CN102603867A - Polyoxin-nikkomycin hybrid antibiotic and preparation method thereof - Google Patents

Abstract

The invention discloses a polyoxin-nikkomycin hybrid antibiotic and a preparation method thereof. Heterologous expression is performed by mutating a biosynthesis gene cluster and introducing a polyoxin industrial strain. Through mass spectrum and nuclear magnetic resonance detection, four polyoxin-nikkomycin hybrid antibiotics are generated. Three of the polyoxin-nikkomycin hybrid antibiotics have more remarkable antibacterial activity on human or plant disease fungi. The hybrid antibiotics are produced by taking the polyoxin industrial strain as a 'cell factory', the antibiotic yield is high, and industrial value is achieved.

Description

Polyoxin and nikemycin hybrid antibiotic and preparation method thereof

Technical field

The present invention relates to utilize the synthetic biology strategy that the Polyoxin industrial strain is carried out the metabolic pathway transformation and produces hybrid antibiotic,, belong to field of biological pharmacy in particular to Polyoxin and nikemycin hybrid antibiotic and preparation method thereof.

Background technology

In recent years; On the one hand because the widespread use of Broad spectrum antibiotics, immunosuppressor, cell toxicity medicament; The incidence of clinical serious diseases such as the universal and malignant tumour of treatment meanss such as conduit intervention, organ transplantation, AIDS constantly rises, and the deep fungal infection case is increasing; On the other hand, the long-term utilization of clinical antifungal drug, the fungi resistance is also more and more serious.Therefore demanding developing novel antifungal drug at present urgently deals with serious day by day deep fungal infection problem.

As the specific inhibitor of chitin synthetase, Polyoxin and nikemycin not only have the anti-mycotic activity of wide spectrum, and lower to plant-animal toxicity.But because external activity is difficult to be converted into activity in vivo, these two microbiotic are not used widely clinically.

Shown in the following structural formula, Polyoxin and nikemycin have very big similarity on chemical structure.They all are to be combined by a nucleosides skeleton and one or two peptidyl.Polyoxin be uridylic or its 5 ' position by the uridylic of methyl, methylol or carboxyl substituted, and the base portion of nikemycin nucleosides skeleton also can be a uridylic, thereby the both can use the base portion of uridylic as the nucleosides skeleton.

On Polyoxin and the nikemycin structure a lot of differences are arranged also.With regard to the base portion of nucleosides skeleton, nikemycin can also use 4-formyl-4-imidazoles-2-ketone except using the uridylic.Concerning the peptide base section, both structural differences are more obvious.The peptidyl of Polyoxin in the R1 position can be to gather oxime acid (POIA), and nikemycin can be a L-glutamic acid in this position; Another peptidyl of Polyoxin is that the carboxamide polyoxy gathers oxaminic acid (CPOAA), and another peptidyl of nikemycin is a pyridone homotype Threonine (HPHT).

Because nikemycin has identical nucleosides skeleton with Polyoxin, thereby their the biosynthetic albumen of responsible nucleosides skeleton has higher homology.Comprise that with the relevant albumen of nucleosides skeleton biosynthesizing PolA, PolD, PolH, PolI, PolJ, Polk have the higher albumen of homology in nikemycin biosynthetic enzyme system.In addition, being responsible for Polyoxin nucleosides skeleton also all has homologous nikemycin biosynthesizing albumen corresponding with the PolR of the PolG of the assembling of peptidyl, the PolQ1 that is responsible for the Polyoxin transportation and PolQ2, responsible Polyoxin biosynthetic controlling.

On Polyoxin and the nikemycin chemical structure certain similarity is arranged, but different; And the genes involved homology is also higher in both biological synthesis gene clusters.This also just means on the one hand can design a series of hybrid antibiotics based on two structural differences of microbiotic; On the other hand because chemical structure is close; Dna homolog property is higher, thereby biosynthesizing albumen also has certain tolerance for the selection of substrate.Thereby can produce hybrid antibiotic through biological synthesis gene cluster transformation to Polyoxin and nikemycin.

Summary of the invention

Technical problem to be solved by this invention provides four hybrid antibiotic Polyoxin N, Nikkoxin B, Nikkoxin C, the Nikkoxin D of Polyoxin and nikemycin, and its structural formula is as follows.

