CN102532275A - Cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses - Google Patents
Cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses Download PDFInfo
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Abstract
The invention relates to a cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses. Amino acid residues at seven sites of a cyclothiazomycin precursor peptide are subjected to point mutation, so that 14 mutant strains are obtained, and 23 novel cyclothiazomycin analogue antibiotics are generated by the heterologous expression of type strain streptomyces lividans 1326 of streptomyces, wherein the products of six mutant strains can inhibit the growth of bipolaris maydis. Compared with the prior art, the cyclothiazomycin analogue can allow the mutant strain building process to be quick and efficient, and random mutant strains can be obtained, the research of biosynthetic mechanism can be conducted by the products generated by the cyclothiazomycin mutant strains so as to provide richer methods and means for the research of the biosynthetic mechanism of sulfur peptide antibiotics. Most importantly, the obtained novel sulfur peptide antibiotics open a way for the artificial design and synthesis of novel sulfur peptide antibiotics.
Description
Technical field
The invention belongs to Microbial resources and genetically engineered field, be specifically related to the sudden change of precursor peptide group and produce ring thiazolidomycin analogue and method and purposes.
Background technology
Microbiotic (antibiotics) is to be had antipathogen or other active one type of secondary metabolites, a chemical substance of other life cell development functions of interfere by what mikrobe (comprising bacterium, fungi, actinomyces) or high plant-animal produced in life process.The forties in 20th century, penicillium mould is applied to clinical marker and antibiotic birth.Find first microbiotic from nineteen twenty-eight Alexander Fleming---since the penicillium mould, scientist has had been found that nearly ten thousand kinds of microbiotic, and the overwhelming majority in them is that toxicity is too big; Be not suitable for the medicine of and communicable disease of animals human as treatment, reach nearly hundred kinds more than but the microbiotic of clinical application is existing, some can suppress microbial growth; Some can suppress parasitic breeding, and some can be used for weeding, and some can be used for treating cardiovascular disorder; Also some can suppress the human immunity reaction; In the human organ transplant operation, so, just because of antibiotic existence; The feasible mankind can defeat each infection, keep fit.
Late 1960s is accompanied by microbiotic and vaccine popularizing fast and development in the world, and infection is controlled by the mankind gradually, during by 1988, and infection even be controlled in outside the 10 the highest big diseases of U.S.'s mortality ratio.Yet, also from that time, along with antibiotic abuse; The antibiotics resistance problem is presented in face of the mankind gradually; But these problems were not paid attention to by the mankind at that time, and this directly causes infection volume soil always, became the high disease of mortality ratio the 3rd; More the terrorise's is abuse and misuses the antibiotic resistance that microbiotic causes and expedited the emergence of " superbacteria " that promptly all microbiotic are all invalid to it.At present, antibiotic resistance becomes " whole world threatens ", and this has also promoted human research and development to new antibiotic more, and the human responsibility of rescue is being born in the research of the microbiotic new drug of especially anti-high drug-resistance pathogenic bacterium.Among numerous new antibiotics, the sulphur peptide antibiotics has been rich in the using value of potentiality by feat of ten minutes particular structure characteristic, has received the people of the world's who comprises the various countries scientist extensive concern.
