CN113337553B - Herbicidin biosynthesis gene cluster and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to a method for improving the yield of fermented herbicidin F and analogues thereof, which comprises the following steps: the HcdR2 gene is overexpressed in host strains for producing herbicidin F and analogues thereof by fermentation. The host strain is streptomyces, and is preferably S. The HcdR2 is a transcription regulatory protein of a LuxR protein family, and the amino acid sequence of the transcription regulatory protein is shown in SEQ ID NO. 1. The invention also relates to a group of novel structurally similar compounds of herbicidin F.

Description

Herbicidin biosynthesis gene cluster and application thereof

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a Herbicidin biosynthesis gene cluster and application thereof.

Background

The application is CN201910471680.8, and the invention name is: the divisional application of Chinese patent application of Herbicidin biosynthesis gene cluster and its application.

Streptomyces mobaraensis US-43 (previously named S.vertecillus var. Pingyangensis n. Var.) is a Streptomyces strain separated from the soil of Pingyang in Zhejiang province, and can produce a series of glycopeptide antibiotics with the same property as bleomycin ^{[ 1-3 ]} Wherein pingyangmycin and boranmycin have been approved by SFDA as anti-tumor agents. As a laboratory deposited strain, we investigated the secondary metabolites of this strain under different fermentation conditions, in the fermentation broth of which piericidin A1 has been found, as well as a series of isocoumarins compounds. In addition, a trace component in the fermentation liquor is detected by an LC-MS method, and the component is identified by UV and MS/MS data and is presumed to be herbicidin F.

Herbicidins (figure 1) are nucleoside antibiotics derived from adenosine, and have a unique furano-pyrano-pyran tricyclic core structure, which is modified by post-processing and can be added with different side chain groups, so that the structure is rich. Reported in s.saponansis ^{[ 4-7 ]} ，S.sp.L-9-10 ^{[ 8 ]} ，S.scopuliridis RB72 ^{[ 9 ]} And s.sp.cb01388 ^{[ 10 ]} From these strains, herbicidins can be isolatedA compound is provided. Herbicidins compounds have a variety of biological activities, and in addition to selective herbicidal activity against dicotyledonous plants ^{[ 4 ]} Also has significant anti-algae and anti-fungal activity ^{[ 6 ]} Recently, a literature has reported that the herbicidin skeleton structure also has anti-Cryptosporidium activity ^{[ 10 ]} . The complex chemical structure and various biological activities of Herbicidins led us to explore structural analogs and biosynthetic mechanisms thereof.

The structure of Herbicidin F has been determined ^{[ 6 ]} However, before the start of the present work, no biosynthetic gene cluster has been reported. Aureonuclein (Figure 1) is a structural analogue of herbicin F, which has the same tricyclic core structure, but has one more tiglyl group and two more methyl groups than aureonuclein in its chemical structure. Subject group of Tang dynasty profit ^{[ 11 ]} A patent reports the biosynthetic gene cluster of aureonuclemycin, which is catalytically synthesized from 4 key genes (anmB, anmC, anmD and anmE), the function of which was verified in a recent paper on the biosynthetic pathway of herbicidins ^{[ 12 ]} The 4 key genes provide important clues for searching and identifying the biosynthesis gene cluster of the herbicidins. Through bioinformatics analysis, we found gene sequences homologous to the above-mentioned 4 key genes in the S.mobaraensis US-43 genome, and successfully located and identified the biosynthetic gene cluster hcd of herbicidin F, which is in turn homologous with her (S.sp.L-9-10) responsible for the biosynthesis of herbicidin F reported ^{[ 13 ]} ) And hbc (S.sp.KIB-027) ^{[ 12 ]} ) The two gene clusters are highly homologous. In addition, overexpression analysis proves that the pathway-specific regulatory factor hcdR2 plays a positive regulatory role in biosynthesis of herbicidin F, and in the hcdR2 overexpression strain, the yield of herbicidin F is remarkably improved, so that convenience is provided for NMR structural identification of herbicidin F.

Meanwhile, we utilize GNPS tool to find novel herbicidins analogues in hcdR2 overexpression strain fermentation broth. Generating a related molecular network diagram according to MS/MS data based on the assumption that similar structures can produce similar fragments in LC-MS ^{[ 14 ]} And visualizing the differences between the molecules ^{[ 15 ]} Thereby more effectively finding similar compounds ^{[ 16-19 ]} . We analyzed the secondary metabolite in the hcdR2 overexpression strain fermentation broth by LC-MS/MS, and further explored herbicidins analogs using GNPS platform. The subject firstly analyzes MS/MS data and nuclear magnetic resonance spectrum (nuclear magnetic resonance spectrum) of 6 novel herbicidins compounds ¹ H NMR) data further confirmed the structure of herbicidin O.

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Disclosure of Invention

The invention firstly relates to a method for improving the yield of fermented herbicidin F and analogues thereof, which comprises the following steps: the HcdR2 gene is overexpressed in host strains for producing herbicidin F and analogues thereof by fermentation.

The host strain is streptomyces, and is preferably S.

The HcdR2 is a transcription regulatory protein of a LuxR protein family, the amino acid sequence of the transcription regulatory protein is shown as SEQ ID NO.1, and the coding nucleotide sequence of the transcription regulatory protein is shown as SEQ ID NO. 2;

SEQ ID NO.1：

MLVGRECELRRLAEVTAGVRQGRSHALVLRGPAGVGKSALLQHLADHAGPGVRVLSTVGVESEAELPFAALHQLLRPLLGAADALPPPQSAALRSAFGMDTAPADRFLVALAALTLLSEASRAEPLILLVDDAHWLDSPSADALLFVARRLGAEGVAVVFAVRDGSQPFPAPGVPDLRLSPLEADAARSLLTHLLPEAAPAVRERLLREAGGNPLALVELSAALSPEQLGGAAALPDELPLTERLQRLFRCRATSLLAEPGNALLLTAAEGDGDLAVIVRAAGDTERALHELSAAATAGLVSLDPERVRFRHPLVRSAVYQGAPLSERRAAHLALADALGEGDDRHVWHLAAAAVGPDDRVAHLLAEMAGRSRRSGGVATATKALRRAAALVSTPRDRARLLVDAAECAWKAAETAQAEALLNEAEPLSDVPALRARLVQVRGAIAHASGDPAVACLILLEGARLVQEDDPRLACETLVMAARSAWVADDPARLAEIAGLLARLPAVTPDVRDRFVAHFRYLAGLSGHAAPDARHDGTALEAHGGGPEGKTGADGKDVPVIWLSGTDPRPWVWPPTFLPYLLHATEPLRDAHQHAVDTLRRNGAIGSLPMSLAPLVALQLVTGPWPTATANGTEALSLALETGQLGAASHLRAMLAWLAAAQGDGDRCHELARESLEISTPRRIASAIALAHWAQGLNALAEGQPLNAVKLLAEVCSPEGGAGHFMLRWIVLPDFVEACVRAGEPERAHHVLTDRDPLHPVPANHPQLRSGWHRSRALLATGEEAEDLFLAALADTGLSPFETGRTHLLYGEWLRRHRRIKPAREQLHLAEAQLDSVGALPWAELARAELRAAGGRSTQGPSTAAVPMGGEQRLTARELQVMRLAAQGLSNGEIAARLFLSPRTVGYHLYKIFPKLGVTSRSQLYGRSSG。

the invention also relates to a group of compounds similar to herbicidin F, and the chemical structure of the compounds is shown as the following formula (1):

r1 is: -CHO-R4, R4 is a straight or branched chain alkane containing 1 to 4C or-C (CH) ₃ )＝CHCH ₃ ；