Prepare the method for above-mentioned hybrid antibiotic, may further comprise the steps:

1) structure of recombinant bacterial strain ZLP2, ZLP3 and ZLP6

Plasmid pJTU5701/ Δ nikB is gone into Polyoxin industrial strain polA transgenation strain SA1 through conjugal transfer,, obtain recombinant bacterial strain ZLP2 through the apramycin resistance screening;

Plasmid pJTU5701/ Δ nikB is gone into Polyoxin industrial strain polA and polF Gene Double mutant strain SA2 through conjugal transfer,, obtain recombinant bacterial strain ZLP3 through the apramycin resistance screening;

NikQ goes into Polyoxin industrial strain polA and polF Gene Double mutant strain SA2 through conjugal transfer with plasmid pJTU5701/ Δ nikB Δ, through the apramycin resistance screening, obtains recombinant bacterial strain ZLP6;

2) fermentation of recombinant bacterial strain ZLP2, ZLP3 and ZLP6 and hybrid antibiotic extract and purifying

Recombinant bacterial strain ZLP2, ZLP3 or ZLP6 are directly carried out fermentation culture, and fermented liquid filters the back with strongly acidic cationic exchange resin absorption with careless acid for adjusting pH value to 3.0, uses the ammoniacal liquor wash-out, the selective collection component, and rotary evaporation concentrates; Detect with HPLC, obtain the pure article of hybrid antibiotic through preparation HPLC again.

Carried out widely using though Polyoxin and nikemycin all are used as anti-mycotic agent, more structural drawbacks limit they clinically as the application of antifungal drug, thereby their structure is transformed very necessary.The present invention is from the chemical structure characteristics of nikemycin and Polyoxin; Designed the hybrid antibiotic of four Polyoxins and nikemycin; And, produce thereby obtained the antibiotic orientation of target from the microbiotic biosynthesis pathway that these chemical structures design.Peptide nucleoside antibiotics (the polyoxin N of four related heterozygosis among the present invention; Nikkoxin B; Nikkoxin C and nikkoxin D) mankind or plant pathogenic fungi had good inhibition effect; Wherein nikkoxin D compares with natural antibiotics for the anti-microbial activity of the pathogenic people's mycoderma shape trichosporon of human condition and is significantly increased, and has the potential clinical development and is worth.In addition, the present invention is that microbiotic output is higher, has industrialization value with the production of Polyoxin industrial strain as " cell factory " hybrid antibiotic.The not only favourable more deep exploration Polyoxin of this research and the biosynthesis mechanism of nikemycin also provide good example for utilizing the synthetic biology strategy to produce more new peptides nucleoside antibioticss.

Description of drawings

The structure synoptic diagram of Fig. 1 mutant plasmid pJTU5701/ Δ nikB and pJTU5701/ Δ nikB Δ nikQ.

The structure synoptic diagram of Fig. 2 mutant strain SA1 and SA2 and electrophoresis checking.

Tunning biological assay of Fig. 3 recombinant bacterial strain and LC-MS analyze.

The high resolution mass spec of Fig. 4 hybrid antibiotic.

Embodiment

The invention provides Polyoxin and nikemycin four hybrid antibiotic Polyoxin N, Nikkoxin B, Nikkoxin C, Nikkoxin D also are provided.The preparation method of four hybrid antibiotics also is provided, has introduced as follows respectively.

1. the structure of recombinant bacterial strain ZLP3, ZLP2 and ZLP6

In order to realize the generation of Polyoxin N and nikkoxin B; The plasmid pJTU5701/ Δ nikB that the biosynthesizing key gene nikB of the peptidyl HPHT of nikemycin is knocked out; See A in the accompanying drawing 1, go into Polyoxin industrial strain uridine skeleton and peptidyl gathers the mutant strain SA2 that oxime acid (POIA) biosynthesis pathway all is blocked, see Fig. 2 through conjugal transfer; Through the apramycin resistance screening, obtain recombinant bacterial strain ZLP3.

In order to realize the generation of nikkoxin D; With plasmid pJTU5701/ Δ nikB, go into the mutant strain SA1 that Polyoxin industrial strain uridine skeleton biosynthesis pathway is blocked through conjugal transfer, see Fig. 2; Through the apramycin resistance screening, obtain recombinant bacterial strain ZLP2.

