has important practical significance for the diagnosis and treatment of maple syrup urine.

Description

Strain delta mfnH for high yield of compound containing L-Val structural unit

The application is as follows: 10/2015, application No.: 201510651516.7, title of the invention: a divisional application of a patent application for the use of aminotransferases and isomerases in the catalysis of the formation of L-allo-Ile.

The technical field is as follows:

the invention belongs to the technical field of genetic engineering and biological catalysis, and particularly relates to an application of an enzyme pair consisting of PLP (pyridoxal 5' -phosphate) dependent aminotransferase and novel isomerase in the formation of L-allo-isoleucine (L-allo-Ile) through cooperative catalysis.

Background art:

isoleucine has two asymmetric centers, so 4 stereoisomers exist: l-isoleucine (L-Ile), D-isoleucine (D-Ile), L-allo-isoleucine (L-allo-Ile) and D-allo-isoleucine (D-allo-Ile), the corresponding relationship is shown in FIG. 1. Besides L-Ile, D-Ile, L-allo-Ile and D-allo-Ile belong to non-protein amino acids, and the existence of the amino acids in the nature is reported. Among them, L-allo-Ile has attracted special attention of scientists due to its wide existence in nature and important scientific significance. L-allo-Ile was first reported in 1985 to be found in subsequent studies that, in addition to being present in plants, could be found to be present as a structural unit in a number of cyclic peptide antibiotics such as aureobasidin A, cordyheptapeptides and aspergillin E from fungi, and globomycin, cyclopecin, desotamides and marformmycins from actinomycetes (structure as shown in FIG. 2). Interestingly, L-allo-Ile has also been found in human plasma, where the concentration of L-allo-Ile is low, at a concentration level that can be nearly detected in the plasma of healthy people; however, in the plasma of patients suffering from maple syrup urine disease (maple syrupus disease), which is an autosomal recessive genetic disease, L-allo-Ile accumulates due to metabolic defects of the patients, and the concentration of L-allo-Ile reaches 5 μ M or more, and therefore, the concentration level of L-allo-Ile in plasma has been one of important means for diagnosing maple syrup urine disease.

The structure of L-allo-Ile is very similar to that of protein amino acid L-Ile, and the difference between L-allo-Ile and L-Ile is the conformation of methyl on carbon atom at beta position is different. Although L-allo-Ile is structurally very similar to L-Ile and is widely found in nature, it is an unsolved puzzle of how life forms biosynthesize L-allo-Ile, a non-protein amino acid, and the enzymatic and enzymatic reaction mechanisms involved in this process.

Desotamides and marformycins are two types of cyclopeptide antibiotics obtained by separating and purifying Streptomyces scopuliris SCSIO ZJ46 and Streptomyces drozdowiczii SCSIO 10141 from deep sea of south sea respectively, and research shows that Desotamides have good inhibitory activity to gram-positive bacteria, while marformycins have good inhibitory action to Propionibacterium acnes (Propionibacterium acnes), and are good lead compounds for treating acne medicines. More importantly, the structures of the two types of cyclic peptide compounds both contain a non-protein amino acid structural unit L-allo-Ile. At present, biosynthetic gene clusters of desotamides and margormycins are cloned, and the research results lay an important foundation for explaining the biosynthetic mechanism of L-allo-Ile on the enzymology level. The explanation of the enzymatic mechanism of L-allo-Ile biosynthesis is to prepare L-allo-Ile by a green enzymatic method and has important practical significance for the diagnosis and treatment of maple syrup urine.

The invention content is as follows:

the object of the present invention is to provide the use of an enzyme pair formed by an aminotransferase and an isomerase to catalyze the formation of L-isoleucine or L-isoleucine.

The aminotransferase and the enzyme pair formed by the isomerase are applied to catalyzing L-isoleucine to form L-alloisoleucine or catalyzing L-alloisoleucine to form L-isoleucine, the aminotransferase is aminotransferase DsaD or aminotransferase MfnO, the isomerase is isomerase DsaE or isomerase MfnH, the amino acid sequence of the aminotransferase DsaD is shown as SEQ ID No.7, the amino acid sequence of the isomerase DsaE is shown as SEQ ID No.8, the amino acid sequence of the aminotransferase MfnO is shown as SEQ ID No.5, and the amino acid sequence of the isomerase MfnH is shown as SEQ ID No. 6.

Preferably, the nucleotide sequence of the coding gene MfnO gene of the aminotransferase MfnO is shown in SEQ ID No. 1.

Preferably, the nucleotide sequence of the coding gene mfnH gene of the isomerase MfnH is shown in SEQ ID NO. 2.

Preferably, the nucleotide sequence of the dsaD gene of the coding gene of the aminotransferase Dsad is shown as SEQ ID NO. 3.

Preferably, the nucleotide sequence of the dsaE gene of the encoding gene of the isomerase DsaE is shown as SEQ ID NO. 4.

The invention mainly relates to three aspects: firstly, identifying aminotransferase/isomerase-DsaD/DsaE and MfnO/MfnH which participate in L-allo-Ile biosynthesis from biosynthetic gene clusters of desonamides and marformmycins respectively by utilizing a bioinformatics analysis method; secondly, carrying out in vivo deletion mutation on aminotransferase/isomerase-Dsad/DsaE and MfnO/MfnH by an in vivo gene knockout method to obtain high-yield strains delta mfnH and Streptomyces coelicolor M1152/07-6H-DKO and Streptomyces coelicolor M1152/07-6H-EKO for producing

compounds

7, 9 and 11 (figure 2) containing L-Val structural units; the invention also relates to application of the identified aminotransferase/isomerase, DsaD/DsaE and MfnO/MfnH in catalyzing L-Ile to convert L-Ile to generate L-allo-Ile, which is characterized in that two enzymes required by the generation of L-allo-Ile act synergistically, the aminotransferase or isomerase alone can not catalyze the generation of L-allo-Ile, and no cofactor is required to be added in the catalysis process.