R2 and R3 are H or CH ₃ ；

Preferably, R1 is:

most preferably, the analog is the following compound:

(1) R1 is

R2 is CH3, R3 is H, molecular weight 522;

(2) R1 is

R2 is CH3, R3 is CH3, and the molecular weight is 510;

(3) R1 is

Molecular weight 510 or 524;

(4) R1 is

Molecular weight 524;

the invention also relates to a method for the fermentative production of said herbicidin F and of compounds analogous to herbicidin F, said method being:

(1) Overexpresses the HcdR2 gene in a host strain;

(2) Producing the host strain by fermentation;

(3) Isolating said herbicidin F and analogs thereof.

The method for producing the host strain by fermentation in the step (2) comprises the following steps: a two-stage liquid fermentation method is adopted, and a fermentation medium comprises: 0.3% high nitrogen corn steep liquor dry powder, 2% soybean cake powder, 2.5% glucose, 2% starch, 2% maltose, 0.2% ₂ HPO ₄ ，0.3％NaCl。

The secondary liquid fermentation parameters are as follows:

inoculating spores of the host strain into seed fermentation liquor, and culturing at 28 ℃ and 220rpm for 48 hours;

inoculating the seed fermentation liquid into a liquid fermentation culture medium, and culturing for 7 days at the temperature of 28 ℃ and the rpm of 220.

The separation in the step (3) is as follows: supernatant of fermentation liquor C ₁₈ The column was enriched and eluted with 50% and 100% methanol solutions, respectively.

The invention also relates to the application of the herbicidin F analogue in preparing a medicament, wherein the medicament is an antifungal or tumor inhibiting medicament.

Drawings

FIG. 1 chemical structures and biosynthetic gene clusters for Aureoneclemycin and herbicidin F.

(A) Chemical structures of Aureoccumulin and herbicidin F (Compound 1).

(B) Biosynthetic gene clusters of Aureoneclemycin and herbicidin F anm and hcd; hbc and her are reported biosynthetic gene clusters of herbicidins. The place marked with lines above the gene cluster is the intergenic region for FIMO analysis. Black lines indicate that the spacer contains a consensus binding sequence; the grey line indicates the consensus sequence for which the spacer has no match.

FIG. 2, effects of overexpression of transcription regulators on the biosynthesis of herbicidin F and gene expression profile.

(A) HPLC analysis of S.mobaraensis US-43 and the yield of herbicidin F in the over-expressing strain. Mobaraensis US-43; ii, US43/pSET152; iii, US43/pL-hcdR2; iv, US43/pL-hcdR1; v, US43/pL-hcdR3.

(B-D) transcriptional expression profiles of different genes in the overexpression strains US43/pL-hcdR2 (B), US43/pL-hcdR1 (C) US43/pL-hcdR3 (D). The data obtained were all from three biological replicates and each sample had two replicate wells. The transcriptional expression level of the gene in the control strain US43/pSET152 was defined as 1.

FIG. 3, using genome mining to find another 15 hcd-like biosynthetic gene clusters in GenBank. The area above the cluster marked by lines is the intergenic region for FIMO analysis. Black lines indicate that the spacer contains a consensus binding sequence; the grey line indicates the consensus sequence for which the spacer has no match.

FIG. 4, results of an alignment analysis of DNA binding domains in LuxR regulatory protein and predicted consensus binding sequences. (A) The DNA binding domain in HcdR2 was aligned to the DNA binding domain of the homologous protein in different strains. The sequence marked by the red box is a consistent sequence; huang Kuang denotes sequences with similar physicochemical properties. The top secondary structure sequence is derived from the three-dimensional structure of TraR (PDB 1L 3L). GenBank accession numbers for these LuxR proteins are as follows S.sp.L-9-10 (Her 12, RYJ 25228.1), S.scopuliridinis RB72 (PVE 09951.1), S.sp.NRRL F-5135 (WP _ 078854789.1), S.acidiscabis strain a10 (GAQ 51936.1), S.sp.V2 (PWG 13438.1), S.ipomoee 91-03 (EKX 64212.1), S.sp.NRRL S-31 (WP _ 079165723.1), S.mobaraensis NBRC 13819 (01239.1), S.sp.CB02959 (PJN 35945.1), S.albulus RC 3214 (EPY 323264), S.zxft 3282, and DSM 3434B 3434. Binding sites for S.sp.sp.sp.NRxR proteins are consistent with each other. The reverse arrows indicate binary symmetry.

FIG. 5, GNPS tools guide the discovery of novel herbicidin analogs. (A) The molecular compounds in the hcdR2 overexpression strain are detected to form a molecular network by an LC-MS method. (B) And (3) searching potential herbicidin analogues by taking herbicidin F as a target, and forming a molecular network diagram. The molecular network diagram contains excimer ions [ M + H ]] ⁺ m/z values from 508 to 536And (4) adding the active ingredients. (C) According to the GNPS analysis result, 10 herbicidin peaks (1-10) are found in the LC-MS spectrum.

FIG. 6, structures of 10 herbicidin analogues and (+) -ESI-MS/MS data. (A) MS/MS analysis results of 10 compounds. The parent ion is indicated by a dashed line and is the same color as the corresponding molecular weight color in GNPS. The figure shows the diagnostic ion peak. (B) The temporary structure of the compound 1-9, the herbicidin analogue with red font is a novel herbicidin analogue.

FIGS. 7 and 7 of Compound 1,2,3 ¹ H NMR comparison (solvent DMSO-d) ₆ )。

Fig. 8, s. Mobaraensis US-43 genome sketch and bioinformatics analysis predicted results.

FIG. 9 MS/MS fragment of compound herbicidin F

FIG. 10 HPLC, UV and HRMS spectra of Compounds 1,2,3

Detailed Description

Material method

Strains, plasmids and culture conditions

Wild-type s.mobaraensis US-43 and also derivatives thereof are listed in table 2 herein.

TABLE 2 strains and plasmids

Am ^r Apramycin (apramycin) resistance; km ^r Kanamycin (kanamycin) resistance; cm ^r Chloramphenicol (Chloramphenicol) resistance

Wild S.mobaraensis US-43 is a streptomycete separated from the Pingyang soil of Zhejiang, china, and the wild S.mobaraensis US-43 and its derivative are cultured at 28 deg.C for 7 days, and solid S5 culture medium is used for propagation culture of the strain ^{[ 32 ]} MS culture for conjugation transferNutrient medium ^{[ 33 ]} The strain uses liquid phi culture medium when extracting genome DNA ^{[ 34 ]} 。

Synthesis of the Compound herbicidin F by two-stage liquid fermentation, liquid seed fermentation Medium (0.3% high-nitrogen corn steep liquor dry powder, 2% Soybean cake powder, 2.5% glucose, 2% starch, 2% maltose, 0.2% K% ₂ HPO ₄ 0.3% NaCl) and fermentation medium (consistent with liquid seed fermentation medium) were used for primary and secondary fermentations, respectively.

