CN101812103A - 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic - Google Patents

Abstract

The invention discloses a new method for biosynthesizing new spirocyclic acetoacetolactone antibiotics with higher activity based on the method of genetic engineering. By site-specific mutation of the keto-reductive (KR) conserved active site of hexa-methylsalicylic acid (6-MSA) synthase, genetically engineered chlorothricin (CHL)-producing strain Streptomyces antibioticus DSM40725 for fermentation New chlorthricin analogues 7 and 8 with improved biological activity. The good biological activities of 7 and 8 have the value of further development into drugs.

Description

Genetic engineering of hexa-methylsalicylate synthase and combinatorial biosynthesis of spiro-acetolactone antibiotics

technical field

The invention belongs to the field of biotechnological engineering, and specifically relates to the function of genetic engineering modification of 6-MSA synthetase, the fermentation, separation, structure identification and activity determination of new compound biosynthesis in recombinant bacterial strain combination.

Background technique

Natural compounds are a major source of drug discovery and development, such as penicillin and erythromycin for anti-infection, bleomycin and etomycin for anti-tumor, avermectin for anti-parasite and immunosuppressant cyclosporine etc., have been the main drugs for human treatment of diseases in the past few decades. Therefore, the research and development of new drugs based on natural compounds has always been a key area of concern in the chemical and medical circles. Among them, the natural products derived from microorganisms account for an important part. There are thousands of microbial natural products reported so far, among which polyketides and non-ribosomal polypeptides account for a considerable proportion.

Spiroacetoacetolactone antibiotic family is a very distinctive class of polyketide natural products, most of which are produced by actinomycetes and have good antibacterial activity. Among them, the representative molecule Chlorothricin (CHL, Figure 1) is a new type of macrolide antibiotic, which was isolated from the strain Streptomyces antibioticus Tü99 for the first time. [6,5] spiro ring (Figure 1): (1) trans decahydronaphthalene ring, (2) spiro ring formed by 4-glycotoacetolactone and a cyclohexene ring (spirocycloacetoacetolactone The natural product of the family gets its name), the side chain sugar group and hexamethylsalicylic acid composition (Helv. Chim. Acta 52 (1969) 127-142). Clothricin has good anti-infective activity, and its antibacterial activity is attributed to the inhibition of anaplerotic _CO2 fixation catalyzed by pyruvate carboxylase. Further studies have shown that CHL interacts with Bacillus subtilis cell membrane phospholipids and has anti-cholesterol activity. The latest research also shows that it has potential antitumor activity (J.Antibiot.34, 1101-1106, J.Antibiot.36, 668-670J.Antibiot.44, 207-212).

The unique chemical structure and rich biological activity of chlorthricin have attracted the attention of the chemical, biological and medical circles. How to use it as a lead compound to develop more valuable clinical drugs has become the direction of efforts of all parties. However, its complex chemical structure has brought great challenges to chemical synthesis and structure derivation. Due to the excessive chiral centers and concentrated functional groups, chemical synthesis has very limited prospects for the production of CHL and its analogues. An important supplement, the development of combinatorial biosynthesis technology provides a new method for obtaining complex natural products and their analogs. Through the cloning of its biosynthetic gene, its biosynthetic mechanism including structural units such as macrocyclic skeleton, side chain sugar group and hexamethylsalicylic acid was revealed (Chemistry & Biology 13, 575-585), on this basis Using the principle of combinatorial biosynthesis, rationally modify its biosynthetic metabolic pathway, and explore and obtain the required new structural analogues with improved biological activity.

Contents of the invention

The object of the present invention is to provide a novel method for producing chlorthricin analogues with improved activity, specifically related to a mutation of amino acid residues in the KR domain of hexa-methylsalicylic acid synthetase, finally in the chlorine Production of Spiroacetoacetolactone Antibiotics with Enhanced Activity in Recombinant Strains of Thricin-Producing Bacteria.

details as follows:

First, the 1.9Kb DNA fragment encoding the KR domain of hexa-methylsalicylic acid synthase was obtained in Streptomyces antibioticus DSM40725 by PCR, and then connected into the vector of pSP72 to obtain a plasmid containing the KR fragment, and the next step was to obtain the plasmid from PCR Cut out the KR domain fragment containing the mutation site, replace the KR domain of the wild-type 6-MSAS and connect it into the expression vector pTGV2, and then introduce the plasmid derived from pTGV2 into the heterologous expression model strain Streptomyces albus Check the product. Results OSA was produced in the recombinant strain in which Y at position 1540 was mutated into F. The above-mentioned method is simple and easy, and the protein with altered function can be obtained quickly with only conventional molecular biology methods, and the mutated product can be detected in a heterologous host. The method for point-mutating proteins provided by the present invention does not depend on any commercial mutation kits at all, but is a simple, cheap and efficient method for point-mutating proteins.

The present invention also provides a strategy and application for producing two new chlorthricin analogues in a Streptomyces antibioticus DSM40725 chlB1 gene knockout mutant strain. The specific method is that we introduced the above-mentioned expression plasmid produced by heterologous expression in Streptomyces albus to produce OSA into the mutant strain of Streptomyces antibioticus DSM40725chlB1 gene knockout, fermentation detection, separation and purification, structural identification, and preliminary biological activity assays obtained two activities Analogues 7 and 8 of doubled chlorthricin.

Description of drawings

Figure 1 shows the structures of Chlorothricin (CHL), deschloro-CHL, and novel compounds 7 and 8 produced by the present invention.

Figure 2 shows several natural products containing aromatic units synthesized by type I repeat PKS, Cholorothricin, Maduroprptin, polyketomycin, Avilamycin, Calicheamicin.

Figure 3 represents the domain composition of fungal and bacterial 6-MSAS and the presumption of products resulting from the selective inactivation of its domains, (A) The composition of the functional domains of 6-MSAS. (B) Synthetic routes of fungal and bacterial 6-MSAS and production of 6-MSA by ChlB1 with DH mutation (H947F); OSA production by ChlB1 with KR mutation (Y1450F); 6-MSAS production by fungal KR mutations (G1387A, G1389P, G1392A) TAL.

Figure 4 shows the HPLC analysis of the heterologous expression products of DH and KR mutated ChlB1 in Streptomyces albus. (I) Streptomyces albus contains pTGV2 empty plasmid; (II) 6-MSA standard substance; (III) Streptomyces albus contains wild-type 6-MSAS (ChlB1) expression plasmid to produce 6-MSA, and the output is about 2mg/ml; (IV) The ChlB1 expression plasmid containing DH mutation (H947A) in Streptomyces albus does not produce 6-MSA; (V) The ChlB1 expression plasmid containing DH mutation (H947F) in Streptomyces albus continues to produce 6-MSA, and the output is about 0.3mg/ml; (VI) TAL standard; (VII) OSA standard; (VIII) Streptomyces albus containing KR mutation (G1389A) ChlB1 expression plasmid does not produce 6-MSA, TAL, OSA; (IV) Streptomyces albus containing KR mutation (G1387A, G1389P, G1392A) The ChlB1 expression plasmid of (X) Streptomyces albus does not produce 6-MSA, TAL, OSA; the ChlB1 expression plasmid of (X) Streptomyces albus containing the KR mutation (Y1540F) produces OSA, and the yield is about 0.3mg/ml.

Figure 5 shows a schematic diagram of the post-modification of the acyl units of 6-MSA and OSA and their transfer to the DMCHL sugar to form deschloro-CHL; CHL; 7 and 8.