In order to realize the generation of nikkoxinC; The plasmid pJTU5701/ Δ nikB Δ nikQ that need nikB and 4-formyl-4-imidazoles-2-ketone biosynthesizing key gene nikQ be knocked out; See B in the accompanying drawing 1, go into mutant strain SA2, see accompanying drawing 2 through conjugal transfer; Through the apramycin resistance screening, obtain recombinant bacterial strain ZLP6.

2. the preparation of hybrid antibiotic

The spore of inoculation recombinant bacterial strain is connected among the TSB, about 30 ℃ of cultivation 36h, is inoculated in the fermentation shake flask, continues to cultivate about 3d, regulates with 1M HCl about the pH value to 6.0 of fermented liquid, and every separated 12h regulates once.With about careless acid for adjusting pH value to 3.0, filter rear filtrate and adsorb, about distilled water wash to pH value to neutrality after the fermentation ends with strongly acidic cationic exchange resin; With 0.1M ammoniacal liquor wash-out, collect that elutriant is rotated evaporation concentration and fermented liquid behind the purifying.

3. the LC-MS of hybrid antibiotic analyzes

ZLP3 fermented liquid behind the purifying carries out LC-MS to be analyzed, and has detected the predicted molecular weight ([M+H] of Polyoxin N and nikkoxin B respectively at RT 13.6min and 20.7min ⁺Ions) m/z 478.1 and m/z 607.2, to be positioned at the base that 287nm shows its nucleosides skeleton be 4-formyl-4-imidazoles-2-ketone but not uridylic to the maximum light absorption value of UV spectrum simultaneously.See accompanying drawing 3A.

In the purified fermentation broth LC-MS of ZLP2 analytical results, except the quasi-molecular ions that PolyoxinN and nikkoxin B occur, detected the predicted molecular weight ([M+H] of nikkoxin D at RT 39.9min ⁺Ions) m/z 587.2, and simultaneously to be positioned at the base that 287nm shows its nucleosides skeleton be 4-formyl-4-imidazoles-2-ketone but not uridylic to the maximum light absorption value of UV spectrum.See accompanying drawing 3A.

Analyze through the ZLP6 purified fermentation broth being carried out LC-MS, detected the predicted molecular weight ([M+H] of nikkoxin C at RT 21.9min place ⁺Ions) m/z 607.2, and simultaneously to be positioned at the base that 262nm shows its nucleosides skeleton be uridylic but not 4-formyl-4-imidazoles-2-ketone to the maximum light absorption value of UV spectrum.See accompanying drawing 3A.

4. the structure elucidation of hybrid antibiotic

To be separated to the pure article of hybrid antibiotic and carry out the high resolution mass spec analysis, the high resolution mass spec of sample the quasi-molecular ions ([M+H] that produces respectively ⁺Ions) m/z 478.1415,607.1843,607.1847 and 587.1946 and corresponding molecular weight theoretical value [M+H] ⁺Ions 478.1416 (polyoxin N, C ₁₆H2 ₃N ₅O ₁₂), 607.1842 (nikkoxin B, C ₂₁H ₃₀N ₆O ₁₅), 607.1842 (nikkoxin C, C ₂₁H ₃₀N ₆O ₁₅) and 587.1944 (nikkoxin D, C ₂₂H ₃₀N ₆O ₁₃) very identical.See accompanying drawing 4.

On NMR, carry out 1D after hybrid antibiotic is dissolved in heavy water ¹The H spectrum, ¹³The detection of C spectrum is carried out structure and is identified.Table 2 (polyoxin N), table 3 (nikkoxin B), table 3 (nikkoxin C) and table 5 (nikkoxin D) are seen in the chemical shift of four compounds

Embodiment 1, and the nikemycin biological synthesis gene cluster is transformed

22F7 is the plasmid of screening from comprising of streptomyces tendae genomic library of complete nikemycin biological synthesis gene cluster, and it has intergrase and integration site, can stably be incorporated in the genome of streptomycete.After 22F7 cut respectively with restriction enzyme XbaI and NheI, mend flat back respectively from connecting, be built into pJTU5701 with Klenow.In order to prove that pJTU5701 comprises complete nikemycin biological synthesis gene cluster, it is gone into to carry out heterogenous expression among the muta lead mycillin TK24 through conjugal transfer, simultaneously pJTU2463 conjugal transfer is gone among the muta lead mycillin TK24 as negative contrast.Zygote ferments after screening with apramycin.Tunning carries out HPLC and detects.Through having integrated the muta lead mycillin TK24 of pJTU2463 with negative contrast and having contrasted over against the fermented liquid HPLC analytical results that produces bacterium streptomyces tendae T ü 01/8c according to nikemycin; Show that the muta lead mycillin TK24 that has integrated pJTU5701 has the ability that produces nikemycin, pJTU5701 has comprised complete nikemycin biological synthesis gene cluster.