The invention discovers that the conformation of methyl on a carbon atom at a beta position is different by observing and comparing structural differences of L-allo-Ile and L-Ile, wherein the carbon atom at the beta position of the L-Ile is 3S type, and the carbon atom at the beta position of the L-allo-Ile is 3R type, so that the inventor speculates that the L-allo-Ile is possibly formed by the conversion of the L-Ile, and the conversion process can be that two enzyme molecules of aminotransferase and isomerase cooperate to complete the conversion between the two enzyme molecules. Firstly, under the action of aminotransferase, L-Ile removes amino group, becomes carbonyl group to fix the carbon atom at alpha position in plane, so that it can not rotate freely. Secondly, under the action of isomerase, the inversion of methyl on the carbon atom at the beta position is completed. The invention carries out bioinformatics analysis on DsaD and MfnO which are respectively annotated as aminotransferases in desotamides and marformmycins biosynthetic gene clusters, and multiple sequence alignment shows that DsaD/MfnO has high sequence homology with reported branched-chain amino acid transferase (BCATs),and has the same characteristic motif "exgxxxnlfxnxtxnxgvxr" as the type IV amino acid transferase, and a catalytic Lysine residue (Lysine) covalently linked to PLP (see fig. 3), suggesting that DsaD/MfnO has PLP-dependent aminotransferase activity. The present invention also carried out structural analysis of DsaE and MfnH annotated as isomerases in biosynthetic gene clusters of desotamides and marformmycins, respectively, using on-line resource HHpred for structural homology analysis of proteins, showing that they have a similar secondary structural folding pattern to proteins belonging to the nuclear transport factor 2 superfamily (NTF 2) which contains many proteins with different functions and low amino acid sequence similarity to each other, including the reported delta⁵-3-ketosteroid isomerase (delta)⁵3-ketosteroid isomerse) capable of catalyzing delta⁵Isomerization of the 3-ketosterol to Δ⁴3-ketosteroids, the inventors speculated that DsaE in the desotamides gene cluster and MfnH in the marformmycins gene cluster may be involved in the isomerization of the methyl group at the beta carbon atom of L-Ile to L-allo-Ile. The aminotransferase/isomerase pairs likely to be involved in L-allo-Ile synthesis were identified in the biosynthetic gene clusters of marformmycins and desotamides, respectively (see FIG. 4), suggesting conservation of the L-allo-Ile biosynthetic machinery.

The invention relates to a method for carrying out knockout mutation on the gene of aminotransferase/isomerase MfnO/MfnH in Streptomyces drozdowiczii SCSIO 10141 which is a wild-type producing strain of marformmycins (as shown in figures 5 and 6), and constructs a high-producing strain delta mfnH for producing a compound 7 containing an L-Val structural unit. Analysis of the fermentation products of the mutants Δ mfnO and Δ mfnH by HPLC revealed that Δ mfnH produced compounds 3 and 4 containing the L-allo-Ile structural unit at all, but produced

compounds

5 and 7 containing the L-Val structural unit, and the yield of compound 7 was increased by about 100-fold as compared with the wild-type strain (see fig. 7); Δ mfnO also produced compound 4 containing the L-allo-Ile structural unit at all (see FIG. 7), but still produced

compounds

5 and 7 containing the L-Val structural unit. These data confirm that MfnO/MfnH plays an essential role in the synthesis of L-allo-Ile, and that L-Val, which has a similar structure to that of L-allo-Ile, can be integrated into the marformmycins peptide chain skeleton during the synthesis of marformycin with no competitive advantage due to deletion mutation of MfnO/MfnH, resulting in the highly productive strain Δ mfnH producing Compound 7 containing an L-Val structural unit.

Therefore, a second object of the present invention is to provide a strain Δ mfnH producing compound 7 at a high yield, wherein the strain Δ mfnH is obtained by knockout deletion mutation of the mfnH gene of wild-type Streptomyces drozdowiczii SCSIO 10141;

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

The present invention also relates to in-frame knockout mutation (in-frame deletion) of the gene for the aminotransferase/isomerase, Dsad/DsaE, in the desotamides gene cluster followed by expression in the heterologous host, Steptomyces coelicolor M1152 (see FIGS. 8 and 9), creating highly productive strains Streptomyces coelicolor M1152/07-6H-EKO and Streptomyces coelicolor M1152/07-6H-DKO that produce

compounds

9 and 10 containing the L-Val building block. Analysis of fermentation products of Streptomyces coelicolor M1152/07-6H-EKO and Streptomyces coelicolor M1152/07-6H-DKO by HPLC revealed that Streptomyces coelicolor M1152/07-6H-EKO in which the dasE gene had been mutated by in-frame deletion did not produce

compounds

8 and 10 containing the L-allo-Ile structural unit at all but produced

compounds

9 and 11 containing the L-Val structural unit, and the production of both compounds was greatly increased as compared with the control strain (about 100-fold for compound 9 and about 140-fold for compound 11) (see FIG. 10). Although the heterologous expression strain Streptomyces coelicolor M1152/07-6H-DKO, in which the dsaD gene has been mutated by an in-frame deletion, still produces

compounds

8 and 10 containing the L-allo-Ile structural unit, the production is greatly reduced, and the production still produces

compounds

9 and 11 containing the L-Val structural unit, but is greatly increased (about 80-fold for compound 9; about 50-fold for compound 11) (see FIG. 10). These data also illustrate the essential role that DsaD/DsaE plays in the synthesis of L-allo-Ile.

Accordingly, a third object of the present invention is to provide a strain Streptomyces coelicolor M1152/07-6H-EKO or Streptomyces coelicolor M1152/07-6H-DKO highly producing

compounds

9 and 11, wherein the strain Streptomyces coelicolor M1152/07-6H-EKO is obtained by introducing and expressing a biosynthetic gene cluster of desamides having a deletion mutation of the DsaE gene in frame into a strain Streptomyces coelicolor M1152, and the strain Streptomyces coelicolor M1152/07-6H-EKO is obtained by introducing and expressing a biosynthetic gene cluster of desaD gene deletion mutated in frame into a strain Streptomyces coelicolor M1152;

the structures of the

compounds

9 and 11 are shown in a formula 2, wherein the compound 9: r₁＝H，R₂＝NH₂(ii) a Compound 11: r₁＝H，R₂＝OH；

The invention also relates to the expression and purification of aminotransferase/isomerase-DsaD/DsaE and MfnO/MfnH in E.coli (DE3) (as shown in FIG. 11), and the obtained enzyme pair can catalyze the conversion of substrate L-Ile into L-allo-Ile without adding any cofactor. Dsad/DsaE or MfnO/MfnH collaborated to catalyze the conversion of substrate L-Ile to L-allo-Ile in 50mM phosphate buffer at pH 8.0 without the addition of any cofactor, with a conversion rate of about 67% (see FIG. 12), but either the aminotransferase DsaD/MfnO alone or the isomerase DsaE/MfnH alone failed to catalyze the conversion of L-Ile to L-allo-Ile. DsaD/DsaE or MfnO/MfnH cooperate to catalyze the conversion of L-Ile to generate L-allo-Ile, belonging to reversible reaction, when L-allo-Ile is taken as a substrate, the product L-Ile can be obtained under the same reaction conditions (as shown in FIG. 14). The equilibrium constant of DsaD/DsaE catalyzed reversible reaction was 1.37 with L-Ile as substrate (FIG. 15). The function complementation of aminotransferase and isomerase in the biosynthesis pathway of Desotamides and marformmycins can be realized, and DsaD/MfnH and MfnO/DsaE can cooperatively catalyze the interconversion between L-Ile and L-allo-Ile (as shown in FIG. 16).