Fig. 6 shows the production of new spirocyclic acetoacetolactone in HPLC analysis Streptomyces antibioticus DSM40725 (ΔchlB1) (I) wild strain Streptomyces antibioticus DSM40725 produces deschloro-CHL, CHL; (II) mutant strain Streptomyces antibioticus DSM40725 (ΔchlB1) produces DMCHL (III) ChlB1 expression plasmid with KR mutation (Y1540F) complementary to Streptomyces antibioticus DSM40725 (ΔchlB1) strain to produce 7, 8 and DMCHL. a, deschloro-CHL; b, CHL; C, DM-CHL; d, 7;

Fig. 7 shows the HMBC diagram of the ¹ H- ¹ H COZY of the new compound 7 and the OSA units of 7 and 8.

Figure 8 shows the spectrum of compound 7 ¹ H-NMR

Figure 9 shows the ¹ H-NMR spectrum of compound 8

Figure 10 shows the H-H COZY of the new compound 7

Figure 11 represents the HSQC of the new compound 7

Figure 12 shows the HMBC of the new compound 7

Figure 13 represents the new compound 7ROESY

Figure 14 shows the HMBC spectrum of the OSA unit of novel compound 8. It shows that the structure of compound 8 is the structure of CHL and an oxygen atom is added at the para-position of the benzene ring of the 6-MSA unit.

Figure 15 shows that MSS4.3 is introduced into the HPLC analysis of fermentation product in Streptomyces antibioticus DSM40725 (ΔchlB1): (I) MSS4.3 plasmid complementary Streptomyces antibioticus DSM40725 (ΔchlB1) strain produces OSA and trace amounts of 7 and 8; (II) OSA standard.

Figure 16 LC-MS of Compound 7

Figure 17 LC-MS of Compound 8

Figure 18 shows the assay of anti-Bacillus subtilis activity of new compounds: 1, deschloro-CHL; 2, CHL; 3,7; 4,8. (I) 5 μg of each compound was dissolved in 200 μl methanol and added to a tendon cup; (II) 10 μg of each compound was dissolved in 200 μl methanol and added to a tendon cup, at 37 degrees for 12 hours. The antibacterial activities of 7 and 8 were about 1 times higher than those of deschloro-CHL and CHL, respectively.

Symbol Description

In Fig. 1, Chlorothricin (CHL): chlorothricin; deschloro-CHL: dechlorothricin; 7 and 8: analogues of chlorothricin.

In Figure 2, PKS: polyketone.

In Figure 3, 6-MSAS, hexa-methylsalicylic acid; AT: acyltransferase; KS: ketosynthase; DH: dehydrogenase; KR: ketoreductase; ACP: acyl carrier protein.

In Figure 4, Streptomyces albus: model strain of heterologous expression, belonging to the genus Streptomyces; ChlB1: hexa-methylsalicylic acid synthase in the chlorthricin biosynthesis gene cluster; HPLC: high performance liquid chromatography; OSA: Orsonic acid; TAL: 2-hydroxy 4-methyl-2-pyrone.

In Fig. 5, CHL: chlorthricin; deschloro-CHL: dechlorothricin; DM-CHL: deschlorothricin deshexa-methylsalicylate.

In accompanying drawing 6, Streptomyces antibioticus DSM40725 chlorthricin-producing bacteria belong to the genus Streptomyces. 7 and 8: New compounds.

In Fig. 7, ¹ H- ¹ H COZY: hydrogen-hydrogen two-dimensional correlation spectrum; HMBC: multi-bond carbon-hydrogen relationship.

Figure 8 ¹ H-NMR: hydrogen nuclear magnetic resonance spectrum.

In Figure 11, HSQC: heteronuclear single quantum coherence spectrum

In Fig. 13, NOESY: two-dimensional nuclear Overhauser enhancement spectrum.

In Fig. 15, MSS4.3: the gene (aviM) of orsonic acid synthase was cloned into the expression vector pWHM3.

In accompanying drawing 17, LC-MS: high performance liquid chromatography and mass spectrometry.

In accompanying drawing 18, Bacillus subtilis: Bacillus subtilis.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Other aspects of the present invention will be apparent to those skilled in the art due to the disclosure herein. The experimental methods in the following examples without specific conditions are generally in accordance with the conditions described in published documents.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described in this article are only for demonstration purposes, but should not limit the content of this aspect.

Example 1 Point mutation and heterologous expression of DH and KR domains of ChlB1

1. Acquisition of ChlB1 with point mutations in DH and KR domains

1) Mutation of the DH domain

Inactivate the DH domain of ChlB1, choose to mutate its 947 histidine residue, and mutate it into glycine or phenylalanine. First, take two pairs of designed primers, and the primer sequences are as follows:

947 position H is mutated to A

Upstream: 5’-C TAC CCC GGC AGC GCC ACC ATC AAC GGC ACG-3’

Downstream: 5’-CGT GCC GTT GAT GGT GGC GCT GCC GGG GTA G-3’

947 position H is mutated to F

Upstream: 5’-C TAC CCC GGC AGC TTC ACC ATC AAC GGC ACG-3’

Downstream: 5’-CGT GCC GTT GAT GGT GAA GCT GCC GGG GTA G-3’

Using pAL1084 (a fragment of the wild-type DH domain cloned into pSP72) as a template template, dNTP, DMSO, enzyme-free water, high-fidelity primestar enzyme and its buffer constitute a PCR reaction system and perform PCR according to the designed program. The PCR product was a plasmid containing a mutated DH domain fragment, which was separated by gel electrophoresis, recovered and purified by cutting the gel, and added restriction endonuclease Dpn I to chop up the plasmid pAL1084 used as a template. Then pipette 5 μl of the enzyme digestion system to transform E.coli DH5, pick a single clone colony and culture it overnight in LB medium (containing Amp antibiotics) until the bacterial solution is thicker. Extract the plasmid and send it for sequencing to detect whether the H at position 947 is mutated into the corresponding amino acid. Then, a 1.0 kb fragment was excised with BglII/AvrII from the sequenced mutated plasmid and connected into the same restriction site of pAL1087 (chlB1 was cloned into pTGV2) to obtain the mutated pTGV2 derived plasmids pAL1088 and pAL1089 of DH.

2) Mutation of KR

To inactivate the KR domain of ChlB1, choose three strategies: ①G at position 1389 is mutated to A, ②G at 1387 is mutated to A, G at 1389 is mutated to P, G at 1392 is mutated to A, ③Y at 1540 position Mutated into F.

Take three pairs of designed primers, the primer sequences are as follows:

① Mutation of G at position 1389 to A

Upstream: 5’-C ACC GGC GGA CTG GCC ACC CTC GGC CTG G-3’

Downstream: 5’-C CAG GCC GAG GGT GGC CAG TCC GCC GGT G-3’

② The G at 1387 is mutated to A, the G at 1389 is mutated to P, and the G at 1392 is mutated to A

Upstream: 5’-C ACC GGC GCA CTG CCC ACC CTC GCC CTG G-3’

Downstream: 5’-C CAG GGC GAG GGT GGG CAG TGC GCC GGT G-3’

③ The Y at position 1540 is mutated into F

Upstream: 5’-GGC CAG GCC GCC TTC GGC TCC GCC AAC G-3’

Downstream: 5’-C GTT GGC GGA GCC GAA GGC GGC CTG GCC-3’

Using pAL1085 (a fragment of the wild-type KR domain cloned into pSP72) as a template template, dNTP, DMSO, enzyme-free water, high-fidelity primestar enzyme and its buffer to form a PCR reaction system, perform PCR according to the designed program. The PCR product was a plasmid containing a mutated KR domain, which was separated by gel electrophoresis, recovered and purified by cutting the gel, and cut up with restriction endonuclease Dpn I as the template plasmid pAL1085. Then pipette 5 ul of the enzyme digestion system to transform E.coli DH5, pick a single clone and drop it into LB culture medium (containing Amp antibiotics) for overnight cultivation until the bacterial solution is thicker. Extract the plasmid and send it for sequencing to detect whether 1387G, 1389G, and 1392G are mutated into the corresponding amino acids. Then, a 1.9kb fragment was excised from the mutated plasmid by AvrII/HindIII and connected to the same restriction site of pAL1087 to obtain KR mutated pTGV2 derived plasmids pAL1090, pAL1091, and pAL1092, respectively.