Escherichia coli DH5α ^{[ 35 ]} Coli ET12567/pUZ8002 as a universal E.coli cloning host ^{[ 36 ]} Is used for the conjugation transfer between the escherichia coli and the streptomycete ^{[ 33 ]} The host of (1), both of which use LB medium ^{[ 35 ]} Culturing at constant temperature of 37 ℃. When antibiotics are required, the working concentrations are as follows: apramycin (apramycin, am, 50. Mu.g/ml), ampicillin (ampicilin, amp, 100. Mu.g/ml), kanamycin (kanamycin, km, 50. Mu.g/ml), chloramphenicol (chloramphenicol, cm, 25. Mu.g/ml).

Construction of hcdR1, hcdR2 and hcdR3 gene overexpression plasmids

To overexpress the hcdR1 gene in s.mobaraensis US-43, the complete hcdR1 gene sequence was amplified using primers pL-hcdR1-F/pL-hcdR 1-R.

The PCR product of the hcdR1 gene was cloned into the NdeI-BamHI site of pL 646. The vector pL646 is derived from the integrative plasmid pSET152, which does not contain a streptomyces replicon, contains a Φ C31 attP site, can integrate into an attB site in the streptomyces genome, and contains the strong promoter ermE × p and the SD sequence of the tuf1 gene upstream of the multiple cloning site.

Using the same strategy, the hcdR2 and hcdR3 genes were cloned into pL646 at NdeI-BamHI and NdeI-XbaI sites, respectively. Thus, over-expression recombinant plasmids pL-hcdR1, pL-hcdR2 and pL-hcdR3 were obtained, and the recombinant plasmids were introduced into E.coli ET12567/pUZ8002 by transformation and respectively introduced into S.mobaraensis US-43 by conjugative transfer. Plasmid pSET152 was simultaneously introduced into S.mobaraensis US-43 as a control.

Analysis of the product herbicidin F

Mobaraensis US-43 wild-type strain and its derivative strain were incubated in solid S5 medium at 28 ℃ for 7 days. Spores of S.mobaraensis US-43 and its derivatives were inoculated into 100mL of seed fermentation broth and cultured at 28 ℃ and 220rpm for 48 hours. 5mL of seed fermentation broth was inoculated into 100mL of liquid fermentation medium. The liquid fermentation culture was carried out at 220rpm at 28 ℃ for 7 days. The supernatant obtained was analyzed by LC-MS and for the analysis of the analogues of herbicidin F, the supernatant of the fermentation broth was collected by Sep-Pak C ₁₈ Classic cards (Waters Associates) were enriched and eluted with 50% and 100% methanol solutions, respectively. The samples were subjected to HPLC detection by Agilent 1100 instruments (Agilent Technologies, santa Clara, calif., USA) using C18 column (Agilent, 150 mm. Times.4.6 mm,5 μm) with detection wavelengths of 210nm and 254nm. The detection conditions were as follows, mobile phase: CH (CH) ₃ OH-H ₂ O; flow rate: 1mL/min; using gradient elution, 0-5min,5% of CH ₃ OH；5-45min，5％-100％CH ₃ OH；45-55min，100％；56-60min，5％CH ₃ OH。

RT-PCR transcriptional analysis

The cells of S.mobaraensis US-43 cultured for 48 hours in the fermentation medium were collected and cryopreserved in liquid nitrogen. According to the extraction method (Promega), the RNA of the strain was extracted with TRIzol reagent, and the remaining chromosomal DNA was removed by DNaseI treatment. The mass and concentration of the extracted RNA was determined using a NanoDrop 8000 spectrophotometer (Thermo Scientific). 2 μ g of total RNA was used as a template for reverse transcription using

One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen) amplified gene fragments from target genes, detected by Real-Time PCR Detection System (Bio-Rad), each reaction System containing 12.5. Mu.L FastStart Universal

Green Master (ROX) (Roche), 2.5. Mu.L of template, 5. Mu.L of primer,5μL RNase-free H ₂ O。

bioinformatics analysis

And (3) digging a potential herbicidin/aureonuclemycin biosynthesis gene cluster by using the hcdB/C/D/E as a target point and utilizing BLASTP. Each gene in each gene cluster is labeled and annotated, and its three-dimensional structure and conserved domains are analyzed using HHpred and BLASTP. The intergenic region in each gene cluster was picked and submitted to the MEME Suite river for MEME-ChIP analysis. The highest scoring sequences in each gene cluster were collected and submitted for MEME analysis to obtain one consensus sequence. To further validate the consensus sequences found, FIMO analysis was performed on all intergenic regions to find individuals that matched the consensus sequence.

Global Natural product Social Molecular Networking(GNPS)

To obtain LC-MS/MS data for GNPS analysis, the US-43/pL-hcdR2 fermentation broth was enriched using a macroporous adsorbent resin 4006 column, and eluted with 30% and 80% aqueous acetone solutions, respectively. The 80% acetone eluate was concentrated under pressure and fractionated by means of ODS column. Fractions containing herbicidins were synthesized as crude extracts. The crude extract was analyzed by LC-MS using a VP-ODS column (150 mm. Times.4.6 mm,5 μm, SHIMADZU) by Agilent 1200 (Agilent Technologies, santa Clara, calif., USA) and an ion trap mass spectrometer (Thermo Fisher Scientific, waltham, MA, USA) at a flow rate of 1mL/min for a gradient elution of 60 minutes (in agreement with the above). LC-ESI (+) MS/MS data was obtained as a 35eV collision energy as a raw file format, converted to a.mzxml file format using MS conversion program of proteo wizard 3.0 and uploaded to massvie server (masse.

According tohttps://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash2.jspThe website provides guidance to analyze the data using the GNPS molecular web tool.

Purification and characterization of Compounds 1-3

In order to separate and purify to obtain the novel herbicidin analogue, the hcdR2 overexpression strain US43/pL-hcdR2 is subjected to expanded fermentation. The broth was centrifuged to remove the mycelium, and the supernatant (about 4L) was passed through a column of macroporous adsorbent resin 4006 (about 400 mL)After washing with water, the active substance was eluted with a gradient (30%, 80%,100% aqueous acetone) to give three fractions, fr 1 to Fr 3. Based on HPLC analysis, we found herbicidin F and its analogs in Fr 2 (1.197 g). We used a reverse phase flash column (RediSep column:40g C) ₁₈ ) Fr 2 was separated with a 14.8% acetonitrile aqueous solution containing 0.01% TFA to obtain 5 fractions Fr 2-1 to Fr 2-5. Fr 2-3 (262 mg) was isolated and purified by semi-preparative HPLC (Repuril-Pur Basic-C18 column,5 μm, 250X 10 mm) to give compounds 1 (11 mg), 2 (1 mg), 3 (0.5 mg). The sample was analyzed using a SHIMADZU LC-20A instrument with a Repsil-Pur Basic-C18 column (5 μm, 150X 4.6 mm) and the same eluent as for semi-preparative HPLC at a detection wavelength of 254nm. HRESIMS data set