NikB is most important for the biosynthesizing of HPHT, the nikB gene on the pJTU5701 just can be blocked the biosynthesizing of HPHT through the method for PCR-targeting with the frame disappearance.Make up the checking of synoptic diagram and electrophoresis and see accompanying drawing 1.

4-formyl-4-imidazoles-2-ketone biosynthesizing is responsible for by NikP1, NikP2 and three albumen of NikQ.From pJTU5701/ Δ nikB, the nikQ gene just can be blocked simultaneously the biosynthesizing of HPHT and 4-formyl-4-imidazoles-2-ketone with the method for PCR-targeting with the frame disappearance.Make up the checking of synoptic diagram and electrophoresis and see accompanying drawing 1.

Embodiment 2, and Polyoxin industrial strain mutant strain makes up

With Polyoxin industrial strain S.aureochromoges YB172 genome is template; Podf and podr are primer; Obtain the sheet cracked ends XbaI enzyme cutting of about 3.0-kb with KOD-plus high-fidelity enzymatic amplification after, be cloned into XbaI and the HpaI site of pOJ446, then this plasmid cut with the BamHI enzyme; The BglII segment that will contain tsr is afterwards inserted this site (direction is consistent with the transcriptional orientation of polA), is built into the interruption carrier pJTU4717 of polA.After checking is correct, should interrupt carrier Transformed E .coliET12567/pUZ8002, the mode through conjugal transfer imports among the S.aureochromoges YB172, screens the interruption mutant strain of polA.

Correct (the Apr of 3 resistances of picking at random ^rThio ^s) zygote be that primer carries out PCR checking with polAeF (R).PCR result shows; The interruption mutant strain of polA (called after SA1) produces the band of 2.2-kb; And the size of the PCR product of YB172 is 1.1-kb, is that correct polA gene interrupts mutant strain thereby prove conclusively three candidate strain, makes up the checking of synoptic diagram and electrophoresis and sees accompanying drawing 2.

Thereby Polyoxin industrial strain polF gene sets out with frame deletion mutantion strain CXR3; Adopt same carrier pJTU4717 and same method that polA gene among the CXR3 is interrupted, constituted Polyoxin industrial strain uridine skeleton and peptidyl and gathered the mutant strain SA2 that oxime acid (POIA) biosynthesis pathway all is blocked.The same SA1 of verification method makes up the checking of synoptic diagram and electrophoresis and sees accompanying drawing 2.

Embodiment 3, the analysis of hybrid antibiotic and preparation

The spore of inoculation recombinant bacterial strain is connected among the TSB; Be to cultivate about 36h on 30 ℃ of shaking tables of 220r/m at rotating speed; Inoculum size according to 2% is inoculated in the fermentation shake flask; Continue to place on 30 ℃ of shaking tables of 220r/m and cultivate about 3d, the HCl with 1M regulates about the pH value to 6.0 of fermented liquid then, and every separated 12h adjusting once.Fermented liquid after fermentation is accomplished with careless acid for adjusting pH value to 3.0 about, it is overanxious to place 70 ℃ of baking ovens to place about 30min the back then, filtrate with strongly acidic cationic exchange resin (homemade 001 * 4, perhaps Dowex 50W * 8 (H ⁺), the ratio of resin demand and fermented liquid is 1: 3, is washed with distilled water to about pH value to neutrality after the absorption fully; Use 0.1M NH ₃H ₂The O wash-out, selective collection component rotary evaporation concentrates.

Use Agilent 1100 series LC/MSD Trap System to carry out LC-MS after liquid concentrator filters and analyze, dry gas flow velocity: 10l/ml wherein, the dry gas temperature: 325 ℃, atomization gas pressure: 30psi, the moving phase ratio is following:

Table 1. moving phase ratio

Time (time)	0.15％TFA	Methyl alcohol
			0	95	5
30	70	30
			50	50	50
51	95	5
			80	95	5

By same condition, LC-8A prepares on the type LC by the condition of PM flow 5ml in Tianjin, island, and the same moving phase ratio is separated and also collected the target hybrid antibiotic.