2. Heterologous expression of ChlB1 with DH and KR domain mutations

1) Intergenus conjugal transfer between strain ET12567 and strain Streptomyces albus J1074.

Transform the above plasmids pAL1088, pAL1089, pAL1090, pAL1091, pAL1092 into ET12567, cultivate ET12567 containing appropriate plasmids to OD ₆₀₀ = 0.4-0.6, collect the bacteria in 25ml culture medium by centrifugation, wash twice with equal volume of LB, resuspend in 1ml LB, as E. coli donor cells. Take 500 μL of 20% glycerol spore suspension of Streptomycesalbus J1074 frozen at -80°C, wash twice with 500 μl of TES buffer, resuspend in an equal volume of TES buffer, heat shock at 50°C for 10 minutes to germinate the spores. Add an equal volume of TSB, incubate at 37°C for 2-5 hours, centrifuge and resuspend in 1.5ml LB medium as Streptomyces acceptor cells. Mix 100 μL of recipient cells of different concentrations with an equal volume of donor cells and spread directly on MS (2.0 g mannitol, 2.0 g soybean cake powder, 2.0 g agar, tap water to 100 ml) plate containing 10 mM magnesium chloride After culturing at 30°C for 20 hours, gently wash the surface of the plate with sterile water to wash away most of Escherichia coli, and cover 1ml of nalidixic acid (final concentration is 50 μg/ml) and corresponding antibiotics ( Tsr 50 μg/ml)) in sterile water. Culture at 30°C for more than 5 days to pick zygotes.

2) Screening of zygotes.

Pick a single colony and culture it in TSB (Tsr 50 μg/ml), and verify the correctness of the introduced plasmid by bacterial liquid PCR.

3) Fermentation and processing:

The bacterium (about 48hr) that is cultivated to the logarithmic growth phase is transferred to 0.5% bacterial liquid in R5A (sucrose 100g/l; Magnesium sulfate 0.25g/l; Magnesium chloride hexahydrate 10.12g/l; Glucose 10g/l; Hy- case amino 0.1g/l; yeast extract 5g/l; Mops 21g/l; 2ml R5 trace elements; adjusted pH=6.85, autoclaved) in liquid medium for 120hrs, and all the fermented products (including bacteria (body and bacteria liquid) pH adjusted to 2-3, ultrasonic 15min (10s/50s); filter out most of the bacteria with filter paper, extract twice with an equal volume of ethyl acetate, spin dry, and re-spin in methanol. HPLC analysis of fermentation products.

HPLC analysis detection conditions:

Instrument: Agilent 1100HPLC system

Column: Phenomenex C18 column (4.63250nm, part number 00F-3300-E0, S/N115575-1

Detection wavelength: UV=220nm

Mobile phase conditions: V=1mL/min; A=H ₂ O(1‰TFA); B=CH ₃ CN(1‰TFA)

Min A%/B%

0, 90%A/10%B

5. 80%A/20%B

25, 30%A/70%B

26, 5%A/95%B

29, 5%A/95%B

30, 90%A/10%B

After LC-MS analysis, it was detected that 6-MSA was not produced in the recombinant strains with mutated H at position 947 to A; 6-MSA was produced in the recombinant strains with mutated H at position 947; G mutation at position 1389 In the recombinant strain that becomes A, OSA, TAL (2-hydroxy 4-methyl-2-pyrone), 6-MSA are not produced; G at 1387 is mutated to A, G at 1389 is mutated to P, and position 1392 OSA, TAL, and 6-MSA were not produced in the recombinant strain in which G was mutated to A; in the recombinant strain in which Y at position 1540 was mutated into F, OSA was produced.

Example 2, KR mutated ChlB1 complementary ChlB1 mutants produce new analogs of CHL

1. Intergeneric conjunctive transfer between E.coli ET12567 and Streptomyces antibioticus DSM40725(ΔchlB1)

Pick a single colony from the transformed E.coli ET12567 culture plate and transfer it to a test tube for overnight culture, pipette 0.5ml of the bacterial liquid into 25ml LB, and culture it in a shaker at 37°C until the OD ₆₀₀ is 0.3-0.4, or 0.4-0.6. The bacteria were collected by centrifugation, washed twice with an equal volume of LB medium, and the bacteria were collected by centrifugation and suspended in 1ml of LB medium. as a DNA donor.

Take out the spore suspension (3×10 ⁹ /mL) of Streptomyces antibioticus DSM40725 (ΔchlB1) stored in 20% glycerol at -80°C, centrifuge at 8,000 rpm for 3 minutes to remove the supernatant, and then wash with 0.5ml TES buffer (0.05M, pH 8.0) Wash twice, resuspend in 500μl TES buffer (0.05M, pH 8.0), heat shock at 50°C for 10 minutes to stimulate spore germination, then add 500μl TSB, mix well, incubate at 37°C for 4-5hr, centrifuge to collect spores and Suspended in 1.5ml LB broth. as recipient bacteria.

Take 100 μl of recipient bacteria and 100 μl of donor bacteria, mix them, and spread them on two AS-1 or MS plates (containing 10 mM MgCl ₂ ), and spread the same treated spores on two AS-1 or MS plates (containing 10 mM MgCl ₂ ), were used as positive control and negative control, respectively. The conjugatively transferred plate was incubated at 30°C for 16-20 hours, and then gently washed the surface of the plate with sterile water to remove most of the _E. ml, the final concentration of Nalidixic acid is 50 μg/ml), and the zygotes are picked after culturing at 30°C for 3-5 days.

The obtained zygotes were inoculated into liquid medium TSB (Tsr=50 μg/ml), and shaken at 30° C. for about 28 hours. 200 μl was taken out and coated in R ₂ YE (no sucrose) (Tsr=50 μg/ml), cultured at 30°C for 5 days, spores were harvested, and stored at -80°C.

2. Fermentation of the recombinant strain of KR mutant ChlB1 complementing Streptomyces antibioticus DSM40725 (ΔchlB1)

Spread 100-200 μl (~109/ml) of Streptomyces spores on MS (20ml, Tsr=50μg/ml) plates, and culture them at 30°C for 10 days; freeze and dry the cultured solid medium, mash it and add 50ml of Water and methanol, sonicate bacteria for 10 minutes (ultrasonic 10 seconds, interval 50 seconds), stir with a stirrer for 1 hour; filter (or centrifuge) to collect the organic phase, and re-extract the medium with 50ml of anhydrous methanol, stir for 1 hour, collect and The two organic phases were combined; the methanol was removed by rotary evaporation to obtain a dark brown residue, which was dissolved in 1 ml of anhydrous methanol and analyzed by HPLC after centrifugation.