Of G2-SQTof

And detecting by UPLC. NMR data are obtained by Bruker spectrometer detection, and the solvent of the compound is DMSO-d ₆ Or CD ₃ OD。

Process for preparation of Compound 1 ¹ H and ¹³ C NMR: ¹ H NMR(600MHz,CD ₃ OD)δ8.36(s,1H,H-2),8.09(s,1H,H-8),6.71(q,J＝7.1Hz,1H,H-3”),6.09(d,J＝1.6Hz,1H,H-1’),5.00(d,J＝3.2Hz,1H,H-8’),4.52(dd,J＝10.3,5.7,1H,H-6’)and 4.50(d,J＝1.8Hz,1H,H-3’),4.45(s,1H,H-10’),4.41(q,J＝2.4Hz,1H,H-4’),4.30(dd,J＝3.2,1.0Hz,1H,H-9’),4.08(d,J＝1.1Hz,1H,H-2’),3.61(s,3H,H-11’-OCH3),3.41(s,3H,H-2’-OCH3),2.30–2.22(m,2H,H-5’),1.90(d,J＝7.0Hz,3H,H-4”),1.85(s,3H,H-5”). ¹³ C NMR(150MHz,CD ₃ OD)δ171.4(C-11’),167.2(C-1”),154.4(C-6),150.3(C-2),149.5(C-4),142.1(C-3”),141.8(C-8),128.6(C-2”),119.9(C-5),93.5(C-7’),91.8(C-2’),89.1(C-1’),79.4(C-4’),78.4(C-10’),74.7(C-3’),72.0(C-8’),70.6(C-9’),66.7(C-6’),58.5(C-2’-OCH3),52.8(C-11’-OCH3),26.7(C-5’),15.2(C-4”),12.5(C-5”)。

Process for preparation of Compound 1 ¹ H NMR (tautomerism of hemiacetal and free carbonyl forms): ¹ H NMR(500MHz,DMSO)δ8.37(s,1H,H-2)/,8.35(s,1H,H-2),8.19(s,2H,H-NH ₂ )7.88(s,1H,H-8),6.63(qd,J＝6.9,1.2Hz,1H,H-3”),5.93(d,J＝1.9Hz,1H,H-1’)/6.00(d,J＝1.6Hz,1H,H-1’),4.91(d,J＝3.1Hz,1H,H-8’),4.46(s,1H,H-10’),4.40(d,J＝2.2Hz,1H,H-3’),4.33(dd,J＝11.6,5.4Hz,1H,H-6’),4.21(d,J＝1.8Hz,1H,H-4’),4.17(s,1H,H-2’)4.16(dd,J＝3.1,0.9Hz,1H,H-9’),3.50(s,3H,H-11’-OCH ₃ )/3.67(s,3H,H-11’-OCH ₃ ),3.32(s,3H,H-2’-OCH ₃ )/3.34(s,3H,H-2’-OCH ₃ ),2.16–2.05(m,2H,H-5’),1.89(dd,J＝7.1,1.0Hz,3H,H-4”),1.81(s,3H,H-5”).

process for preparation of Compound 2 ¹ H NMR (tautomerism of hemiacetal and free carbonyl forms): ¹ H NMR(600MHz,DMSO-d ₆ )δ8.33(s,1H,H-2)/8.30(s,1H,H-2),8.04(s,2H,H-NH ₂ )7.90(s,1H,H-8)/7.88(s,1H,H-8),6.61(dd,J＝13.7,6.8Hz,1H,H-3”),5.91(s,1H,H-1’)/5.88(s,1H,H-1’),4.97(d,J＝4.1Hz,1H,H-8’)/4.90(d,J＝3.1Hz,1H,H-8’),4.64-4.18(m,6H,H-4’,H-6’,H-10’,H-2’,H-3’,H-9’,signals from hemiacetal form and free carbonyl form overlapped with each other),3.50(s,3H,H-11’-OCH ₃ )/3.48(s,3H,H-11’-OCH ₃ ),2.24–1.93(m,2H,H-5’),1.88(d,J＝7.1Hz,3H,H-4”)/1.85(d,J＝7.1Hz,3H,H-4”),1.82(s,3H,H-5”)/1.81(s,3H,H-5”H).

process for preparation of Compound 3 ¹ H NMR (tautomerism of hemiacetal and free carbonyl forms): ¹ H NMR(500MHz,DMSO-d ₆ )δ13.00(s,1H,H-COOH),8.26(s,1H,H-2),7.81(s,1H,H-8),7.72(s,2H,H-NH ₂ ),6.72(m,1H,H-3”),5.95(d,J＝1.9Hz,1H,H-1’)/5.92(d,J＝1.9Hz,1H,H-1’),4.97(d,J＝4.1Hz,1H,H-8’)/4.92(d,J＝3.1Hz,1H,H-8’),4.57-4.14(m,6H,H-4’,H-6’,H-10’,H-2’,H-3’,H-9’,signals from hemiacetal form and free carbonyl form overlapped with each other),3.34(s,3H,H-2’-OCH ₃ )/3.32(s,1H,H-2’-OCH ₃ ),2.20–1.93(m,2H,H-5’),1.89(dd,J＝7.0,0.9Hz,2H,H-4”)/1.87(dd,J＝7.1,0.8Hz,2H,H-4”),1.81(d,J＝1.0Hz,3H,H-5”).

example 1 identification of the herbicidin F biosynthetic Gene Cluster (hcd) in S.mobaraensis US-43 by bioinformatics analysis

Mobaraensis US-43 is a mature strain (CPCC 204095) studied in the laboratory, and the research on metabolites of the strain for many years shows that the strain can produce a large amount of other types of compounds besides glycopeptide antibiotics. In order to systematically and deeply discover other types of metabolites, we determined the whole genome sequence of S.mobaraensis US-43, and predicted that its genome contains at least 47 secondary metabolite biosynthetic gene clusters by using the anti SMASH 4.0.0 software ^[20] . By performing LC-MS analysis on the strain fermentation liquor, a trace component compound 1 is detected, and through UV and MS/MS data analysis, the compound 1 is presumed to be herbicidin F.

The structure of Herbicidin F has been determined, but its biosynthetic pathway has not been reported. The compound aureonuclemycin (fig. 1A) has the same three-ring core skeleton as herbicidin F, and in order to study the biosynthetic gene cluster of herbicidin F, the key genes (anmB, anmC, anmD and anmE) (fig. 1B) for aureonuclemycin biosynthesis were targeted, and the genome of s.mobaraensis US-43 was searched for the possible presence of herbicidin biosynthetic gene cluster. We found the corresponding homologous sequences of the four key genes (anmcbde) in one gene cluster of s.mobaraensis US-43 (fig. 1B) by BLASTP analysis and anti smash prediction. Two methyltransferases downstream of the homologous sequence, which we speculate are responsible for methylation modifications in the herbicidin F structure; upstream of this sequence there is a β -ketoacyl synthetase, which we speculate to be involved in the biosynthesis of the side chain tiglyl group in the herbicidin F structure; besides, the near-field of the gene chip is also provided with a transport protein, three transcription regulatory factors and other proteins. We named this gene cluster hcd, presumably responsible for the biosynthesis of herbicidin F. Functional annotations of the gene cluster hcd are shown in Table 2.