The invention discloses application of an enzyme pair consisting of aminotransferase and isomerase in catalyzing and forming L-allo-isoleucine (L-allo-Ile) or L-isoleucine. Thus, an important foundation is laid for explaining the biosynthesis mechanism of L-allo-Ile on the enzymology level. The explanation of the enzymatic mechanism of L-allo-Ile biosynthesis is to prepare L-allo-Ile by a green enzymatic method and has important practical significance for the diagnosis and treatment of maple syrup urine.

The Streptomyces scopulieridis SCSIO ZJ46 of the invention is disclosed in the literature: 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 scopuliris SCSIO ZJ46Active ingredient pathway-reactive Gram-reactive bacteria.J.Nat.prod.,2014,77(8), pp 1937-K1. The strain the applicant also holds, warranting supply to the public since 20 years.

The Streptomyces drozdowiczii SCSIO 10141 strain of the present invention is disclosed in Xiao Zhou, Hongbo Huang, Jie Lia, Yongxiang Song, Renwang Jiang, Jig Liu, Si Zhang, Yan Hua, Jianhua Ju.New anti-reactive cyclopeptide conjugates and absolute stereospecies from the deleted sea-derived Streptomyces scSIO 10141.tetrahedron Volume 70, Issue 42,21 October 2014, Pages 7795-. The strain the applicant also holds, warranting supply to the public since 20 years.

Streptomyces coelicolor M1152 disclosed in Master thesis: xiasan, the biosynthesis research of Drimentines compounds in Streptomyces sp.OUC6819 from mangrove forest, China university of oceans, 2013. The strain the applicant also holds, warranting supply to the public since 20 years.

Description of the drawings:

FIG. 1 is a chemical structural formula of L-isoleucine (L-Ile), D-isoleucine (D-Ile), L-allo-isoleucine (L-allo-Ile) and D-allo-isoleucine (D-allo-Ile).

FIG. 2 is the chemical structures of marformmycins and desotamides, wherein 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 represent

compounds

1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, respectively.

FIG. 3 is a multiple sequence alignment of the aminotransferase Dsad/MfnO with branched-chain amino acid transferases that have been reported. The arrow indicates the conserved lysine catalytic participation covalently bound to PLP, and the box indicates the characteristic motif "EXGXXNLFXnLXXnLXXGXR" of IV-aminotransferase.

FIG. 4 is the location of the aminotransferase and isomerase enzymes in the biosynthetic gene clusters for maformmycins and desotamides. Aminotransferase DsaD/MfnO is marked in red; the isomerase DsaE/MfnH is marked in green.

FIG. 5 shows the deletion mutation of the aminotransferase gene mfnO in marformcins producing bacteria using PCR-targeting technology. (A) Schematic representation of the mutation process; (B) performing PCR identification on the mutant strain delta mfnO, wherein W: taking the genome DNA of a wild strain Streptomyces drozdowiczii SCSIO 10141 as a template; m: using mutant delta mfnO genome DNA as template, marker NDA molecular weight standard.

FIG. 6 shows the deletion mutation of the aminotransferase gene mfnH in marformcins producing strain by PCR-targeting. (A) Schematic representation of the mutation process; (B) PCR identification of the mutant strain Δ mfnH, W: taking the genome DNA of a wild strain Streptomyces drozdowiczii SCSIO 10141 as a template; m: using mutant delta mfnH genome DNA as template, marker NDA molecular weight standard.

FIG. 7 is an HPLC analysis of the mutant Δ mfnO and Δ mfnH fermentation products.

FIG. 8 is the in-frame deletion mutation of the dsaD gene and expression in the heterologous host Streptomyces coelicolor M1152. (A) A schematic diagram; (B) PCR identification of deletion mutations, WT: taking the genomic DNA of a control strain Streptomyces coelicolor M1152 as a template; DKO: heterologous expression strain Streptomyces coelicolor M1152/07-6H-DKO genome DNA is taken as a template, and M is a DNA molecular weight standard substance.

FIG. 9 is the in-frame deletion mutation of the dsaE gene and expression in the heterologous host Streptomyces coelicolor M1152. (A) A schematic diagram; (B) PCR identification of deletion mutations, WT: taking the genomic DNA of a control strain Streptomyces coelicolor M1152 as a template; EKO: heterologous expression strain Streptomyces coelicolor M1152/07-6H-EKO genome DNA is taken as a template, and M is a DNA molecular weight standard substance.

FIG. 10 is an HPLC analysis of fermentation products of the heterologous expression strains Streptomyces coelicolor M1152/07-6H-DKO and Streptomyces coelicolor M1152/07-6H-EKO, where the dsaD and dsaE genes have been mutated by in-frame deletion. i: control strain Streptomyces coelicolor m 1152; ii: a heterologous expression strain Streptomyces coelicolor M1152/07-6H containing a biosynthetic gene cluster of desotamides; iii: a heterologous expression strain, Streptomyces coelicolor M1152/07-6H-DKO, having dsaD mutated by an in-frame deletion; iv: heterologous expression strain Streptomyces coelicolor M1152/07-6H-EKO, in which dsaE has been mutated by an in-frame deletion.

FIG. 11 shows SDS-PAGE analysis of expression and purification of DsaD/DsaE and MfnO/MfnH in E.coli (DE 3).

FIG. 12 shows that purified recombinant DsaD/DsaE and MfnO/MfnH catalyze the conversion of L-Ile in vitro to L-allo-Ile.

FIG. 13 is a diagram of the preparation of the enzymatic product L-allo-Ile from Dsad/Dsae¹H NMR spectrum. A: of the enzymatic product L-allo-Ile¹H NMR spectrum; b: of L-allo-Ile standards¹H NMR spectrum

FIG. 14 shows that purified recombinant DsaD/DsaE and MfnO/MfnH catalyze the conversion of L-allo-Ile in vitro to L-Ile.

FIG. 15 is a determination of equilibrium constants for DsaD/DsaE catalyzed reversible reactions. However, when L-Ile is used as a substrate, the equilibrium constant Keq is calculated according to the formula to give (2.89/2.11) ═ 1.37.