Analysis of the mutant strains produced two brand new peaks, and LC-MS detected that the two compounds were probably analogues of CHL, named 7 and 8.

Example 3, fermentation isolation and purification of new compounds of mutant strains, structural identification and determination of biological activity

When the mutant strain was fermented in a small amount, two possible new compounds 7 and 8 were detected by HPLC and LC-MS. In order to identify the structures of these two compounds, we carried out a large amount of fermentation on the mutant strain. See Example 2 for the fermentation method. After the culture medium is freeze-dried, mash it and add an equal volume of anhydrous methanol, sonicate for 10 minutes (ultrasound for 10 seconds, interval of 50 seconds), stir with a stirrer for 1 hour, filter (or centrifuge) to collect methanol, and rotate to remove methanol , dispersed with 1/5 volume of water, adjusted to pH 2.0-3.0, extracted three times with an equal volume of ethyl acetate, and dried under reduced pressure to obtain a dark brown paste. The first step of the paste is coarse separation, mix the paste with 100-200 mesh coarse silica gel, drain it with an oil pump, and put it on a normal phase column pre-packed with 300-400 mesh silica gel. The gradient elution conditions are:

Eluent ratio

Ethyl acetate: Petroleum ether

80%/20%

60%/40%

40%/60%

20%/80%

pure ethyl acetate

100%

Dichloromethane: Methanol

95%/5%

90%/10%

pure acetone

100%

Two new compounds were found to appear in the elution fractions of 40% ethyl acetate/60% petroleum ether; 20% ethyl acetate/80% petroleum ether and 100% ethyl acetate. Drain and dissolve in 3ml of methanol, and put it on a high-pressure reverse-phase column of C18 material. The elution order is:

Water: methanol ratio

95%/5%

90%/10%

85%/15%

80%/20%

60%/40%

50%/50%

40%/60%

100%

Two new compounds appear in 80% water/20% methanol, 60% water/40% methanol these two fractions, collect these two fractions, suck dry under reduced pressure, easily in 3ml methanol, then HPLC semi-preparative, according to the above-mentioned HPLC Elution conditions: collect two new peaks, and finally dissolve the HPLC-prepared sample in 3ml methanol, load it on a superdex gel column, and elute with 100% methanol to finally obtain two pure new compounds 7 and 8. The ESI-MS/MS spectra of the new compounds 7, 8 and CHL showed that the molecular ion peak of 7 was 959.17; the molecular ion peak of 8 was 993.05; the molecular ion peak of CHL was 977.36. 7, 8 and CHL Both contain characteristic fragment peaks: 535,665,777; 7 has fragment peak 447; 8 has fragment peak 481; CHL has fragment peak 465; indicating that the structures of new compounds 7 and 8 are increased in the 6-methyl salicylic acid unit an oxygen atom. The new compound was pumped dry and dissolved in 500 μl deuterated methanol or deuterated chloroform for ¹ H-NMR, ¹³ C-NMR, HMBC, HSQC, CONSY, NOESY data collection, and analyzed the ¹ H- The NMR spectrum shows that the two single-peak chemical shifts in the 7 structure are 6.24and 6.29, indicating that it contains two aromatic hydrogens; the single-peak chemical shift in the 8 structure is 6.53, indicating that it contains an aromatic hydrogen; the single-peak chemical shift in the CHL structure 6.76, a doublet chemical shift of 7.37 (coupling constant 8.9 Hz), indicating the presence of three aromatic hydrogens. The comparison of the three results shows that the difference between the structures of 7 and 8 and CHL is that an oxygen atom is added to the benzene ring of the 6-MSA unit. ¹³ C-NMR spectrum. The spectrum shows that 7 and CHL have the same carbon skeleton. The HH COZY (Fig. 10), HSQC (Fig. 11), HMBC (Fig. 12), and ROESY (Fig. 13) of 7 indicate that the structure of 7 is that of deschloro-CHL with an increase in the para position of the benzene ring of the 6-MSA unit Oxygen atom. The HMBC (Figure 14) spectrum of the OSA unit of 8 shows that the structure of compound 8 is the structure of CHL and an oxygen atom is added at the para-position of the benzene ring of the 6-MSA unit. In summary, the structure of the compound is confirmed. The antibacterial activity of compounds 7 and 8 was tested, and it was found that the activity of new compounds 7 and 8 against Gram-positive bacteria Bacillus subtilis was about twice that of deschloro-CHL and CHL, respectively. It shows that the hydroxyl group introduced by 7 and 8 at the para-position of 6-MSA unit increases the water solubility of the compound and improves its biological activity.

The following genes and protein sequences provided according to the content of the present invention:

Amino Acid/Nucleotide Sequence Listing:

SEQUENCE LISTING

<110>Shanghai Institute of Organic Chemistry

<120> Genetic engineering of 6-methylsalicylic acid synthase and combinatorial biosynthesis of spiro-acetoacetolactone antibiotics

<130> specification, claims

<160>2

<170>PatentIn version 3.3

<210>1

<211>5271

<212>DNA

<213>Streptomyces antibioticus DSM40725

<220>

<221> CDS

<222>(1)..(5271)

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Ser Phe Asp Ala Ser Ala Asp Gly Tyr Gly Arg Gly Glu Gly Cys Gly