TABLE 2 functional notes on the Gene cluster hcd

Example 2 confirmation of hcd midpathway-specific regulatory protein

The predicted hcd gene cluster contains three transcriptional regulatory genes hcdR1, hcdR2 and hcdR3, an over-expression strain of each transcriptional regulatory gene is constructed, the yield change of herbicidin F in fermentation liquor of the over-expression strain is detected by HPLC (high performance liquid chromatography), whether the predicted gene cluster hcd is responsible for the biosynthesis of the herbicidin F is judged, and whether the hcdR1, the hcdR2 and the hcdR3 are specific regulatory genes of the biosynthesis intermediate path of the herbicidin F is further determined. We cloned each transcriptional regulatory gene into pSET152 ^{[ 21 ]} Derived expression plasmid vector pL646 ^{[ 22 ]} It contains a φ C31 attP site, which can integrate into the attB site in the Streptomyces genome, and a strong promoter ermE. P and the SD sequence of the tuf1 gene upstream of the multiple cloning site. Respectively introducing the constructed overexpression plasmids pL-hcdR1, pL-hcdR2 and pL-hcdR3 into a wild type S.mobaraensis US-43 by means of conjugative transfer to obtain overexpression strains US43/pL-hcdR1, US43/pL-hcdR2 and US43/pL-hcdR3. Simultaneously, plasmid pSET152 was introduced into S.mobaraensis US-43 wild-type strain to obtain US43/pSET152 strain as an experimental control strain. Wild type strain S.mobaraensis US-43, control strain US43/pSET152, over-expression strain US43/pL-hcdR1, US43/pL-hcdR2 and US43/pL-hcdR3 were fermented under the same conditions, the fermentation broth was treated in the same manner, and the supernatant of the fermentation broth was analyzed by HPLC detection.

The HPLC analysis result shows that the yield of the compound 1 is remarkably improved only in the over-expression strain US43/pL-hcdR2 (figure 2A) relative to the wild strain S.mobaraensis US-43 and the control strain US43/pSET152, thereby providing convenience for the separation and purification of the compound 1. Compound 1 was analyzed by MS/MS data and compared to the reported NMR spectra to determine Compound 1 as herbicidin F.

The trace components in the strain fermentation liquor are confirmed to be herbicidin F, and only in the hcdR2 overexpression strain US43/pL-hcdR2 fermentation liquor, the yield of the herbicidin F is obviously improved, which indicates that the hcdR2 may be a pathway-specific forward regulatory factor in the biosynthesis of the herbicidin F.

Next, we used quantitative RT-qPCR technology to detect the transcriptional expression of genes putatively responsible for the biosynthesis of herbicidin F in over-expressed strains. Relative expression of each gene in the gene cluster hcd at 48h of fermentation is detected, and analysis results show that compared with a control strain US43/pSET152, the relative expression levels of regulatory genes hcdR2 and hcdB-T are obviously improved in an hcdR2 over-expression strain US43/pL-hcdR2; there was essentially no change in the relative expression levels of hcd1, hcd2 and hcd3 (FIG. 2B).

In US43/pL-hcdR1 and US43/pL-hcdR3, the relative expression levels of the regulatory genes hcdR1 and hcdR3 were significantly upregulated, and the relative expression levels of the other genes were not significantly changed (FIG. 2C, 2D). The relative expression of the gene detected by quantitative RT-qPCR was consistent with the level of synthesis of herbicidin F in the over-expressed strain detected by HPLC, indicating that only hcdR2 was the pathway-specific forward regulatory gene in the biosynthesis of herbicidin F among hcdR1, hcdR2 and hcdR3.

Meanwhile, the relative expression level of the hcd gene in the over-expression strain US43/pL-hcdR2 shows that hcdB-H and hcdT are responsible for biosynthesis of the compound herbicidin F; the relative expression levels of hcd 1-3 did not change significantly, indicating that hcd 1-3 may not be involved in the biosynthesis of herbicidin F.

Example 3 HcdR2 is a relatively conserved pathway-specific regulatory protein in herbicidin biosynthesis

The pathway-specific regulatory protein HcdR2 belongs to the LuxR family and has a canonical helix-turn-helix (HTH) DNA Binding Domain (DBD) at the C-terminus of the regulatory protein by BLASTp analysis. We searched GenBank for similar sequences targeting hcdB/C/D/E and found that at least 15 actinomycetes contained the hcd/hbc/her/anm similar gene cluster (FIG. 3), and among them 11 gene clusters contained transcriptional regulatory proteins belonging to the LuxR protein family. Of these LuxR family regulatory proteins, the remaining LuxR regulatory proteins, except for the LuxR protein within the strain s. Mobaraensis NBRC 13819 gene cluster (99.78% amino acid similarity to HcdR2 alignment analysis), showed 30-43% amino acid similarity over the full-length range as compared to HcdR 2. HHpred and BLASTp analyses of these LuxR regulatory proteins showed that their three-dimensional structures share high similarity, including two relatively conserved regions, the N-terminal AAA ATPase domain and the C-terminal helix-turn-helix (HTH) DNA Binding Domain (DBD).

Furthermore, the analysis of the DBD domain of these HcdR2 homologous proteins aligned with the DBD domain of the LuxR family regulator TraR showed overall homology and used online ESPript ^{[ 23 ]} The platform displays the four-helical domain of HTH motif. The HTH domain regulates gene expression by binding to specific DNA sites near the promoter of interest. We used online MEME Suite 5.0.4 ^{[ 24-26 ]} The program analyzed the intergenic region in each gene cluster to find possible HcdR2 consensus binding sequences.

Firstly, the MEME-ChIP of MEME-Suite is used for analyzing the selected 48 intergenic regions, and the HcdR2 consistent binding sequence with the highest score is selected from the analysis results. This consensus binding sequence has a potential palindromic sequence with LuxR ^{[ 27 ]} ，TraR ^{[ 28 ]} ，LasR ^{[ 29 ]} ，QscR ^{[ 30 ]} The known binding sites of isoregulatory proteins are identical. We then scanned 48 intergenic regions using FIMO to find sequences that could match motif. The search resulted in 30 promoter regions that could be matched to motifs and ordered by p-value (fig. 1 and 3). In a search of 14 gene clusters including hcd, her, anm, at least one of these gene clusters was found to have a 21-bp consensus binding sequence and was mostly located in the promoter region of hcdB (fig. 4B). These results suggest that homologous proteins of HcdR2 might regulate the synthesis of herbicidin analogs by binding to consensus sequences in different strains. Interestingly, we found concordant knots in 4 specific gene clustersThe homologous protein of HcdR2 is not found near the sequence, but the homologous protein of HcdR2 is found at other positions in the genome of 3 strains. We also found binding sites in the anmB promoter region of the gene cluster anm. These results show that the mechanism of specific regulation of the herbicidins biosynthetic pathway may be conserved among different strains containing the hcd/hbc/her/anm gene cluster. This suggests that we could activate a new hcd/hbc/her/anm gene cluster by over-expressing the HcdR2 homologous protein.