FIG. 16 is a graph showing that aminotransferase/isomerase pairs derived from different biosynthetic pathways, DsaD/MfnH and MfnO/DsaE, can cooperate in catalyzing the interconversion between L-Ile and L-allo-Ile.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

Deletion mutation of mfnO gene (the nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence of the amino transferase MfnO coded by the gene is shown as SEQ ID NO. 5) and mfnH gene (the nucleotide sequence is shown as SEQ ID NO.2, the amino acid sequence of the isomerase MfnH coded by the gene is shown as SEQ ID NO. 6) in a wild type producing strain Streptomyces hydro

Obtaining an in vitro knockout mutant strain by utilizing a PCR-targeting method. According to the obtained biosynthetic gene cluster sequence of marformmycins, a pair of mfnO and mfnH gene knockout primers are designed according to a PCR-targeting system reported in the literature, and the primer sequences are shown in the mfnO and mfnH knockout primers in the table 1. Then constructing an in-vitro knockout plasmid by referring to a PCR-targeting method and transferring the knockout plasmid into a conjugately transferred donor bacterium. The method comprises the following specific steps: (1) the cosmid plasmid containing the biosynthesis gene cluster of marformmycins (plasmid cosmid 247E, GenBank accession number of nucleotide sequence of the biosynthesis gene cluster of marformmycins: KP715145.1) is transferred into Escherichia coli E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790 strain containing target plasmid, L-arabinose with 10mM is used for inducing expression of lambda/red recombination system, and the recombinant system is prepared into electroporation competent cells for standby. (2) The plasmid pIJ778 was digested with the endonucleases EcoR I and Hind III, and a DNA fragment of about 1.4kb containing the origin of transfer (oriT) and spectinomycin (spectinomycin) resistance gene was recovered and used as a PCR template, and 1.4kb of PCR product was amplified by PCR using the primers mfnOdelF/mfnOdelR and mfnHdelF/mfnHdelR, respectively, and 50. mu.L of PCR reaction was performed: 3U of high-fidelity DNA polymerase, 5 mu L of 10 multiplied by Buffer, 0.5mmol/L of dNTPs0.5mmol/L, 2.5 mu L of DMSO, 0.5 mu mol/L of each primer, about 1ng of DNA template and water for supplementing to 50 mu L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; the amplification cycle is denaturation at 94 ℃ for 45s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 90s for 30 cycles; finally, extension is carried out for 10min at 72 ℃. The 1.4kb PCR product was recovered and purified separately for further use. (3) The PCR products were each electroporated into competent cells E.coli BW25113/pIJ790 prepared in step (1) to be recombined, plated on LB screening plates (containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin, 50. mu.g/mL spectinomycin), and cultured overnight at 37 ℃. Positive single clones were picked up from the plate, plasmids were extracted, and recombinant plasmids designated delmfnO and delmfnH, in which partial fragments of mfnO and mfnO genes were replaced with the transfer origin and spectinomycin-resistant gene, respectively. (4) The constructed recombinant mutant plasmids delmfnO and delmfnH are respectively transformed into E.coli ET12567/pUZ8002 to obtain recombinant strains E.coli ET12567/pUZ8002/delmfnO and E.coli ET12567/pUZ8002/delmfnH which are used as donor bacteria for conjugal transfer.

The wild type Streptomyces drozdowiczii SCSIO 10141 strain was streaked on ISP2 medium (malt extract 4g, yeast extract 4g, glucose 4g, sea salt 30g, agar powder 20g, water added to 1L, pH 7.2) plates for 3-5 days, and the grown spores were collected on TSB medium with a sterile cotton swab and vortexed to disperse the spores. The mycelia and spores were separated by filtration, suspended in 5mL of TSB medium, heat-shocked at 50 ℃ for 10min, and then germinated at 28 ℃ for 2-4 hours as a conjugative transfer recipient bacterium. When donor bacteria E.coli ET12567/pUZ8002/delmfnO and E.coli ET12567/pUZ8002/delmfnH were grown at 37 ℃ in 50mL LB liquid medium containing 50. mu.g/mL kanamycin, 25. mu.g/mL chloramphenicol and 50. mu.g/mL spectinomycin, respectively, to an OD600 value of about 0.6, the cells were collected by centrifugation (4000rpm, 10min), washed 3 times with LB, suspended in 300. mu.L LB medium, and used as conjugately transferred donor bacteria. Mixing 400 μ L of the above recipient bacterium and 100 μ L of donor bacterium, spreading on M-ISP4 solid culture medium (soluble starch 10g, yeast extract 0.5g, peptone 1g, NaCl 1g, MgSO 1) without containing any antibiotic₄·7H₂O 1g，(NH₄)₂SO₄2g，K₂HPO₄1g，CaCO₃2g, sea salt 30g and trace elements 100 mu L, adding water to 1L, pH 7.2), drying, and culturing at 28 ℃ for 18-20 h. Then the plates were taken out, covered with water containing antibiotics to a final concentration of 100. mu.g/mL spectinomycin and 50. mu.g/mL trimethoprim, blow-dried, placed in an incubator at 28 ℃ and incubated for 3-4 days and observed.

After the small bacteria grow on the conjugal transfer plate, the small bacteria are transferred to an ISP2 plate containing 100 mug/mL spectinomycin and 50 mug/mL trimethoprim by using a sterile toothpick, after 2-3 days of culture at 28 ℃, genome DNA of each mutant strain is extracted, and positive clone is obtained by PCR detection by using detection primers (the primer sequences are shown in detection primer sequences of mfnO and mfnH in a table 2), namely, the aminotransferase mfnO gene knockout double-crossover mutant strain delta mfnO and the isomerase mfnH gene knockout double-crossover mutant strain delta mfnH are obtained.

Table 1: name and sequence of knockout primer required for constructing mutant strains of mfnO and mfnH genes

Table 2: name and sequence of detection primer required for constructing mfnO and mfnH gene mutant strain

Example 2

The same-frame deletion mutation of dsaD gene (the nucleotide sequence of which is shown as SEQ ID NO.3 and the amino acid sequence of the coded aminotransferase DsaD is shown as SEQ ID NO. 7) and dsaE gene (the nucleotide sequence of which is shown as SEQ ID NO.4 and the amino acid sequence of the coded isomerase DsaE is shown as SEQ ID NO. 8) and the expression in heterologous host Streptomyces coelicolor M1152