245 250 255

ctg ctc gtg ctg aag cgg ttg tcg gac gcg gtg cgg gac ggg gat ccg 816

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

260 265 270

gtg ctg gcg gtg atc cgg ggc agt tcg gtg aac cag gac ggg aag acg 864

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

275 280 285

aac ggg atc atg gcg ccg agt ggt tcg gcg cag gag cat gtg ctg gat 912

Asn Gly Ile Met Ala Pro Ser Gly Ser Ala Gln Glu His Val Leu Asp

290 295 300

ctg gcg tgc cgg cgg gcg ggg gtg gat ccg gcg tcg gtg gat tac gtc 960

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

305 310 315 320

gag gcg cat ggc acg ggg acg cgg ctt gga gac ccg ttg gaa gcg ggt 1008

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

325 330 335

gcg ctg agc gcg gtg ttc ggg cgg ggg cgg ccc aag gat gag ccg tgt 1056

Ala Leu Ser Ala Val Phe Gly Arg Gly Arg Pro Lys Asp Glu Pro Cys

340 345 350

ctg atc ggt tcg gtg aag tcg aac atc ggg cat ctg gag gcg gcg gcg 1104

Leu Ile Gly Ser Val Lys Ser Asn Ile Gly His Leu Glu Ala Ala Ala

355 360 365

ggg att gcg agc ctg atc aag gcg acg ctg gcg ttg agc aag gga gag 1152

Gly Ile Ala Ser Leu Ile Lys Ala Thr Leu Ala Leu Ser Lys Gly Glu

370 375 380

atc ccg ccg agt ctg aac ttc tcg cag ggc aat ccg gcg atc gac tgg 1200

Ile Pro Pro Ser Leu Asn Phe Ser Gln Gly Asn Pro Ala Ile Asp Trp

385 390 395 400

gcg gag tcc ggg ctg cgg gtg gtg acc gag cgg acg gcc tgg ccc gag 1248

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

405 410 415

cgg gag gac cga ccg gtc cgt gcg ggc gtt tcc ggc ttc ggc tat ggc 1296

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

420 425 430

ggc acc atc gcg cat gtg gtc atg gag cag gcg cct gag gtg agt cgg 1344

Gly Thr Ile Ala His Val Val Met Glu Gln Ala Pro Glu Val Ser Arg

435 440 445

ccc gat gac gcg gcg ggt gat gag ggg tct gcc gag gtc gtg acg gag 1392

Pro Asp Asp Ala Ala Gly Asp Glu Gly Ser Ala Glu Val Val Thr Glu

450 455 460

cgg ctg ttc ccg ctc tcg ggt gga acg cag gcc gga ctc cgg gcg tat 1440

Arg Leu Phe Pro Leu Ser Gly Gly Thr Gln Ala Gly Leu Arg Ala Tyr

465 470 475 480

gcg gga cgc ctc gcg gac cgg ctg tcg gac gac gac gcc gag gaa ctg 1488

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

485 490 495

ccc ctg gag tcg gtc ggg cac acc ctg gcc ttg cgc agg tcg gcg ctg 1536

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

500 505 510

gcg cac cgg gcc gcc gtc gtg gcc tcg gac cgc aag gac ctg gtg gcc 1584

Ala His Arg Ala Ala Val Val Ala Ser Asp Arg Lys Asp Leu Val Ala

515 520 525

aag ctg cgg ttg atc acg ctg ggg gag cag acc cgg gaa gcc gtg atc 1632

Lys Leu Arg Leu Ile Thr Leu Gly Glu Gln Thr Arg Glu Ala Val Ile

530 535 540

ggg tcg gta ccc tcc gat gcc ggt gcg ggg ccg gtg tgg gtg ttc tcc 1680

Gly Ser Val Pro Ser Asp Ala Gly Ala Gly Pro Val Trp Val Phe Ser

545 550 555 560

ggg cat ggt tcg cag tgg tcg ggg atg ggg cgt gaa ctg ctg gcg tcc 1728

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

565 570 575

gag ccc gcg ttc gca gcg gtg atc gac gag atc gat ccc gtt ttc cgt 1776

Glu Pro Ala Phe Ala Ala Val Ile Asp Glu Ile Asp Pro Val Phe Arg

580 585 590

gcg gag atc ggg ttc tcg gcc cgg cag gct ctg ctc gac ggt gac ttc 1824

Ala Glu Ile Gly Phe Ser Ala Arg Gln Ala Leu Leu Asp Gly Asp Phe

595 600 605

gac acc gtc gac cgt gtt cag acg atg att ttc gcg gtg cag gtc gcg 1872

Asp Thr Val Asp Arg Val Gln Thr Met Ile Phe Ala Val Gln Val Ala

610 615 620

ctg gcg gcg gtc tgg cac tct tat ggt gcc gcc ccg tcg gcg gtg atc 1920

Leu Ala Ala Val Trp His Ser Tyr Gly Ala Ala Pro Ser Ala Val Ile

625 630 635 640

ggg cac tcc gtg ggg gag atc gcg gcg gct gtg gcg gcg ggt gcg ctg 1968

Gly His Ser Val Gly Glu Ile Ala Ala Ala Val Ala Ala Gly Ala Leu

645 650 655

tcg ctg acg gac gga gcg cgg ctg atc tgc cgc cgc tcc cga ctc ttg 2016

Ser Leu Thr Asp Gly Ala Arg Leu Ile Cys Arg Arg Ser Arg Leu Leu

660 665 670

cgg cgg gtg gcc ggc cag gga gcg atg gct atg gcg agc atc tcc ttc 2064

Arg Arg Val Ala Gly Gln Gly Ala Met Ala Met Ala SerIle Ser Phe

675 680 685

gag gag gcg gcc gag cgg ctg gcg ggc cgt acg gat gtg gtg ccg gcg 2112

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

690 695 700

att gcc gcg tcc ccg ctc tcc gcg gtc gtg gca ggt gac cct gca gcg 2160

Ile Ala Ala Ser Pro Leu Ser Ala Val Val Ala Gly Asp Pro Ala Ala

705 710 715 720

atc aac gcg ctg atc gac gag tgg cag gca cag gac atc cag atg cgc 2208

Ile Asn Ala Leu Ile Asp Glu Trp Gln Ala Gln Asp Ile Gln Met Arg

725 730 735

cgg gtc gcc tcg gac gtg gcc ttc cac agc ccg cac atg gac ccg ctg 2256

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

740 745 750

ctc acc gaa atc gcg gcc gct gcc gag gac ttg acg ccg cgc cag ccc 2304

Leu Thr Glu Ile Ala Ala Ala Ala Glu Asp Leu Thr Pro Arg Gln Pro

755 760 765

gaa ctc ccg gtg tac tcc acg gcc atg gag gac ccc cgc tcc cag gcg 2352

Glu Leu Pro Val Tyr Ser Thr Ala Met Glu Asp Pro Arg Ser Gln Ala

770 775 780

acc ctc gac ggc tcc tac tgg gcc gcc aac ctg cgt aac ccg gtg cgg 2400

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

785 790 795 800

ttg cag ccg gcg gtg acg gcg gcg gtc gag gac ggc cac cgc gcg ttc 2448

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

805 810 815

atc gaa gtg tcc gcg cat ccc gtg gtc acg cac tcc atc ggc gag acg 2496

Ile Glu Val Ser Ala His Pro Val Val Thr His Ser Ile Gly Glu Thr

820 825 830

ctc tcc gag ctc ggc cag gag gac gcc ttc acc ggc tcc tcc ctg cgc 2544

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

835 840 845

cgc aac cag ccc gaa cgc gcc acc ctc ctg tcc gcc gtc ggc gcg gcg 2592

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

850 855 860

cac tgc cat ggc atc gcg gtg gac tgg gcg cgt ctg cac ccg acc ggt 2640

His Cys His Gly Ile Ala Val Asp Trp Ala Arg Leu His Pro Thr Gly

865 870 875 880

gac ctg gtc gcc ctg ccg ctg gtg gcc tgg cag cgc agc ccg cac tgg 2688

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

885 890 895

cac gag cgg gcc tcc gcc gcc acc ggc cag ggc ttg cag cac gac ctt 2736

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

900 905 910

gac tcc cac gcg ctg ctc ggg ccg cgc gtc ccg gtc gcg gga cgg ccg 2784