Embodiment 4, the structure elucidation of hybrid antibiotic

To be separated to the pure article of hybrid antibiotic and carry out the high resolution mass spec analysis, hybrid antibiotic the quasi-molecular ions ([M+H] that produces respectively ⁺Ions) m/z 478.1415,607.1843,607.1847 and 587.1946 and corresponding molecular weight theoretical value [M+H] ⁺Ions 478.1416 (polyoxin N, C ₁₆H2 ₃N ₅O ₁₂), 607.1842 (nikkoxin B, C ₂₁H ₃₀N ₆O ₁₅), 607.1842 (nikkoxin C, C ₂₁H ₃₀N ₆O ₁₅) and 587.1944 (nikkoxin D, C ₂₂H ₃₀N ₆O ₁₃) very identical.And the segment peak that second order ms produced of four compounds also can enough corresponding chemical structures the peak shape that possibly form make an explanation.

The further parsing of the structure of four chemicals is got hybrid antibiotic respectively and on the BrukerAV400MHz NMR, to be carried out 1D after 20mg is dissolved in heavy water ¹The H spectrum, ¹³The detection of C spectrum.

Produce Polyoxin N from that look Streptomycin sulphate bacterium ZLP3 (S.aureochromogenes ZLP3) is separated to and be separated to Polyoxin N gained nuclear magnetic resonance spectrum from streptomyces ansochromogenes Δ sanN/pPOL2 (S.ansochromogens Δ sanN/pPOL2) and match from gold.(table 2)

The spectrum that Nikkoxin B is produced is similar with polyoxin N major part, but also exists ¹The δ of H hydrogen spectrum _H4.99 (dd, J=9.0,5.0Hz, 1H), 2.22 (m, 1H), 2.04 (m, 1H), 2.47 (t, J=7.3Hz, 2H) position with ¹³The δ of C carbon spectrum _C176.8,58.7,28.5,32.4, produce new signal on and 179.6 positions, this L-glutamic acid group with nikemycin J component is consistent.In addition, the structure of nikkoxin B has also obtained H-2 " and C-6 ' between the confirmation of dependency.(table 3)

Nikkoxin C's ¹H hydrogen spectrum with ¹³The chemical shift that C carbon spectrum is is compared with nikkoxin B, except at δ _H7.67 (d, J=8.0Hz, 1H) and 5.90 (d.J=8.0Hz, 1H) outside, other place is all very similar.And these are uridylic replacement 4-formyl-4-imidazoles-2-ketone characteristic signals as the base portion of nucleosides.(table 4)

Nikkoxin D has also produced the characteristic signal of Polyoxin peptidyl POIA except having produced the signal similar with polyxin N.The structure elucidation of Nikkoxin D has also used two dimensional NMR H-H COSY and HMBC, H-2 simultaneously " show that with the dependency of C-6 ' POIA links to each other with polyoxin N through peptide bond to form nikkoxin D.(table 5)

In sum, these four antibiotic chemical structures are in full accord with the structure of predicting according to the Polyoxin in the recombinant bacterial strain and nikemycin biosynthesis gene.

Table 2polyoxin N is that solvent is when 400MHz (δ in ppm, mult., J in Hz) with the heavy water ¹³C with ¹The H chemical shift

Position	1H(δ，mult.，J)	13C(δ)
			1		156.1
3		126.9
			4	7.61(s)	128.5
6	9.24(s)	183.0
			1’	5.59(d，4.8)	90.4
2’	4.55(t，5.5)	75.1
			3’	4.51(t，5.5)	72.8
4’	4.30(t，5.0)	85.3
			5’	4.84(d，4.8)	57.0
6’		173.7
			1”		170.5
2”	4.24(d，5.5)	58.7
			3”	4.12(m)	71.2
4”’	4.07(m)	72.2
			5”’	4.07(m)	67.9
6”’		161.7

Table 3Nikkoxin B is that solvent is when 400MHz (δ in ppm, mult., J in Hz) with the heavy water ¹³C with ¹The H chemical shift