Example 4 discovery of novel herbicidin analogs in hcdR2 overexpressing Strain fermentation broths Using molecular network tools

In the hcdR2 overexpression strain US43/pL-hcdR2, the yield of herbicidin F is obviously improved, and meanwhile, some herbicidin trace analogues which cannot be detected in the fermentation liquor of S.mobaraensis US-43 wild strain are found in the fermentation liquor of the overexpression strain US43/pL-hcdR 2. GNPS is an effective tool for finding new compounds, and we use it to analyze and search herbicidin analogues in fermentation broth of hcdR2 overexpression strain. Crude extracts of the fermentation broth of the US43/pL-hcdR2 strain were analyzed by Agilent 1200 (Agilent Technology, santa Clara, calif., USA) coupled LTQ XL ion trap mass spectrometer. And uploading the obtained LC-MS/MS data to a MassiVE platform (MassIVE. Ucsd. Edu), and analyzing by using a molecular network tool based on GNPS to generate a molecular network diagram. The use of Cytoscape V3.5.1 ^{[ 31 ]} The tool visualizes molecular network relationships. By analysis of crude extracts of the fermentation broth of strain US43/pL-hcdR2, a network of molecules centered on the compound herbicidin F was plotted, which contained 10 different molecules with m/z values from 508 to 536 (FIG. 5). We analyzed the LC-ESI (+) MS (FIG. 5) and ESI (+) -MS/MS (FIG. 6A) data for these compounds, and identified 4 known herbicidins compounds ( compounds 1,2/3,4 and 5) and 6 novel herbicidins structures (compound 3/2,6-10) (FIG. 6B).

Compound 1, molecular weight 536, was confirmed to be herbicidin F according to its MS/MS fragmentation pattern (FIGS. 6, 9). In the ESI (+) -MS/MS profile of Compound 1, the two peaks at m/z values 418 (F3) and 283 (F6) are respectivelyCorresponding to [ M-tigly-2H ₂ O+H] ⁺ And [ M-tegly-alkenyl-2H ] ₂ O+H] ⁺ These two peaks can be used as diagnostic ion peaks. The peaks having M/z values of 518, 500, 454 (F1), 436 (F2), 319 (F4) and 301 (F5) correspond to [ M-H ] respectively ₂ O+H] ⁺ ，[M-2H ₂ O+H] ⁺ ，[M-tigly+H] ⁺ ，[M-tigly-H ₂ O+H] ⁺ ，[M-tigly-adenyl+H] ⁺ And [ M-tigly-alkenyl-H ₂ O+H] ⁺ And the compounds, together with F3 and F6, form characteristic peaks in the MS/MS spectrum of the herbicidins compound.

Compounds 2 and 3 have similar MS/MS spectra as herbicidin F (1), with compounds 2 and 3 having the same excimer ion peak and fragment and 14Da less than compound 1 (FIG. 6), indicating that at R ₂ Or R ₃ Methyl groups may not be present. To further analyze their structures, compounds 2 (1 mg) and 3 (0.5 mg) were isolated and purified. Compounds 2 and 3 have a maximum absorption wavelength at 260nm, consistent with the nucleoside characteristic absorption spectrum (FIG. 10). According to high-resolution electrospray ionization mass spectrum (HR-ESIMS) [ M + H ]] ⁺ m/z value of 522.1857, confirming that compounds 2 and 3 have the same formula C ₂₂ H ₂₇ O ₁₀ N ₅ One CH less than herbicidin F ₂ This further confirms the structure at R ₂ Or R ₃ Presume that the position is one less methyl. By using ¹ Further determination of the position of the methyl group by H NMR spectra, by collection of Compounds 2 and 3 ¹ H NMR spectrum, getting hydroxyl proton signal which can help to determine methyl position. In DMSO-d6, there are two groups for both compounds 2 and 3 ¹ H NMR signals, which are probably constructed by the presence of hemiketal structures and the presence of intervarietal forms of the free carbonyl forms in herbicidins, are reported ^[13] At D ₂ In O, tautomerism exists between a hemiketal structure forming a B ring in a herbicidin structure and a free carbonyl form thereof, and a map can be caused to present two sets of nuclear magnetic signals. For ease of comparative analysis, the solvent for Compound 1 was changed to DMSO-d ₆ . By comparing compounds 2 and 1 ¹ H NMR spectrum (FIG. 7) and found to lack H-2' -OCH in Compound 2 ₃ Signal, confirmation of the Compound2 at R ₂ Lacks a methyl group and has the same structure as herbicidin K. By comparing compounds 3 with 1,2 ¹ H NMR spectrum (FIG. 7) and found that Compound 3 lacks H-11' -OCH ₃ Signal, containing H-11' -COOH signal, confirmed Compound 3 at R ₃ Lacks a methyl group and contains a free carboxyl group at C-11', and compound 3 was analytically identified as a novel analog of herbicidin F, designated herbicidin O.

The MS/MS fragmentation of compounds 4-10 compared to compounds 1-3 (FIG. 6) was analyzed, and the structures of compounds 4-10 were preliminarily deduced. Compound 4 was identified as herbicidin G and compound 5 as herbicidin B based on the excimer peaks (28 Da and 82Da smaller than Compound 1, respectively) and the diagnostic fragment of Compounds 4 and 5. Compounds 6 and 7 have the same F3 ([ M-tigy-2H ] as compounds 2 and 3 ₂ O+H] ⁺ ,m/z 404)、F6([M-tigly-adenyl-2H ₂ O+H] ⁺ M/z 269) fragment indicating that compounds 6, 7 and compounds 2,3 are at R ₁ The substituents at (A) are different. The molecular weight of Compound 6 was 14Da less than that of Compound 7, indicating that R for Compound 6 ₁ Which lacks a methyl group and contains an isobutyl group, is the same as the substituent for herbicidin E. Since the amount of compounds 6 and 7 obtained by purification is relatively small, it has not been possible to confirm that the methyl group is positioned at R ₂ Or R ₃ . Compounds 8 and 9 have the same F3 ([ M-tigy-2H) as Compound 1 (herbicidin F) ₂ O+H] ⁺ ,m/z 418)、F6([M-tigly-adenyl-2H ₂ O+H] ⁺ M/z 283) lysis fragment, except R ₁ The substituents at (A) are different. The molecular weight of Compound 8 was 26Da less than that of herbicidin F, indicating that Compound 8 is at R ₁ The substituent at a position is propionyl. The excimer ion of Compound 9 was 12Da smaller than herbicidin F, indicating that R of Compound 9 ₁ May contain an isobutyl group, the same substituent as herbicidin E and Compound 6. The excimer ion of Compound 10 is 18Da smaller than herbicidin F and has the same diagnostic ion fragment F3 ([ M-tigy-2H) as herbicidin F ₂ O+H] ⁺ ,m/z 418)、F6([M-tigly-adenyl-2H ₂ O+H] ⁺ M/z 283) indicating that there is one H in Compound 10 ₂ The O molecule is lost.The structures of these herbicidins analogues cannot be completely defined due to the relatively small amounts of the compounds obtained by isolation and purification, but GNPS does exert a great advantage as an effective tool for the discovery of herbicidins analogues.

Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of the present invention.