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

915 920 925

ctg gaa ctg tgg cgc aca ctg ctc gac gac gag acg cgc ccc tac ccc 2832

Leu Glu Leu Trp Arg Thr Leu Leu Asp Asp Glu Thr Arg Pro Tyr Pro

930 935 940

ggc agc gcc acc atc aac ggc acg gag atc gtg ccc gcc gcc gtc ctg 2880

Gly Ser Ala ThrIle Asn Gly Thr Glu Ile Val Pro Ala Ala Val Leu

945 950 955 960

atc aac acg ttc ctc gac gcg gca cgc gcc gcc gac ggg gcc cgc ccg 2928

Ile Asn Thr Phe Leu Asp Ala Ala Arg Ala Ala Asp Gly Ala Arg Pro

965 970 975

gtc ctg cgg gac atg gcg ctg cgg ctg ccg ctg atc acc acc gag cgg 2976

Val Leu Arg Asp Met Ala Leu Arg Leu Pro Leu Ile Thr Thr Glu Arg

980 985 990

cgc gaa ctc cag gtc gtc agg gac gac aac tcc ttg cgt ctg gcc tcg 3024

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

995 1000 1005

cgt tca ctg gag gac ggt gcc gcg tgg ctg acc cac acc acc gcc 3069

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

1010 1015 1020

acc gcc gca ccg gcg ggc agc ggc gaa gcg ctc cag gac ctg gcc 3114

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

1025 1030 1035

gcc ggt gcc gtg ttg cgc ccg gcg gac ccg ggt gat gtg cag cgc 3159

Ala Gly Ala Val Leu Arg Pro Ala Asp Pro Gly Asp Val Gln Arg

1040 1045 1050

cac ctg acc tcg gtg ggc gtg ccg acc atg gga ttt gag tgg acc 3204

His Leu Thr Ser Val Gly Val Pro Thr Met Gly Phe Glu Trp Thr

1055 1060 1065

atc gag gaa ctc gcc cgg agc gag ggc atg ttg gcc gca cgt gtg 3249

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

1070 1075 1080

agt gtc gag cgg ccg cag cgg gcc cag gag acg tgg gcg ccc ttg 3294

Ser Val Glu Arg Pro Gln Arg Ala Gln Glu Thr Trp Ala Pro Leu

1085 1090 1095

ctg gac gcc gcg ctg tcc atc gcg ccg acg gcc atc ccc ggc ccg 3339

Leu Asp Ala Ala Leu Ser Ile Ala Pro Thr Ala Ile Pro Gly Pro

1100 1105 1110

ccg gcc ctg cgc atg gtg gcc tcc ttc gag gag atc gtc acc gaa 3384

Pro Ala Leu Arg Met Val Ala Ser Phe Glu Glu Ile Val Thr Glu

1115 1120 1125

ggc gcc ccg ccg gcc ggt ccg gcg acc atc cag gtc gcg gcc gac 3429

Gly Ala Pro Pro Ala Gly Pro Ala Thr Ile Gln Val Ala Ala Asp

1130 1135 1140

ccg gtc cac gag aac acc gtc gac gtc cgg atc gcc gac acc gac 3474

Pro Val His Glu Asn Thr Val Asp Val Arg Ile Ala Asp Thr Asp

1145 1150 1155

ggg cag gcc gtg gcg tgg gtg cgc ggc ctg cgc tac gac ggc atg 3519

Gly Gln Ala Val Ala Trp Val Arg Gly Leu Arg Tyr Asp Gly Met

1160 1165 1170

gac cag ggc ggc atg acg gcg gcg cac ccc cgc gac ctg gtc ttc 3564

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

1175 1180 1185

gag atg gcc tgg cgg ccc ttc gag gcc ccc gcg ccg cag gac gtg 3609

Glu Met Ala Trp Arg Pro Phe Glu Ala Pro Ala Pro Gln Asp Val

1190 1195 1200

tcc gcc cgc cgg atc gtc ctg atc gcc gca cac gac gtg aag ccc 3654

Ser Ala Arg Arg Ile Val Leu Ile Ala Ala His Asp Val Lys Pro

1205 1210 1215

ctg cgc acg gcc ctc acc cgt gcc ggc gct cac gtc gac gtc ggg 3699

Leu Arg Thr Ala Leu Thr Arg Ala Gly Ala His Val Asp Val Gly

1220 1225 1230

ctg gac ggc acg cte gac gag aac acc gac gtc gtc gtg gtg ccc 3744

Leu Asp Gly Thr Leu Asp Glu Asn Thr Asp Val Val Val Val Pro

1235 1240 1245

gac ctc acc gcg gac atc ccc gtc ccc gag gcc gca gcc cgt tcc 3789

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

1250 1255 1260

gca tgg ctg ctg ctg agc acc gcg cag cgc atc gcc gcc ctg gac 3834

Ala Trp Leu Leu Leu Ser Thr Ala Gln Arg Ile Ala Ala Leu Asp

1265 1270 1275

acc ctg cgc ttc ccc cgc ctg tgg tgc ctg acc acc gca gtc cgt 3879

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

1280 1285 1290

gaa agc cag gcc gaa acc cac ctc gcg cag tcc acc ctg tgg ggc 3924

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

1295 1300 1305

ctg ggc cgg gtg atc gcg ggc gag cac agc gaa ctg tgg ggc ggc 3969

Leu Gly Arg Val Ile Ala Gly Glu His Ser Glu Leu Trp Gly Gly

1310 1315 1320

gtc atc gac ctg gcc ccc ggc acc ccg gac gcc acc acc ctg ctc 4014

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

1325 1330 1335

agc gtc ctg cac acc ggc ggc ggc gag gac gtc atc gcc ctc cgc 4059

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

1340 1345 1350

gac ggc acc gcc acc acg gcc cgc ctc acc acg acg caa cgc gag 4104

Asp Gly Thr Ala Thr Thr Ala Arg Leu Thr Thr Thr Gln Arg Glu

1355 1360 1365

ccc act ggc acc ccg ctg gaa tgc cgg gcg gac gga acg tac ctg 4149

Pro Thr Gly Thr Pro Leu Glu Cys Arg Ala Asp Gly Thr Tyr Leu

1370 1375 1380

atc acc ggc gga ctg ggc acc ctc ggc ctg gaa gtc gcc ggc cgg 4194

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

1385 1390 1395

ctc gcc gaa cgc ggc gcc cgc cgt ctc gtc ctc gcc gga cgc acc 4239

Leu Ala Glu Arg Gly Ala Arg Arg Leu Val Leu Ala Gly Arg Thr

1400 1405 1410

gga ctg cca ccc cgc tcc acc tgg ggc gag acc acc gac acg cac 4284

Gly Leu Pro Pro Arg Ser Thr Trp Gly Glu Thr Thr Asp Thr His

1415 1420 1425

acc agg cag cgc atc gag gcc gtc aag gcc ctc gaa gac cag ggc 4329

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

1430 1435 1440

gtc acc gtc cgt gtc atc ccc ctc gac atc acc gac ag gcc aag 4374

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

1445 1450 1455

gcc gcc gaa cag ctc acc ccc gac gcc ctg ggc ctg cca ccc atc 4419

Ala Ala Glu Gln Leu Thr Pro Asp Ala Leu Gly Leu Pro Pro Ile

1460 1465 1470

cgc ggc atc gtc cac ctc gcc ggc gtc ctc gac aac cgc atg gtg 4464

Arg Gly Ile Val His Leu Ala Gly Val Leu Asp Asn Arg Met Val

1475 1480 1485

acc gcg gtc gac gag aca tcc ctg cgc acc gtg ctg cgg ccc aag 4509

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

1490 1495 1500

gcc gac ggc gcc tgg acc ctg cac acc ctc ttc ccg ccc ggc acc 4554

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

1505 1510 1515

atc gac ttc ctg atc ctg ttc tcc tcc tgc ggc cag ctc ctc ggc 4599

Ile Asp Phe Leu Ile Leu Phe Ser Ser Cys Gly Gln Leu Leu Gly

1520 1525 1530

ctg ccc ggc cag gcc gcc tac ggc tcc gcc aac gcc ttc ctc gac 4644

Leu Pro Gly Gln Ala Ala Tyr Gly Ser Ala Asn Ala Phe Leu Asp

1535 1540 1545

gcc ctc gcc gtc cac cgc aac acc acc acc acc ccg acc gcc gcc gac 4689

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