Position	1H(δ，mult.，J)	? 13C(δ)
			1		156.2
3		126.7
			4	7.72(s)	129.4
6	9.28(s)	183.0
			1’	5.61(d，6.1)	90.9
2’	4.75 (with H 2O is overlapping)	74.3
			3’	4.39(dd，5.0，3.5)	73.7
4’	4.27(m)	85.5
			5’	4.78 (with H 2O is overlapping)	57.3
6’		172.1
			1”		176.8
2”	4.46(dd，9.0，5.0)	58.7
			3”	2.17(m)，2.00(m)	28.5
4”	2.42(t，7.3)	32.4
			5”		179.6
1”’		170.4
			2”’	4.25(m)	58.7
3”’	4.12(m)	71.4
			4”’	3.99(m)	72.2

5”’	3.65(m)	67.9
			6”’		161.7

Table 4Nikkoxin C is that solvent is when 400MHz (δ in ppm, mult., J in Hz) with the heavy water ¹³C with ¹The H chemical shift

Position	1H(δ，mult.，J)	13C(δ)
			1		154.4
3		168.8
			4	5.90(d，8.0)	105.2
5	7.67(d，8.1)	146.0
			1’	5.78(d，5.4)	94.2
2’	4.60(t，5.5)	74.5
			3’	4.39(t，4.9)	73.3
4’	4.25(dd，7.3，4.5)	85.1
			5’	4.81 (with H 2O is overlapping)	57.3
6’		172.1
			1”		176.9
2”	4.49(dd，9.0，5.0)	54.8
			3”	2.22(m)，2.04(m)	28.4
4”	2.47(t，7.3)	32.5
			5”		179.6
1”’		170.4
			2”’	4.29(d，5.5)	58.7
3”’	4.15(m)	71.4
			4”’	4.02(m)	72.2
5”’	4.13(m)	67.9
			6”’		161.7

Table 5Nikkoxin D is that solvent is when 400MHz (δ in ppm, mult., J in Hz) with the heavy water ¹³C with ¹The H chemical shift

Position	1H(δ，mult.，J)	13C(δ)
			1		156.1
3		126.9
			4	7.69(s)	128.7
6	9.26(s)	182.9
			1’	5.67(d，4.9)	90.5
2’	4.49(t，4.9)	75.4
			3’	4.48(t，5.6)	73.3
4’	4.34(t，5.7)	84.4
			5’	4.81 (with H 2O is overlapping)	53.7
6’		170.9
			1”		173.9
2”	5.59(brs)	70.2
			3”		125.5
4”	4.94(brs)	62.0

5”	(5.66 overlapping)	125.5
			6”	1.67(d，6.3)	15.4
1”’		170.5
			2”’	4.29(d，4.9)	58.7
3”’	4.19(m)	71.2
			4”’	4.01(m)	72.4
5”’	4.10-4.18(m)	67.9
			6”’		161.6

Embodiment 5, recombinant bacterial strain fermented liquid biological activity determination

Choosing recombinant bacterial strain ZLP2, ZLP3 and ZLP6 ferments; Simultaneously with Polyoxin industrial strain gold produce look streptomycete YB 172 (S.aureochromoges YB 172) and nikemycin generation bacterium streptomyces tendae T ü 01/8c (S.tendae T ü 01/8c) as positive control; Ferment together as negative control with mutant strain SA1, SA2; Behind the filtering fermentation liquor, carry out biological activity determination.

Get biological activity determination and will indicate fungi skin shape trichosporon to be inoculated in the YEME substratum, subsequent use behind the cultivation 24h, get the PDA that melts about 15ml and cultivate based on placing 55 ℃ of about 20min of baking oven in the Universal bottle; Treat that the temperature of substratum reduces to about 55 ℃; The indicator culture that adds 50-100 μ l then places aseptic Oxford cup in the good position of mark, and adds the fermentating liquid filtrate of 20 μ l; Insert 30 ℃ of incubators then and cultivate after 24 hours observations.

Experimental result shows, compares with the mutant strain SA1, the SA2 that lose anti-mycotic activity, and recombinant bacterial strain ZLP2, ZLP3 and ZLP6 have recovered anti-mycotic activity, and wherein, ZLP2 shows stronger anti-mycotic activity, and ZLP6 has only demonstrated faint anti-mycotic activity.(seeing accompanying drawing 3B)

Embodiment 6, and the hybrid antibiotic anti-mycotic activity is measured

In order further to study the anti-microbial activity of these four kinds of hybrid antibiotics to the mankind or plant pathogenic fungi; We have chosen five representational indicators and have carried out the test of minimal inhibitory concentration, comprising: Candida albicans Candida albicans, skin shape trichosporon T.cutaneum (condition pathomycete), long handle alternaric bacteria Alternaria alternate (the former bacterium of Alternaria alternate), dry thread Pyrenomycetes Rhizoctonia solani Ktihn (rice banded sclerotial blight cause of disease bacterium) and grey Magnaporthe grisea Magnaporthe grisea (rice blast pathogenic bacteria).The contrast that this institute selects for use is respectively Polyoxin B component and K component and Nikemycin Z component and X component.