The first is the in-frame deletion mutation of the dsaD and dsaE genes. The specific operation process is as follows: (1) referring to the PCR-targeting system reported in the literature, plasmid pIJ773 was first digested with EcoR I and Hind III enzymes, and a DNA fragment of about 1.4kb containing the origin of transfer (oriT) and apramycin (apramycin) resistance genes was recovered as a DNA fragment required for PCR amplification to knock-out the dsaD and dsaE genes. (2) Based on the sequence of dsaD and dsaE genes, a pair of knock-out primers were designed, which were characterized by 39 nucleotides homologous to the deletion mutant gene of interest (see Table 3, in uppercase letters), 19 or 20 nucleotides homologous to the left or right ends of the resistance gene fragment (see Table 3, in lowercase letters), and, in addition, an endonuclease SpeI site (see Table 3, SpeI site underlined) between 39 nucleotides and 19/20 nucleotides. Using this primer, PCR amplification was carried out using the recovered 1.4kb DNA fragment containing the origin of transfer (oriT) and the apramycin (apramycin) resistance gene as a template to obtain a 1.4kb PCR product, 50. mu.L of a PCR reaction: 3U of high-fidelity DNA polymerase, 5 mu L of 10 multiplied by Buffer, 0.5mmol/L of dNTPs, 2.5 mu L of DMSO, 0.5 mu mol/L of each primer and about 1ng of DNA template, and water is added to supplement the volume to 50 mu L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; the amplification cycle is denaturation at 94 ℃ for 45s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 90s for 30 cycles; finally, extension is carried out for 10min at 72 ℃. The 1.4kb PCR product was recovered and purified separately for further use. (3) Next, cosmid 07-6H from SuperCos1 plasmid was selected as the starting cosmid for the in-frame deletion mutation of dsaD and dsaE, which contained the biosynthetic gene cluster of desotamides (GenBank accession number: KP769807.1 for the nucleotide sequence of the biosynthetic gene cluster of desotamides). Transferring the cosmid 07-6H into Escherichia coli E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/07-6H, inducing the expression of the lambda/red recombination system by using 10mM L-arabinose, and preparing the recombinant into an electrotransformation competent cell for later use. (4) The 1.4kb PCR products obtained in step (2) were individually electroporated into competent cells E.coli BW25113/pIJ790/07-6H prepared in step (3) to be recombined, plated on LB screening plates (containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin, 50. mu.g/mL apramycin), and cultured overnight at 37 ℃. Positive single clones were picked from the plates, plasmids were extracted, and recombinant cosmids, named 07-6H-DKO and 07-6H-EKO, replaced with a partial fragment of the dsaD and dsaE genes in 07-6H-DKO and 07-6H-EKO, respectively, with the transfer origin and the apramycin resistance gene. (5) The recombinant cosmids 07-6H-DKO and 07-6H-EKO were digested with SpeI, phenol: chloroform extraction, ethanol precipitation, and then use T4 ligase were connected, transformed competent cells E.coli DH5, spread on LB plate containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin, at 37 ℃ overnight culture. Clones were identified by PCR using detection primers (see Table 4), and cosmids which lost the transfer origin and the DNA fragment of the apramycin resistance gene and self-ligated were selected and named 07-6H-DKO-IF and 07-6H-EKO-IF.

Followed by the introduction of cosmids 07-6H-DKO-IF and 07-6H-EKO-IF, whose dsaD and dsaE genes had been mutated by in-frame deletion, into a heterologous host Streptomyces coelicolor M1152 for expression. Before introducing Streptomyces coelicolor M1152, cosmids 07-6H-DKO-IF and 07-6H-EKO-IF were first engineered to be suitable for heterologous expression. The strategy for the transformation was to replace the kanamycin resistance gene derived from plasmid SuperCos1 on cosmids 07-6H-DKO-IF and 07-6H-EKO-IF with NDA fragments containing the apramycin resistance gene aac (3) IV, the junction transfer origin original oriT, the integrase gene and the integration site of int. psi. C31, respectively, using the lambda/red recombination system of E.coli. The DNA fragment of aac (3) IV-oriT-int. psi.C 3 was derived from the plasmid pSET152AB constructed in this laboratory, and after the plasmid pSET152AB was completely digested with BamH I/EcoR I, a fragment of about 5.5kb was recovered, which contained the aac (3) IV-oriT-int. psi.C 3DNA fragment and the 1.0kb DNA fragment flanking the replaced kanamycin gene. Transferring 07-6H-DKO-IF and 07-6H-EKO-IF into E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/07-6H-DKO-IF and E.coli BW25113/pIJ 790/07-6H-EKO-IF. About 100mg of the recovered 5.5kb DNA fragment was added to E.coli BW25113/pIJ790/07-6H-DKO-IF and E.coli BW25113/pIJ790/07-6H-EKO-IF competent cells, respectively, and transferred to an electric cuvette for electrotransformation at a voltage of 1.4 kv. After completion of the electric shock, a pre-cooled 0.5mL LB medium was quickly added, and after 1 hour of resuscitation at 37 ℃ the plates were plated on LB plates containing 100. mu.g/mL ampicillin and 50. mu.g/mL apramycin. After 12 hours, after the transformants grew, positive recombinant plasmids were verified by PCR using detection primers (Table 4), and the positive recombinant plasmids were named 07-6H-DKO-AB and 07-6H-EKO-AB. The constructed recombinant plasmid is electrically transferred into E.coli ET12567/pUZ8002 to obtain E.coli ET12567/pUZ8002/07-6H-DKO-AB and E.coli ET12567/pUZ8002/07-6H-EKO-AB as donor bacteria for conjugal transfer.

Next, E.coli ET12567/pUZ8002/07-6H-DKO-AB and E.coli ET12567/pUZ8002/07-6H-EKO-AB were transferred by conjugation with Streptomyces coelicolor M1152. Strains E.coli ET12567/pUZ8002/07-6H-DKO-AB and 07-6H-EKO-AB are respectively connectedAfter culturing in 3mL of LB liquid medium (containing 100. mu.g/mL ampicillin, 50. mu.g/mL apramycin, 25. mu.g/mL chloramphenicol, and 50. mu.g/mL kanamycin) at 37 ℃ for 12 hours, 40. mu.L of the suspension was inoculated into 4mL of the same medium and cultured until the OD was 0.6, the cells were collected by centrifugation, washed 2 times with LB liquid medium containing no antibiotic, washed off the antibiotic, and concentrated by centrifugation for use. Meanwhile, s.coelicolor m1152 spores were collected with 10% glycerol, filtered through a filter, centrifuged at 3600rpm for 8min, the supernatant was discarded, an appropriate amount of LB medium was added to suspend the spores, and the spores were heat-shocked in a water bath at 50 ℃ for 10 min. The transformed strains E.coli ET12567/pUZ8002/07-6H-DKO-AB and 07-6H-EKO-AB are respectively mixed with S.coelicolor M1152 spores according to the volume ratio of 2:1, and the mixture is coated on MS + MgCl₂(final concentration 10mM) on solid plates. After 20-24 hours, the plates are taken out, water containing antibiotics is used for covering the plates, the final concentration of the water is 100 mug/mL of apramycin and 50 mug/mL of trimethoprim, after the plates are dried by blowing, the plates are placed in an incubator at the temperature of 28 ℃, and the plates are cultured for 3-4 days and then observed. After the growth of the microspores on the conjugal transfer plate, the microspores are transferred to an MS culture medium (20 g of soybean meal, 20g of mannitol and 20g of agar powder, water is added to 1L, and the pH value is 7.2) plate containing 100 mug/mL of apramycin and 50 mug/mL of trimethoprim by using a sterile toothpick, after the MS culture medium is cultured for 2 to 3 days at the temperature of 28 ℃, genome DNA of each mutant strain is extracted, and positive clones are obtained by PCR detection by using detection primers (the primer sequences are shown in a table 4), so that desosamide biosynthetic gene cluster heterologous expression strains Streptomyces coelicolor 115M 2/07-6H-O and Streptomyces coelicolor M1152/07-6H-EKO, of which dsaD and dsaE genes are subjected to in-frame deletion mutation, are respectively obtained.