The sulphur peptide antibiotics is one type and comes from mikrobe, is rich in element sulphur, structure by the important natural product of highly modifying, up to the present, have been found that 80 surplus kind.Although they exist textural difference on the whole, they have typical constitutional features jointly: (1) sulphur peptide antibiotics all is rich in annular (macrocyclic) polypeptide through highly modifying and contain sulphur; (2) on the central position of space structure be one by three or four substituted nitrogen heterocyclic rings of thiazole ring (like pyridine pyridine and dehydration piperidines dehydrapiperidine); (3) around this center heterocyclic, distributing numerous by the different molecule cyclic amino acid residue (heterocyclic residues) of modified; Comprising thiazole (thiazoles) oxazole (oxazoles); And dehydroamino acid (dehydroamino acids) [Chem Rev (2005) 105,685-714].Except these many structure general character; Most of sulphur peptide antibioticses also have common characteristic aspect biological activity: (1) sulphur peptide antibiotics medicine and agriculture aspect have a wide range of applications; Thiostrepton (thiostrepton) can suppress rrna synthetic proteins [J Mol Biol (1998) 276; 391-404], can also induce TipA proteic a large amount of synthetic [J Bacteriol (1989) 171,1459-1466]; (2) ring thiazolidomycin (cyclothiazomycin) then is a kind of renin inhibitor (renin inhibitor) with potential application foreground; Meanwhile farm crop pathomycetes such as paddy rice flax also there are the germ-resistant effect of inhibition [J Antibiot (1991) 44,582-8; Bioorg Med Chem (2006) 14,8259-70]; (3) in sulphur peptide antibiotics, there is considerable microbiotic that various high drug-resistance pathogenic strainss are all had restraining effect; Comprise MRSA (methicillin-resistant Staphylococcus aureus;); PRSP (penicillin-resistant Streptococcus pneumoniae) and VRE (vancomycin-resistant enterococci) [Chem Rev (2005) 105,685-714; Chem Biol (2009) 16,141-147].These characteristics have greatly excited the research enthusiasm of various countries scientist to the sulphur peptide antibiotics, make that scientist is filled with unbounded confidence to the new drug that can develop anti-high drug-resistance pathogenic strains.
In the last few years; The scientist in the whole world is always in a series of interesting problem about the sulphur peptide antibiotics; Comprise that common amino-acid residue is that which kind of biochemical reaction through which enzyme forms complicated spatial structure like this, be again how to demonstrate so different bioactive.The organic chemist resolves in method high modified polypeptides [the Angew Chem Int Ed Engl (2007) 46 among external synthesizing through organic synthetic; 7930-7954], and the biologist also began to have the important breakthrough progress for the research of sulphur peptide antibiotics in 09 year.Domestic and international several laboratory is almost angled simultaneously and has been got sulphur peptide antibiotics biological synthesis gene cluster, has realized the breakthrough of zero.This type of antibiotic biosynthesizing mechanism is opened step by step.Continuous development along with the gene order-checking technology; We believe that the research of sulphur peptide antibiotics will constantly go deep into; Open the answer to a riddle of the problems that perplexing scientist, thereby be that the human new drug of developing novel anti-high drug-resistance pathogenic strains has been established sturdy basis.
(Cyclothiazomycin CLT) obtained [J Antibiot (1991) 44,582-8s] by Japanese scientist from Streptomyces sp.NR0516 separation in 1991 to the ring thiazolidomycin, was the later microbiotic of discovery time among the sulphur peptide antibiotics.It is many epithios peptide antibiotics of a kind of Cheng Qiaozhuan, and external toxicological experiment shows that it has human cell's feritin (human plasma renin) of inhibition activity, IC50=1.7mM.Compare and other sulphur peptide antibioticses from molecular structure, it has numerous structural uniquenesses: (1) unique sulphur peptide antibiotics that has tricyclic structure; (2) have non-2, the substituted center of thiazole, 3-position pyridine ring; (3) the free carboxyl terminal demonstrates half unbound state because of the existence of thioether bond, and intramolecularly is not all to be ubiquitous thiazole ring in the sulphur peptide, but has comprised 3 thiazole rings and 3 thiazolines; (4) have a thioether bond on the tertiary carbon position.Have tricyclic structure and contain a plurality of thiazole rings or thiazoline just because of the ring thiazolidomycin, make that its solvability is lower, resolve its space structure to scientist and brought great challenge.2006; The Japan scientist successfully describes out the three-dimensional arrangement of ring thiazolidomycin through the method for NMR; And isolated two kinds of not derive isomer B1 and B2 of the ring thiazolidomycin of isomorphic map in the tunning, they find these two kinds isomer very stable under solid state of deriving, but in solution, can transform each other; Be conversion rate slow [Bioorg Med Chem (2006) 14,8259-70] very.And they find that also the ring thiazolidomycin is a kind of suppressor factor [Bioorg Med Chem (2006) 14,8259-70] of RNA polymerase.In addition, because the structure singularity of ring thiazolidomycin, the structurizing scholar has also produced keen interest [Org Lett (2004) 6:3401-4] for external synthetic similar molecule.