1550 1555 1560

acc acc agc ttc ggc tgg acc tcc tgg cgc ggc cag ggc atg gcc 4734

Thr Thr Ser Phe Gly Trp Thr Ser Trp Arg Gly Gln Gly Met Ala

1565 1570 1575

gtc aac gac gtc gtc gac gcc gaa ctg cgc gcc cga ggc gtc acc 4779

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

1580 1585 1590

gac atc acc acc cag gaa gcc ttc gcc gcc tgg gac ttc gcc gca 4824

Asp Ile Thr Thr Gln Glu Ala Phe Ala Ala Trp Asp Phe Ala Ala

1595 1600 1605

caa cac ggc ccc gga aac tac ccc gtc cta cgc cgg ctg ccc cac 4869

Gln His Gly Pro Gly Asn Tyr Pro Val Leu Arg Arg Leu Pro His

1610 1615 1620

gag ccg gac atg gac cag ctc ccc ctc ctc agc gag atc cac cac 4914

Glu Pro Asp Met Asp Gln Leu Pro Leu Leu Ser Glu Ile His His

1625 1630 1635

acc cag ccc acc gcc ccc acc tcc ggc gcc gca acc gac tcc tac 4959

Thr Gln Pro Thr Ala Pro Thr Ser Gly Ala Ala Thr Asp Ser Tyr

1640 1645 1650

gcg ggc ctc gcc ccc gac gaa ctg cgc gcc cgc ctc atc gac gag 5004

Ala Gly Leu Ala Pro Asp Glu Leu Arg Ala Arg Leu Ile Asp Glu

1655 1660 1665

gtc gcc gca cac atc tcg gcc gag atg aaa ctc gcc gcc tcc cag 5049

Val Ala Ala His Ile Ser Ala Glu Met Lys Leu Ala Ala Ser Gln

1670 1675 1680

ctc gac cac cgc aag tcc ctg gtc gag cag ggc ctg gac tcg gtg 5094

Leu Asp His Arg Lys Ser Leu Val Glu Gln Gly Leu Asp Ser Val

1685 1690 1695

atg acg atc gtg atc cgg cgc cgc ctg gag aag tgg ttc ggt cac 5139

Met Thr Ile Val Ile Arg Arg Arg Leu Glu Lys Trp Phe Gly His

1700 1705 1710

aaa ctc ccc gcg acc ctg ctg tgg cac cag ccc acc gtc acc gcc 5184

Lys Leu Pro Ala Thr Leu Leu Trp His Gln Pro Thr Val Thr Ala

1715 1720 1725

atc agc gaa cac ctg gcc gaa ctc ctg gcc ccc acc acg tcc cag 5229

Ile Ser Glu His Leu Ala Glu Leu Leu Ala Pro Thr Thr Ser Gln

1730 1735 1740

ccc gac aac acg gca ccc gcc gaa ccg gcg gca acg gcc tga 5271

Pro Asp Asn Thr Ala Pro Ala Glu Pro Ala Ala Thr Ala

1745 1750 1755

<210>2

<211>1756

<212>PRT

<213>Streptomyces antibioticus DSM40725

<400>2

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

1 5 10 15

Glu Pro Ile Ala Val Leu Gly Met Ala Cys Arg Phe Ala Gly Gly Ala

20 25 30

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

35 40 45

Ile Gly Glu Val Pro Glu Lys Arg Trp Arg Ala Tyr Glu Glu Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ala Trp Glu Ala Leu Glu His Ala Gly Ile Ala Pro Arg Glu Leu Ala

115 120 125

Gly Thr Asp Ala Gly Val Phe Val Gly Ile Gly Ser Asp Asp Tyr Gly

130 135 140

Arg Arg Leu Leu Glu Asp Leu Pro Gly Ile Glu Ala Trp Thr Gly Ile

145 150 155 160

Gly Ser Ala Met Cys Ala Ala Ala Asn Arg Ile Ser Tyr Ala Leu Asp

165 170 175

Leu Lys Gly Pro Ser Leu Ala Val Asp Thr Ala Cys Ser Ala Ser Leu

180 185 190

Val Ala Val His Leu Ala Cys Gln Ser Leu Arg Ala Gly Glu Ser Glu

195 200 205

Val Ser Leu Ala Ala Gly Val Asn Leu Met Ile Ser Pro Gly Leu Thr

210 215 220

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

225 230 235 240

Ser Phe Asp Ala Ser Ala Asp Gly Tyr Gly Arg Gly Glu Gly Cys Gly

245 250 255

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

260 265 270

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

275 280 285

Asn Gly Ile Met Ala Pro Ser Gly Ser Ala Gln Glu His Val Leu Asp

290 295 300

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

305 310 315 320

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

325 330 335

Ala Leu Ser Ala Val Phe Gly Arg Gly Arg Pro Lys Asp Glu Pro Cys

340 345 350

Leu Ile Gly Ser Val Lys Ser Asn Ile Gly His Leu Glu Ala Ala Ala

355 360 365

Gly Ile Ala Ser Leu Ile Lys Ala Thr Leu Ala Leu Ser Lys Gly Glu

370 375 380

Ile Pro Pro Ser Leu Asn Phe Ser Gln Gly Asn Pro Ala Ile Asp Trp

385 390 395 400

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

405 410 415

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

420 425 430

Gly Thr Ile Ala His Val Val Met Glu Gln Ala Pro Glu Val Ser Arg

435 440 445

Pro Asp Asp Ala Ala Gly Asp Glu Gly Ser Ala Glu Val Val Thr Glu

450 455 460

Arg Leu Phe Pro Leu Ser Gly Gly Thr Gln Ala Gly Leu Arg Ala Tyr

465 470 475 480

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

485 490 495

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

500 505 510

Ala His Arg Ala Ala Val Val Ala Ser Asp Arg Lys Asp Leu Val Ala

515 520 525

Lys Leu Arg Leu Ile Thr Leu Gly Glu Gln Thr Arg Glu Ala Val Ile

530 535 540

Gly Ser Val Pro Ser Asp Ala Gly Ala Gly Pro Val Trp Val Phe Ser

545 550 555 560

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

565 570 575

Glu Pro Ala Phe Ala Ala Val Ile Asp Glu Ile Asp Pro Val Phe Arg

580 585 590

Ala Glu Ile Gly Phe Ser Ala Arg Gln Ala Leu Leu Asp Gly Asp Phe

595 600 605

Asp Thr Val Asp Arg Val Gln Thr Met Ile Phe Ala Val Gln Val Ala

610 615 620

Leu Ala Ala Val Trp His Ser Tyr Gly Ala Ala Pro Ser Ala Val Ile

625 630 635 640

Gly His Ser Val Gly Glu Ile Ala Ala Ala Val Ala Ala Gly Ala Leu

645 650 655

Ser Leu Thr Asp Gly Ala Arg Leu Ile Cys Arg Arg Ser Arg Leu Leu

660 665 670

Arg Arg Val Ala Gly Gln Gly Ala Met Ala Met Ala Ser Ile Ser Phe

675 680 685

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

690 695 700

Ile Ala Ala Ser Pro Leu Ser Ala Val Val Ala Gly Asp Pro Ala Ala

705 710 715 720

Ile Asn Ala Leu Ile Asp Glu Trp Gln Ala Gln Asp Ile Gln Met Arg

725 730 735

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

740 745 750

Leu Thr Glu Ile Ala Ala Ala Ala Glu Asp Leu Thr Pro Arg Gln Pro

755 760 765

Glu Leu Pro Val Tyr Ser Thr Ala Met Glu Asp Pro Arg Ser Gln Ala

770 775 780

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

785 790 795 800

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

805 810 815

Ile Glu Val Ser Ala His Pro Val Val Thr His Ser Ile Gly Glu Thr

820 825 830

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

835 840 845

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

850 855 860

His Cys His Gly Ile Ala Val Asp Trp Ala Arg Leu His Pro Thr Gly

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Leu Glu Leu Trp Arg Thr Leu Leu Asp Asp Glu Thr Arg Pro Tyr Pro