Table 3: knockout primer names and sequences required for construction of dsaD and dsaE gene mutants

Table 4: detection primer names and sequences required for constructing dsaD and dsaE gene mutant strains

Example 3

Biological fermentation and detection of marformmycins:

activating wild Streptomyces drozdowiczii SCSIO 10141 or mutant strains delta mfnO and delta mfnH on an ISP2 culture medium (4 g of malt extract, 4g of yeast extract, 4g of glucose, 30g of sea salt, 20g of agar powder, adding water to 1L, pH 7.2) for spore production, and respectively inoculating equal amount of spores to 50mL of m-AM2ab fermentation medium (10 g of soybean meal, 5g of starch, 2g of peptone, 20g of glucose, 2g of yeast extract and K in a 250mL triangular flask₂HPO₄0.5g，MgSO₄·7H₂0.5g of O, 2g of calcium carbonate and 30g of sea salt, adding water to 1L, and carrying out shake fermentation at 28 ℃ and 200rpm under the condition of pH 7.0). After the culture for 7 days, 100mL of butanone is added for extraction, the cells are broken by ultrasonic treatment for 30min, and then the mixture is kept stand for layering. After the butanone extraction liquid and the water phase are separated, sucking the supernatant extraction liquid, evaporating butanone by using a rotary evaporator, dissolving residues in methanol to form a sample, and performing High Performance Liquid Chromatography (HPLC) detection.

The detection conditions are as follows: alttima C18(250 × 4.6mm, 5 μm) reverse phase column, mobile phase a 15% acetonitrile containing 0.03% acetic acid, mobile phase B85% acetonitrile; the flow rate was 1mL/min, and the detection wavelengths were 215nm and 275 nm.

HPLC sample injection program: 0-20min, 0% -100% of phase B; 20-25min, 100% phase B; 25.01-30min, 100% -0% of phase B.

As a result, as shown in FIG. 7, the wild-type producer Streptomyces drozdowiczii SCSIO 10141 produced

compounds

3, 4 and 5, and Δ mfnH produced compounds 3 and 4 containing the L-allo-Ile structural unit but produced

compounds

5 and 7 containing the L-Val structural unit, and the yield of compound 7 was increased by about 100-fold as compared with the wild-type strain (see FIG. 7); Δ mfnO also produced compound 4 containing the L-allo-Ile structural unit at all, but still produced

compounds

5 and 7 containing the L-Val structural unit (see fig. 7).

Example 4

Biological fermentation and detection of desotamides:

will deThe sotamides wild-type producer Streptomyces scopuliperidinis SCSIO ZJ46 was spread on ISP4 medium plates to activate spore production, and the heterologous expression control strains Streptomyces coelicolor M1152, dsaD and dsaE deletion mutant heterologous expression strains Streptomyces coelicolor M1152/07-6H-DKO and Streptomyces coelicolor M1152/07-6H-EKO were spread on MS medium (soybean meal 20g, mannitol 20g, agar powder 20g, water added to 1L, pH 7.2) plates to activate spore production. Equal amount of spores were inoculated into 50mL of m-AM2ab fermentation medium (10 g of soybean powder, 5g of starch, 2g of peptone, 20g of glucose, 2g of yeast extract, K) in a 250mL triangular flask, respectively₂HPO₄0.5g，MgSO₄·7H₂0.5g of O, 2g of calcium carbonate and 30g of sea salt, adding water to 1L, and carrying out shake fermentation at 28 ℃ and 200rpm under the condition of pH 7.0). After the culture for 7 days, 100mL of butanone is added for extraction, the cells are broken by ultrasonic treatment for 30min, and then the mixture is kept stand for layering. After the butanone extraction liquid is separated from the water phase, the butanone extraction liquid on the upper layer is absorbed and evaporated to dryness by a rotary evaporator, and the residue is dissolved in methanol to form a sample for High Performance Liquid Chromatography (HPLC) detection.

HPLC sample injection program: 0-20min, 0% -100% of phase B; 20-25min, 100% phase B; 25.01-30min, 100% -0% of phase B;

as a result, as shown in FIG. 10, Streptomyces coelicolor M1152/07-6H-EKO in which the dasE gene had been mutated by an in-frame deletion did not produce

compounds

8 and 10 containing the L-allo-Ile structural unit at all, but produced

compounds

9 and 11 containing the L-Val structural unit, and the yields of both compounds were greatly increased as compared with the control strain (about 100-fold for compound 9 and about 140-fold for compound 11) (see FIG. 10). Heterologous expression strain Streptomyces coelicolor M1152/07-6H-DKO in which dsaD gene has been mutated by in-frame deletion, although it still produces

compounds

8 and 10 containing L-allo-Ile building block, its yield is greatly reduced; in addition, it still produces

compounds

9 and 11 containing the L-Val structural unit with a greatly improved yield (about 80-fold for compound 9; about 50-fold for compound 11).