SEQUENCE LISTING

<110> institute of medical and Biotechnology of Chinese academy of medical sciences

<120> Herbicidin biosynthesis gene cluster and application thereof

<130> CP121020046C（CP11902318C-DIV）

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 930

<212> PRT

<213> HcdR2 amino acid sequence

<400> 1

Met Leu Val Gly Arg Glu Cys Glu Leu Arg Arg Leu Ala Glu Val Thr

1 5 10 15

Ala Gly Val Arg Gln Gly Arg Ser His Ala Leu Val Leu Arg Gly Pro

20 25 30

Ala Gly Val Gly Lys Ser Ala Leu Leu Gln His Leu Ala Asp His Ala

35 40 45

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

50 55 60

Glu Leu Pro Phe Ala Ala Leu His Gln Leu Leu Arg Pro Leu Leu Gly

65 70 75 80

Ala Ala Asp Ala Leu Pro Pro Pro Gln Ser Ala Ala Leu Arg Ser Ala

85 90 95

Phe Gly Met Asp Thr Ala Pro Ala Asp Arg Phe Leu Val Ala Leu Ala

100 105 110

Ala Leu Thr Leu Leu Ser Glu Ala Ser Arg Ala Glu Pro Leu Ile Leu

115 120 125

Leu Val Asp Asp Ala His Trp Leu Asp Ser Pro Ser Ala Asp Ala Leu

130 135 140

Leu Phe Val Ala Arg Arg Leu Gly Ala Glu Gly Val Ala Val Val Phe

145 150 155 160

Ala Val Arg Asp Gly Ser Gln Pro Phe Pro Ala Pro Gly Val Pro Asp

165 170 175

Leu Arg Leu Ser Pro Leu Glu Ala Asp Ala Ala Arg Ser Leu Leu Thr

180 185 190

His Leu Leu Pro Glu Ala Ala Pro Ala Val Arg Glu Arg Leu Leu Arg

195 200 205

Glu Ala Gly Gly Asn Pro Leu Ala Leu Val Glu Leu Ser Ala Ala Leu

210 215 220

Ser Pro Glu Gln Leu Gly Gly Ala Ala Ala Leu Pro Asp Glu Leu Pro

225 230 235 240

Leu Thr Glu Arg Leu Gln Arg Leu Phe Arg Cys Arg Ala Thr Ser Leu

245 250 255

Leu Ala Glu Pro Gly Asn Ala Leu Leu Leu Thr Ala Ala Glu Gly Asp

260 265 270

Gly Asp Leu Ala Val Ile Val Arg Ala Ala Gly Asp Thr Glu Arg Ala

275 280 285

Leu His Glu Leu Ser Ala Ala Ala Thr Ala Gly Leu Val Ser Leu Asp

290 295 300

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

305 310 315 320

Gln Gly Ala Pro Leu Ser Glu Arg Arg Ala Ala His Leu Ala Leu Ala

325 330 335

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

340 345 350

Ala Ala Val Gly Pro Asp Asp Arg Val Ala His Leu Leu Ala Glu Met

355 360 365

Ala Gly Arg Ser Arg Arg Ser Gly Gly Val Ala Thr Ala Thr Lys Ala

370 375 380

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

385 390 395 400

Leu Leu Val Asp Ala Ala Glu Cys Ala Trp Lys Ala Ala Glu Thr Ala

405 410 415

Gln Ala Glu Ala Leu Leu Asn Glu Ala Glu Pro Leu Ser Asp Val Pro

420 425 430

Ala Leu Arg Ala Arg Leu Val Gln Val Arg Gly Ala Ile Ala His Ala

435 440 445

Ser Gly Asp Pro Ala Val Ala Cys Leu Ile Leu Leu Glu Gly Ala Arg

450 455 460

Leu Val Gln Glu Asp Asp Pro Arg Leu Ala Cys Glu Thr Leu Val Met

465 470 475 480

Ala Ala Arg Ser Ala Trp Val Ala Asp Asp Pro Ala Arg Leu Ala Glu

485 490 495

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

500 505 510

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

515 520 525

Ala Ala Pro Asp Ala Arg His Asp Gly Thr Ala Leu Glu Ala His Gly

530 535 540

Gly Gly Pro Glu Gly Lys Thr Gly Ala Asp Gly Lys Asp Val Pro Val

545 550 555 560

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

565 570 575

Phe Leu Pro Tyr Leu Leu His Ala Thr Glu Pro Leu Arg Asp Ala His

580 585 590

Gln His Ala Val Asp Thr Leu Arg Arg Asn Gly Ala Ile Gly Ser Leu

595 600 605

Pro Met Ser Leu Ala Pro Leu Val Ala Leu Gln Leu Val Thr Gly Pro

610 615 620

Trp Pro Thr Ala Thr Ala Asn Gly Thr Glu Ala Leu Ser Leu Ala Leu

625 630 635 640

Glu Thr Gly Gln Leu Gly Ala Ala Ser His Leu Arg Ala Met Leu Ala

645 650 655

Trp Leu Ala Ala Ala Gln Gly Asp Gly Asp Arg Cys His Glu Leu Ala

660 665 670

Arg Glu Ser Leu Glu Ile Ser Thr Pro Arg Arg Ile Ala Ser Ala Ile

675 680 685

Ala Leu Ala His Trp Ala Gln Gly Leu Asn Ala Leu Ala Glu Gly Gln

690 695 700

Pro Leu Asn Ala Val Lys Leu Leu Ala Glu Val Cys Ser Pro Glu Gly

705 710 715 720

Gly Ala Gly His Phe Met Leu Arg Trp Ile Val Leu Pro Asp Phe Val

725 730 735

Glu Ala Cys Val Arg Ala Gly Glu Pro Glu Arg Ala His His Val Leu

740 745 750

Thr Asp Arg Asp Pro Leu His Pro Val Pro Ala Asn His Pro Gln Leu

755 760 765

Arg Ser Gly Trp His Arg Ser Arg Ala Leu Leu Ala Thr Gly Glu Glu

770 775 780

Ala Glu Asp Leu Phe Leu Ala Ala Leu Ala Asp Thr Gly Leu Ser Pro

785 790 795 800

Phe Glu Thr Gly Arg Thr His Leu Leu Tyr Gly Glu Trp Leu Arg Arg

805 810 815

His Arg Arg Ile Lys Pro Ala Arg Glu Gln Leu His Leu Ala Glu Ala

820 825 830

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

835 840 845

Glu Leu Arg Ala Ala Gly Gly Arg Ser Thr Gln Gly Pro Ser Thr Ala

850 855 860

Ala Val Pro Met Gly Gly Glu Gln Arg Leu Thr Ala Arg Glu Leu Gln

865 870 875 880

Val Met Arg Leu Ala Ala Gln Gly Leu Ser Asn Gly Glu Ile Ala Ala

885 890 895

Arg Leu Phe Leu Ser Pro Arg Thr Val Gly Tyr His Leu Tyr Lys Ile

900 905 910

Phe Pro Lys Leu Gly Val Thr Ser Arg Ser Gln Leu Tyr Gly Arg Ser

915 920 925

Ser Gly

930

<210> 2

<211> 2793

<212> DNA

<213> HcdR 2-encoding nucleotide sequence

<400> 2

atgctggtcg gacgggagtg tgagttacgg cggttggcgg aggtcacggc cggcgtacgc 60

caggggcgca gccacgccct cgtactgcgc ggcccggccg gggtgggcaa gagcgcgttg 120