With being diluted to 2560 μ g ml after the microbiotic weighing to be tested ^-1Mother liquor, then it half-and-half is diluted to 0.5-2560 μ gml ^-1The test soup of 10 concentration gradients.

For filamentous fungus, the soup of 10 μ l is joined 96 orifice plates that inject 100 μ l PDA substratum, be placed on 4 ℃ afterwards and let the microbiotic diffusion in 24 hours.Fungi is seeded in the surface of substratum, observations after cultivating 48 hours under 28 ℃.

For yeast, the soup of 10 μ l is joined 96 orifice plates that inject 100 μ l YEME substratum, wherein added 1-5 * 10 in the substratum ²The yeast of CFU.Observations after cultivating 24 hours under 28 ℃.

According to the test result of minimal inhibitory concentration, hybrid antibiotic shown diversified anti-mycotic activity, some in addition compare the raising that highly significant is arranged with the activity of contrast.For human pathogenic bacteria Candida albicans (Candida albicans), the anti-mycotic activity of polyoxin N similar with Nikemycin Z (MIC=16 μ g/ml), and contrast Polyoxin B and other three hybrid antibiotics do not show tangible anti-microbial activity.And for the pathogenic mycoderma shape trichosporon (T.cutaneum) of human condition; Except Polyoxin B and polyoxin N; All components all have stronger restraining effect to it, and wherein the antibacterial work of nikkoxin D (MIC=0.5 μ g/ml) is about four times of hybrid antibiotics such as Polyoxin K component and other nikkoxin C.For two important plant pathogenic fungi long handle alternaric bacterias (Alternaria alternate) and grey Magnaporthe grisea (Magnaporthe grisea), polyoxin N and nikkoxin B have demonstrated the strongest activity.And a plant pathogenic fungi dry thread Pyrenomycetes (Rhizoctonia solani Ktihn) is all not too responsive to specimen, but hybrid antibiotic nikkoxin B and C have also shown bacteriostatic activity (MIC=16 μ g/ml) preferably.

Antibiotic output of table 6. and minimal inhibitory concentration (μ g ml ^-1)

Claims (3)

1. the hybrid antibiotic of Polyoxin and nikemycin from left to right is respectively nikkoxin B, nikkoxin C and nikkoxin D, and its structure is as follows:

、 、 。

2. prepare the method for the said hybrid antibiotic of claim 1, may further comprise the steps:

1) structure of recombinant bacterial strain ZLP2, ZLP3 and ZLP6

With plasmid pJTU5701/ NikBGo into the Polyoxin industrial strain through conjugal transfer PolATransgenation strain SA1 through the apramycin resistance screening, obtains recombinant bacterial strain ZLP2;

With plasmid pJTU5701/ NikBGo into the Polyoxin industrial strain through conjugal transfer PolAWith PolFGene Double mutant strain SA2 through the apramycin resistance screening, obtains recombinant bacterial strain ZLP3;

With plasmid pJTU5701/ NikB nikQGo into the Polyoxin industrial strain through conjugal transfer PolAWith PolFGene Double mutant strain SA2 through the apramycin resistance screening, obtains recombinant bacterial strain ZLP6;

2) fermentation of recombinant bacterial strain ZLP2, ZLP3 and ZLP6 and hybrid antibiotic extract and purifying

Recombinant bacterial strain ZLP2, ZLP3 or ZLP6 are directly carried out fermentation culture, and fermented liquid filters the back with strongly acidic cationic exchange resin absorption with careless acid for adjusting pH value to 3.0, uses the ammoniacal liquor wash-out, the selective collection component, and rotary evaporation concentrates; Detect with HPLC, obtain the pure article of hybrid antibiotic through preparation HPLC again.

3. the described hybrid antibiotic of claim 1 is as the application of lead compound in the development and preparation active drug.

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