Example 5

Expression and purification of aminotransferase DsaD (amino acid sequence of which is shown in SEQ ID No. 7), isomerase DsaE (amino acid sequence of which is shown in SEQ ID No. 8), aminotransferase MfnO (amino acid sequence of which is shown in SEQ ID No. 5), and isomerase MfnH (amino acid sequence of which is shown in SEQ ID No. 6) in e.coli BL21(DE 3):

the dsaD (nucleotide sequence of which is shown in SEQ ID NO. 3), dsaE (nucleotide sequence of which is shown in SEQ ID NO. 4), mfnO (nucleotide sequence of which is shown in SEQ ID NO. 1) and mfnH (nucleotide sequence of which is shown in SEQ ID NO. 2) genes were cloned between NdeI and EcoRI sites of the vector pET28a (+) according to a conventional method to obtain pET28a (+)/dsaD, pET28a (+)/dsaE, pET28a (+)/mfnO and pET28a (+)/mfnH, which were sequenced correctly and then transformed into E.coli BL21(DE3) for expression. The resulting transformant strain was selected for overnight culture of a single clone, inoculated into 200mL of LB medium containing 50. mu.g/mL of kanamycin in a 1L Erlenmeyer flask (1L of LB medium was inoculated in total to each strain) at an inoculum size of 1%, cultured at 37 ℃ with a shaker at 200rpm/min until the OD600 was about 0.6, added with isopropyl-. beta. -D-thiogalactopyranoside (IPTG) at a final concentration of 0.1mM, and induced to express at 25 ℃ for a further 12 to 15 hours. The cells were collected by centrifugation, washed 2 times with 50mL of binding buffer (50mM sodium phosphate, 500mM NaCl, 10mM imidazole, pH 8.0), resuspended in 30mL of binding buffer, sonicated to release the protein, and then centrifuged at low temperature with high speed freezing to remove insoluble fractions. The soluble supernatant fraction was applied to a nickel column HisTrap HT column (1mL, GE Healthcare), and after the filtrate was filtered off, it was washed with 10mL wash buffer 1(50mM sodium phosphate, 500mM NaCl, 50mM imidazole, pH 8.0), then 2mL wash buffer 2(50mM sodium phosphate, 500mM NaCl, 90mM imidazole, pH 8.0), and then eluted with 5mL eluonbuffer (50mM sodium phosphate, 500mM NaCl, 250mM imidazole, pH 8.0). The resulting mixture was concentrated to 2.5mL using an ultrafiltration tube (Millipore,10mL, 3kD), desalted using a PD-10 desalting column (GE Healthcare), stored in 10% glycerol-containing sodium phosphate buffer (50mM, pH 8.0), assayed for protein concentration by the Bradford method, and stored at-80 ℃ for further use, to thereby obtain purified aminotransferase DsaD, isomerase DsaE, aminotransferase MfnO and isomerase MfnH, respectively. The electrophoretogram of the purified protein is shown in FIG. 11.

Example 6

DsaD/DsaE and MfnO/MfnH in vitro enzymatic reactions and detection:

(1) detecting the enzymatic activities of aminotransferase DsaD/isomerase DsaE and aminotransferase MfnO/isomerase MfnH when L-Ile is taken as a substrate: in 100. mu.L of a sodium phosphate buffer (50mM, pH 8.0), 1mM of substrate L-Ile, 5. mu.M of aminotransferase DsaD or MfnO, 5. mu.M of isomerase DsaE or MfnH were added and the mixture was reacted at 30 ℃ for 4 hours.

(2) Detecting the enzymatic activities of aminotransferase DsaD/isomerase DsaE and aminotransferase MfnO/isomerase when L-allo-Ile is taken as a substrate: to 100. mu.L of a sodium phosphate buffer (50mM, pH 8.0), 1mM of substrate L-allo-Ile, 5. mu.M of aminotransferase DsaD or MfnO, 5. mu.M of isomerase DsaE or MfnH were added and the mixture was reacted at 30 ℃ for 4 hours.

After the reaction was completed, 200. mu.L of methanol was added to terminate the reaction, vortexed, left to stand at room temperature for 20 minutes, and then 1,2000 Xg was centrifuged for 20 minutes, the supernatant was evaporated to dryness by a rotary evaporator, and the residue was dissolved in 40. mu.L of 2mM CuSO₄The solution was analyzed by chiral HPLC at 25. mu.L to detect the enzymatic reaction. The chiral HPLC analysis conditions were: a MCI GEL CRS10W column (Mitsubishi, 50X 4.6mm,3 μm) chiral column was used; flow 2mM CuSO₄A solution; the flow rate was 1mL/min, the detection time was 30 minutes, and the detection wavelength was 254 nm.

Example 7

I, catalyzing by using L-Ile as a substrate

(1) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of substrate L-Ile, 5. mu.M of aminotransferase DsaD, 5. mu.M of isomerase DsaE was added and the mixture was allowed to react at 30 ℃ for 4 hours. After the reaction was completed, 20mL of methanol was added to terminate the reaction, vortexed, left to stand at room temperature for 20 minutes, and then 1,2000 Xg was centrifuged for 20 minutes, the supernatant was evaporated to dryness, and the residue was dissolved in 2mM CuSO₄Solution, purified by chiral HPLC, with the conditions: a MCI GEL CRS10W column (Mitsubishi, 50X 4.6mm,3 μm) chiral analysis was used(ii) a The mobile phase is 2mM CuSO₄A solution; the flow rate was 1mL/min, the detection time was 30 minutes, and the detection wavelength was 254 nm. After fractions having a retention time of 13 minutes were pooled and added Ethylene Diamine Tetraacetic Acid (EDTA) to a final concentration of 2mM, the pH of the mixture was adjusted to 4.0. The above pH 4.0 solution was extracted twice with an equal volume of a mixture of n-heptane and di- (2-ethylhexylphosphonic acid) (7:3) and the combined organic phases were collected. The combined organic phases collected above were back-extracted twice with an equal volume of 5% HCl solution and the combined aqueous phases were collected. The combined aqueous fractions were evaporated to dryness and the residue was further purified on a forward silica gel column (isocratic elution with n-butanol-glacial acetic acid-water (4:1: 5)) to obtain the pure enzymatic reaction product L-allo-Ile (V in fig. 12A, i and ii in fig. 12A and 12B are standards for L-Ile and L-allo-Ile, respectively). It is composed of¹The H NMR spectrum is shown in FIG. 13.

Enzymatic reaction product l-allo-Ile HR-ESI-MS [ M + H ]]⁺＝132.1038(calc.for C₆H₁₃NO₂,132.1019)；¹H NMR(500MHz,D₂O),δ0.86(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:¹H NMR(500MHz,D₂O),δ0.86(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 of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of aminotransferase DsaD was added and the mixture was allowed to react at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), the enzymatic reaction product L-allo-Ile (iii in FIG. 12A) was not obtained.

(3) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of substrate L-Ile, 5. mu.M of isomerase DsaE was added, and the mixture was allowed to react at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), the enzymatic reaction product L-allo-Ile (iV in FIG. 12A) was not obtained.

(4) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile was added without adding any enzyme, and the mixture was allowed to react at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), the enzymatic reaction product L-allo-Ile (Vi in FIG. 12A) was not obtained.

(5) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of the aminotransferase MfnO, and 5. mu.M of the isomerase MfnH were added and reacted at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), a pure enzymatic reaction product L-allo-Ile (V in FIG. 12B) was obtained.