930 935 940

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

945 950 955 960

Ile Asn Thr Phe Leu Asp Ala Ala Arg Ala Ala Asp Gly Ala Arg Pro

965 970 975

Val Leu Arg Asp Met Ala Leu Arg Leu Pro Leu Ile Thr Thr Glu Arg

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

Ala Gly Ala Val Leu Arg Pro Ala Asp Pro Gly Asp Val Gln Arg

1040 1045 1050

His Leu Thr Ser Val Gly Val Pro Thr Met Gly Phe Glu Trp Thr

1055 1060 1065

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

1070 1075 1080

Ser Val Glu Arg Pro Gln Arg Ala Gln Glu Thr Trp Ala Pro Leu

1085 1090 1095

Leu Asp Ala Ala Leu Ser Ile Ala Pro Thr Ala Ile Pro Gly Pro

1100 1105 1110

Pro Ala Leu Arg Met Val Ala Ser Phe Glu Glu Ile Val Thr Glu

1115 1120 1125

Gly Ala Pro Pro Ala Gly Pro Ala Thr Ile Gln Val Ala Ala Asp

1130 1135 1140

Pro Val His Glu Asn Thr Val Asp Val Arg Ile Ala Asp Thr Asp

1145 1150 1155

Gly Gln Ala Val Ala Trp Val Arg Gly Leu Arg Tyr Asp Gly Met

1160 1165 1170

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

1175 1180 1185

Glu Met Ala Trp Arg Pro Phe Glu Ala Pro Ala Pro Gln Asp Val

1190 1195 1200

Ser Ala Arg Arg Ile Val Leu Ile Ala Ala His Asp Val Lys Pro

1205 1210 1215

Leu Arg Thr Ala Leu Thr Arg Ala Gly Ala His Val Asp Val Gly

1220 1225 1230

Leu Asp Gly Thr Leu Asp Glu Asn Thr Asp Val Val Val Val Pro

1235 1240 1245

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

1250 1255 1260

Ala Trp Leu Leu Leu Ser Thr Ala Gln Arg Ile Ala Ala Leu Asp

1265 1270 1275

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

1280 1285 1290

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

1295 1300 1305

Leu Gly Arg Val Ile Ala Gly Glu His Ser Glu Leu Trp Gly Gly

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Asp Gly Thr Ala Thr Thr Ala Arg Leu Thr Thr Thr Gln Arg Glu

1355 1360 1365

Pro Thr Gly Thr Pro Leu Glu Cys Arg Ala Asp Gly Thr Tyr Leu

1370 1375 1380

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

1385 1390 1395

Leu Ala Glu Arg Gly Ala Arg Arg Leu Val Leu Ala Gly Arg Thr

1400 1405 1410

Gly Leu Pro Pro Arg Ser Thr Trp Gly Glu Thr Thr Asp Thr His

1415 1420 1425

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

1430 1435 1440

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

1445 1450 1455

Ala Ala Glu Gln Leu Thr Pro Asp Ala Leu Gly Leu Pro Pro Ile

1460 1465 1470

Arg Gly Ile Val His Leu Ala Gly Val Leu Asp Asn Arg Met Val

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

Ile Asp Phe Leu Ile Leu Phe Ser Ser Cys Gly Gln Leu Leu Gly

1520 1525 1530

Leu Pro Gly Gln Ala Ala Tyr Gly Ser Ala Asn Ala Phe Leu Asp

1535 1540 1545

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

1550 1555 1560

Thr Thr Ser Phe Gly Trp Thr Ser Trp Arg Gly Gln Gly Met Ala

1565 1570 1575

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

1580 1585 1590

Asp Ile Thr Thr Gln Glu Ala Phe Ala Ala Trp Asp Phe Ala Ala

1595 1600 1605

Gln His Gly Pro Gly Asn Tyr Pro Val Leu Arg Arg Leu Pro His

1610 1615 1620

Glu Pro Asp Met Asp Gln Leu Pro Leu Leu Ser Glu Ile His His

1625 1630 1635

Thr Gln Pro Thr Ala Pro Thr Ser Gly Ala Ala Thr Asp Ser Tyr

1640 1645 1650

Ala Gly Leu Ala Pro Asp Glu Leu Arg Ala Arg Leu Ile Asp Glu

1655 1660 1665

Val Ala Ala His Ile Ser Ala Glu Met Lys Leu Ala Ala Ser Gln

1670 1675 1680

Leu Asp His Arg Lys Ser Leu Val Glu Gln Gly Leu Asp Ser Val

1685 1690 1695

Met Thr Ile Val Ile Arg Arg Arg Leu Glu Lys Trp Phe Gly His

1700 1705 1710

Lys Leu Pro Ala Thr Leu Leu Trp His Gln Pro Thr Val Thr Ala

1715 1720 1725

Ile Ser Glu His Leu Ala Glu Leu Leu Ala Pro Thr Thr Ser Gln

1730 1735 1740

Pro Asp Asn Thr Ala Pro Ala Glu Pro Ala Ala Thr Ala

1745 1750 1755

Claims (5)

1. volution etheric acid lactone microbiotic is characterized in that structural formula is as follows:

R in the formula ₁=OH, R ₂=H;

Perhaps R ₁=OH, R ₂=Cl.

2. genetic engineering modified 6-MSAS and the antibiotic method of combinatorial biosynthesis of spirocyclic etheric acid lactone is characterized in that this method comprises:

1) Y in catalytic activity site that has introduced the KR structural domain of 6-MSAS in the PCR primer is mutated into the mutational site of F, to contain the segmental plasmid of KR is template, PCR obtains corresponding KR fragment, cut connection by enzyme and substitute original KR structural domain, and finally be cloned among the expression vector pTGV2, be built into the expression plasmid of the 6-MSAS of KR point mutation;

2) expression plasmid with the 6-MSAS of KR structural domain rite-directed mutagenesis imports to heterogenous expression among the Streptomyces albus, produces OSA in the recombinant bacterial strain that obtains;

3) expression plasmid that produces OSA after the point mutation of 6-MSA KR structural domain is imported among the chlorothricin generation bacterium Streptomyces antibioticus DSM40725 of six-cresotinic acid acid synthase gene interruption, the recombinant bacterial strain fermentation that obtains, tunning obtains the described volution etheric acid of claim 1 lactone microbiotic through separation and purification;

Wherein 6-MSAS represents six-cresotinic acid acid enzyme, and OSA represents orsellinic acid.

3. a 6-MSAS makes Nucleotide and the protein sequence of OSAS into, it is characterized in that the Y in 1540 in the KR structural domain catalytic activity site of 6-MSAS is mutated into the sequence in the corresponding mutational site of F; Wherein OSAS represents the orsellinic acid synthetic enzyme.

4. a 6-MSAS as claimed in claim 3 makes Nucleotide and the protein sequence of OSAS into, and the Y that it is characterized in that 1540 in the KR structural domain catalytic activity site of described 6-MSAS is mutated into the mutant strain that Streptomycesantibioticus DSM40725 that expression plasmid that the sequence in the corresponding mutational site of F is built into for the PCR primer imports to Streptomyces albus and chlBl gene knockout obtains.

5. be used to prepare antibiotic or antitumor drug as claim 1 volution etheric acid lactone microbiotic.

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