ctccagcacc tcgcggacca cgccggaccc ggggtgcggg tgctgtccac cgtcggcgtg 180

gagagcgagg ccgagctgcc cttcgccgcc ctgcaccagt tgctgcgccc gttgctcggc 240

gcggccgacg ccctgccgcc cccgcagagc gcggcgctgc gctcggcctt cggcatggac 300

accgcacccg ccgaccggtt cctggtggcg ctcgccgcgc tgacgctcct gtcggaggcg 360

tcccgcgcgg aaccgctgat cctgctggtg gacgacgccc actggctgga ctccccctcc 420

gccgacgccc tcctcttcgt ggcccggcgg ctcggcgccg aaggcgtcgc ggtggtcttc 480

gccgtacggg acgggtccca gccctttccg gcgccgggcg tgcccgacct gcgcctctcc 540

cccctggagg cggacgcggc ccggtcgctg ctcacccacc tgctgcccga agccgcgccc 600

gcggtgcgcg agcgactgct gcgggaggcg ggcggcaacc ccctcgccct ggtcgaactg 660

tccgccgccc tcagccccga acagctcggc ggcgcggcgg ccctaccgga cgaactgccg 720

ctgaccgagc ggctgcagcg gctcttccgc tgccgggcca ccagcctgct ggccgagccc 780

ggcaacgccc tgctgctgac cgccgccgag ggcgacggag acctggcggt catcgtccgc 840

gccgccgggg acaccgaacg cgccctgcac gagctgtccg ccgcggccac cgcggggctg 900

gtctccctgg acccggaacg ggtccggttc cgccaccccc tggtccgctc ggccgtctac 960

cagggcgcgc cgctcagcga acggcgcgcc gcgcacctcg ccctggccga cgcgctgggc 1020

gagggcgacg accggcacgt ctggcacctg gccgccgccg cggtgggccc cgacgacaga 1080

gtcgcgcacc tgctggccga gatggccggc cgctcccggc gctccggtgg agtggccacc 1140

gccaccaagg cactgcgccg ggccgccgca ctggtctcca ccccgcgcga ccgcgcccgg 1200

ttgctcgtcg acgccgccga gtgcgcctgg aaggccgccg agaccgccca ggccgaggcg 1260

ctgctcaacg aggccgagcc gctgtccgac gtgcccgcgc tgcgcgcccg gctcgtccag 1320

gtgcgcgggg ccatcgccca cgcctccggc gatccggccg tcgcctgcct gatcctgctg 1380

gagggtgcgc gcctcgtaca ggaggacgat ccccggctgg cctgcgagac gctggtgatg 1440

gcggcgcgtt cggcctgggt ggccgacgac ccggcgcggc tggcggagat cgccggtctg 1500

ctggcgcggc tcccggcggt gacaccggac gtccgggacc gcttcgtggc gcatttccgg 1560

tacctcgcgg gcctgtccgg ccacgcggcc ccggacgccc gccacgacgg tacggctctg 1620

gaggcgcacg gcggcgggcc ggaggggaag acgggcgccg acggcaagga cgtccccgtc 1680

atctggctgt ccggcaccga ccccagaccg tgggtgtggc cgcccacctt cctgccgtac 1740

ctcctccatg ccaccgagcc gttgcgcgac gcccatcagc acgcggtcga cacgctgcgc 1800

cgcaacggcg cgatcggctc cctcccgatg tccctggccc ctctcgtggc gctccagctc 1860

gtcaccggcc cctggcccac cgccaccgcc aacggcaccg aggccctctc cctggccctg 1920

gagaccggcc agctgggcgc cgcctcccac ctgcgggcga tgctcgcctg gctggccgcg 1980

gcacagggcg acggcgaccg ctgccacgaa ctggcccgcg agtcgctgga gatctccacc 2040

ccgcgccgga tcgcctccgc catcgccctg gcccactggg cgcagggcct caacgccctg 2100

gccgaggggc agcccctgaa cgcggtcaag ctgctggccg aggtctgttc ccccgagggc 2160

ggtgccggac acttcatgct gcgctggatc gtcctgcccg acttcgtcga ggcgtgcgtc 2220

agagccggcg agcccgagcg cgcccaccac gtgctgaccg accgcgaccc cctccacccg 2280

gtgccggcga accatccgca gctgcgctcc ggctggcacc gcagccgggc cctgctcgcc 2340

accggcgagg aagcggagga cctgttcctg gccgccctgg ccgacaccgg cctctccccc 2400

ttcgagaccg gccgcaccca cctgctctac ggcgagtggc tccgccgcca ccgccgcatc 2460

aaaccggccc gcgagcaact gcacctcgcc gaggcccaac tggacagcgt gggcgcgctg 2520

ccatgggcgg aactggcacg cgccgaacta cgggccgcgg gcggccgctc cacccagggg 2580

ccttcgacgg cggccgttcc catgggcggg gaacagcgtc tgacagcacg cgaactccag 2640

gtcatgcggc tcgccgcaca gggcctgagc aacggcgaga tcgccgcccg gctgttcctg 2700

agcccccgca ccgtcggcta tcacctctac aagatcttcc cgaagctggg ggtcacgtcg 2760

cggtcccagt tgtacggacg ctcctccggc tga 2793

Claims (4)

1. A method for increasing the yield of a fermented herbicidin F analogue, said method comprising:

(1) Overexpresses the HcdR2 gene in a host strain;

(2) Producing the host strain by fermentation;

(3) Isolating said herbicidin F and analogs thereof;

the host strain is S.mobaraensis US-43 strain;

the HcdR2 is a transcription regulatory protein of a LuxR protein family, and the amino acid sequence of the transcription regulatory protein is shown in SEQ ID NO. 1;

the chemical structure of the herbicidin F analogue is shown as the following formula (1):

(1) R1 is

R2 is CH3, R3 is H, molecular weight 522;

(2) R1 is

R2 is CH3, R3 is CH3, and the molecular weight is 510;

(3) R1 is

R2 is H or CH3, R3 is H or CH3, the molecular weight is 510 or 524;

(4) R1 is

R2 is CH3, R3 is CH3, molecular weight 524.

2. The method of claim 1,

the method for producing the host strain by fermentation in the step (2) comprises the following steps: a two-stage liquid fermentation method is adopted, and a fermentation medium comprises: 0.3% of dry high nitrogen corn steep liquor powder, 2% of soybean cake powder, 2.5% of glucose, 2% of starch, 2% of maltose, 0.2% ₂ HPO ₄ ，0.3％NaCl。

3. The method of claim 2, wherein the secondary liquid fermentation parameters are:

inoculating spores of the host strain into seed fermentation liquor, and culturing at 28 ℃ and 220rpm for 48 hours;

the seed fermentation liquid is inoculated in a liquid fermentation culture medium, and the liquid fermentation culture medium is cultured for 7 days at the temperature of 28 ℃ and the rpm of 220.

4. The method according to any one of claims 1 to 3,

the separation in the step (3) is as follows: supernatant of fermentation liquor C ₁₈ The column was enriched and eluted with 50% and 100% methanol solutions, respectively.

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