(6) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of aminotransferase MfnO was added and the mixture was allowed to react at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), the enzymatic reaction product L-allo-Ile (iii in FIG. 12B) was not obtained.

(7) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of substrate L-Ile, 5. mu.M of isomerase MfnH was added, and the mixture was allowed to react at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), the enzymatic reaction product L-allo-Ile (iV in FIG. 12B) was not obtained.

(8) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile was added without adding any enzyme, and the mixture was allowed to react at 30 ℃ for 4 hours. According to the analysis and structural identification in step (1), the enzymatic reaction product L-allo-Ile (Vi in FIG. 12B) was not obtained.

II, using L-allo-Ile as substrate

(1) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of a substrate L-allo-Ile, 5. mu.M of aminotransferase DsaD, 5. mu.M of isomerase DsaE was added and allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-Ile (V in FIG. 14A, i and ii in FIGS. 14A and 14B are standards for L-Ile and L-allo-Ile, respectively).

(2) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of aminotransferase DsaD was added and the mixture was allowed to react at 30 ℃ for 4 hours, and no enzymatic reaction product L-Ile was obtained (iii in FIG. 14A).

(3) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of the isomerase DsaE was added and the mixture was allowed to react at 30 ℃ for 4 hours, whereby the enzymatic reaction product L-Ile was not obtained (iV in FIG. 14A).

(4) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile was added, and the mixture was left at 30 ℃ without adding any enzyme and reacted for 4 hours, whereby an enzymatic reaction product L-Ile (Vi in FIG. 14A) was not obtained.

(5) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of the aminotransferase MfnO, 5. mu.M of the isomerase MfnH were added and allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-Ile (V in FIG. 14B).

(6) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of aminotransferase MfnO was added and the mixture was allowed to react at 30 ℃ for 4 hours, and no enzymatic reaction product L-Ile was obtained (iii in FIG. 14B).

(7) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of the isomerase MfnH was added and the mixture was allowed to react at 30 ℃ for 4 hours, whereby the enzymatic reaction product L-Ile (iV in FIG. 14B) was not obtained.

(8) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile was added, and the mixture was left at 30 ℃ without adding any enzyme and reacted for 4 hours, whereby an enzymatic reaction product L-Ile (Vi in FIG. 14B) was not obtained.

III,

(1) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of the aminotransferase DsaD, 5. mu.M of the isomerase DsaE was added and the mixture was allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-allo-Ile (iii in FIG. 16A, i and ii in FIGS. 16A and 16B are standards for L-Ile and L-allo-Ile, respectively).

(2) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of the aminotransferase DsaD, 5. mu.M of the isomerase MfnH were added and reacted at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-allo-Ile (iV in FIG. 16A).

(3) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of the aminotransferase MfnO, 5. mu.M of the isomerase MfnH were added and reacted at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-allo-Ile (V in FIG. 16A).

(4) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile, 5. mu.M of the aminotransferase MfnO, 5. mu.M of the isomerase DsaE were added and reacted at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-allo-Ile (Vi in FIG. 16A).

(5) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-Ile was added, and the mixture was left at 30 ℃ for 4 hours without adding any enzyme, whereby an enzymatic reaction product L-allo-Ile (Vii in FIG. 16A) was not obtained.

(6) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of aminotransferase DsaD, 5. mu.M of isomerase DsaE was added and allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-Ile (iii in FIG. 16B).

(7) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of aminotransferase DsaD, 5. mu.M of isomerase MfnH was added and allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-Ile (iV in FIG. 16B).

(8) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of the aminotransferase MfnO, 5. mu.M of the isomerase MfnH were added and allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-Ile (V in FIG. 16B).

(9) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile, 5. mu.M of the aminotransferase MfnO, 5. mu.M of the isomerase DsaE were added and allowed to react at 30 ℃ for 4 hours to obtain a pure enzymatic reaction product L-Ile (Vi in FIG. 16B).

(10) In 10mL of a sodium phosphate buffer (50mM, pH 8.0), 1mM of the substrate L-allo-Ile was added, and the mixture was left at 30 ℃ without adding any enzyme and reacted for 4 hours, whereby an enzymatic reaction product L-Ile (Vii in FIG. 16B) was not obtained.

Example 8

DsaD/DsaE catalyzed reversible reaction equilibrium constant determination:

to 50. mu.L of sodium phosphate buffer (50mM, pH 8.0), add2.5mM of the substrate L-Ile or L-allo-Ile, 0.1mM of PLP, 5. mu.M of aminotransferase DsaD, 5. mu.M of isomerase DsaE were added and the reaction was carried out at 30 ℃ for 4 hours. After the reaction was completed, 200. mu.L of methanol was added to terminate the reaction, vortexed, left to stand at room temperature for 20 minutes, and then 1,2000 Xg was centrifuged for 20 minutes, the supernatant was evaporated to dryness by a rotary evaporator, and the residue was dissolved in 40. mu.L of 2mM CuSO₄The solution was analyzed by chiral HPLC at 25. mu.L to detect the enzymatic reaction. The chiral HPLC analysis conditions were: a MCI GEL CRS10W column (Mitsubishi, 50X 4.6mm,3 μm) chiral column was used; flow 2mM CuSO₄A solution; the flow rate was 1mL/min, the detection time was 30 minutes, and the detection wavelength was 254 nm. The final concentrations of substrate and product were calculated by integrating the corresponding HPLC peaks. The equilibrium constant (Keq) was calculated from the following calculation formula to obtain Keq ([ product concentration ]]/[ substrate concentration)])。

The equilibrium constant of DsaD/DsaE catalyzed reversible reaction was found to be 1.37 when L-Ile was used as substrate (FIG. 15).

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> a strain Δ mfnH for highly producing a compound containing an L-Val structural unit

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1122

<212> DNA

<213> actinomycetes 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> actinomycetes 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> actinomycetes SCSIO ZJ46(Streptomyces scopulieridis 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> actinomycetes SCSIO ZJ46(Streptomyces scopulieridis 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> actinomycetes 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> actinomycetes 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> actinomycetes SCSIO ZJ46(Streptomyces scopulieridis 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> actinomycetes SCSIO ZJ46(Streptomyces scopulieridis 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. The strain delta mfnH for highly producing the compound 7 is characterized in that the strain delta mfnH is obtained by mixing wild streptomyces delbrueckii (Streptomyces delbrueckii) (A. delbrueckii, A. clarkii, AStreptomyces drozdowiczii) The mfnH gene of SCSIO 10141 is obtained by knockout deletion mutation; the structural formula of the compound 7 is shown as a formula 1, wherein R₁＝H，R₂＝CH₃，R₃＝OH；

The nucleotide sequence of the mfnH gene is shown as SEQ ID NO. 2;