2009; Doctor Wang Jiang of Shanghai Communications University successfully angles male Ke silk plasmid (cosmid) from streptomyces hygroscopicus Yingcheng City mutation 10-22 genomic library; A polypeptide that is wherein comprising a bit of dna sequence encoding 60aa; The structure deduced amino acid sequence of the sequence of the 18aa of C end and ring thiazolidomycin wherein, and can both prove that through follow-up biological activity test and the analysis of LC-EI-Q-TOF molecular weight detection and the analysis of MS/MS fragment the 5102-II microbiotic encircles thiazolidomycin exactly.In addition; Through making up a plurality of heterogenous expression carriers; In streptomycete type strain muta lead mycillin (Streptomyces lividans) 1326, successfully realize the heterogenous expression of sulphur peptide antibiotics first; And through different ORF integrated positioning a complete ring thiazolidomycin biological synthesis gene cluster, comprised 15 ORFs, length is the dna sequence dna of 22.7kb, shows that through bioinformatic analysis wherein unique structure gene cltA coding produces the precursor of ring thiazolidomycin; This precursor polypeptide carries out posttranslational modification through other expressed in gene cluster albumen, is released to outside [AEM (2010) p.2335-2344] after the last maturation.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of precursor group sudden change to produce ring thiazolidomycin analogue and method and purposes.The present invention is a kind of method and application thereof that utilizes point mutation and heterogenous expression to obtain novel ring thiazolidomycin analogue; Through the suddenlyd change amino-acid residue in 7 sites of ring thiazolidomycin precursor peptide of selected element; Then through the heterogenous expression in the type strain muta lead mycillin Streptomyces of streptomycete lividans 1326; 14 mutant strains have been obtained; Produced 23 kinds of novel ring thiazolidomycin analogues, wherein, the product that 6 mutant strains are arranged has restraining effect to the growth of corn stigma (Bipolaris maydis).
The objective of the invention is to realize through following technical scheme:
The present invention relates to the ring thiazolidomycin analogue that a kind of precursor peptide group sudden change produces, obtain: through in ring thiazolidomycin precursor peptide gene cltA, inserting the restriction enzyme site of two BglII and SpeI restriction enzyme through following method; Be connected with the double digestion product to make up with mutant primer and realize former site mutation ring thiazolidomycin precursor peptide gene cltA; Obtain ring thiazolidomycin analogue through the heterogenous expression in the pattern of streptomycete then.
Preferably, the said method of in ring thiazolidomycin precursor peptide gene cltA, inserting the restriction enzyme site of two BglII and SpeI restriction enzyme specifically comprises the steps:
(1) selects the pMD-18T carrier for use; The 3kb fragment of coming out to contain the part ring thiazolidomycin gene cluster of ring thiazolidomycin precursor peptide gene cltA with pcr amplification is connected; Restriction enzyme SnaBI and SacI restriction enzyme site are contained in the fragment two ends that pcr amplification produces, and downcut to be connected with pJTU4892 again;
(2) method through PCR-Targeting replaces to the neo kalamycin resistance gene with the aac among the plasmid pJTU4892 (3) IV apramycin resistant gene, obtains plasmid pJTU4895;
The fragment of (3) downcutting 3kb from the recombinant plasmid pMD-18T-3kb that contains BglII and SpeI recognition site with restriction enzyme SnaBI and SacI; With cut the carrier pJTU4895 that handled with restriction enzyme SnaBI with the SacI enzyme and be connected; Transformed into escherichia coli DH10B, acquisition also has the plasmid pJTU4896 of restriction enzyme BglII and SpeI recognition site;
(4) cutting the fragment of handling pJTU4896 with XbaI and EcoRI enzyme is connected structure with the fragment of cutting processing pSET152 with XbaI and EcoRI enzyme and obtains plasmid pJTU4897; This plasmid pJTU4897 contains aac (3) IV apramycin resistant gene, and contains restriction enzyme BglII and SpeI recognition site.
Preferably; Said former site mutation ring thiazolidomycin precursor peptide gene cltA is specially the amino-acid residue in arbitrary site in 7 sites of the ring thiazolidomycin precursor peptide gene that suddenlyd change or two sites, and said 7 sites are C3, T4, S5, T6, G7, T8 and P9 position.
Preferably, said heterogenous expression is the heterogenous expression in the type strain muta lead mycillin Streptomyces of streptomycete lividans 1326.
Preferably, the mutant strain that said ring thiazolidomycin analogue is corresponding is C3S, T4D, T6D, T6V, T6G, T6S, T6C, G7A, G7S, T8D, T8S, P9H, P9A or T68D.
Preferably, the mutant strain that said ring thiazolidomycin analogue is corresponding is T6D, T6S, T6V, T6G, T8S or P9A.
Preferably, the mutant strain that said ring thiazolidomycin analogue is corresponding is T6S, T6V, T6G or T8S.
Preferably, the structural formula of said ring thiazolidomycin analogue is shown in formula (I), formula (II), formula (III), formula (IV) or the formula V,
The invention still further relates to a kind of the above ring thiazolidomycin analogue and suppress the purposes in the suppressor factor of corn stigma growth in preparation.
Preferably, the corresponding mutant strain of ring thiazolidomycin analogue of said suppressor factor employing is T6S, T6V, T6G or T8S.
Compared with prior art; The present invention has following beneficial effect: the present invention not only lets structure mutant strain process become fast effectively; Can obtain the random mutation strain in theory; And, can carry out the biosynthesizing Study on Mechanism through the product that ring thiazolidomycin mutant strain generates, abundant more ways and means is provided for sulphur peptide antibiotics biosynthesizing Study on Mechanism.The most important thing is that the numerous novel sulphur peptide antibiotics of acquisition has been opened up road for manual work designs synthesizing new sulphur peptide antibiotics.
Description of drawings
The process synoptic diagram of Fig. 1, structure heterogenous expression mutant strain.
The LC-EI-Q-TOF analysis of Fig. 2, the various mutant strain solid fermentation things of generation; Marked the molecular ion peak [M+2] of the staple of the ring thiazolidomycin analogue that ten mutant strains produce among the figure
2+
The LC-EI-Q-TOF and the MS/MS mass spectroscopy figure of Fig. 3, mutant strain T4D solid fermentation thing methanol extract; Wherein, A, T4D LC-EI-Q-TOF analyze and isotopic peak; The chemical structure of B, ring thiazolidomycin with and the positive ion state, the solid line of black connects and to be peptide bond in the structural formula, dotted line is connected to the covalent linkage of other types, on behalf of this amino-acid residue, last target asterisk passed through back modification; C, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of D, T4D product.
The LC-EI-Q-TOF of Fig. 4, T6D and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, T6D product.
The LC-EI-Q-TOF of Fig. 5, T6V and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, T6V product.
The LC-EI-Q-TOF of Fig. 6 .T6G and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, T6G product.
The LC-EI-Q-TOF of Fig. 7, G7A and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, G7A product.
The LC-EI-Q-TOF of Fig. 8, G7S and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, G7S product.
The LC-EI-Q-TOF of Fig. 9, T8D and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, T8D product.
The LC-EI-Q-TOF of Figure 10, T8S and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, T8S product.
The LC-EI-Q-TOF of Figure 11, P9H and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, P9H product.
The LC-EI-Q-TOF of Figure 12, T68D and MS/MS mass spectroscopy figure; Wherein, A, LC-EI-Q-TOF analyze and isotopic peak; B, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of C, T68D product.
The LC-EI-Q-TOF of Figure 13, T6S and MS/MS mass spectroscopy figure; Wherein, A, the LC-EI-Q-TOF that are respectively two ring thiazolidomycin analogues analyze and isotopic peaks; The relative content that B, isotropic substance peak height characterize; C, bivalent molecule ionic MS/MS fragment are analyzed; The chemical structure of D, T6S product.
The LC-EI-Q-TOF of Figure 14, C3S and MS/MS mass spectroscopy figure; Wherein, the LC-EI-Q-TOF of A, four ring thiazolidomycin analogues analyzes and isotopic peak; The relative content that B, isotropic substance peak height characterize; C, bivalent molecule ionic MS/MS fragment are analyzed a-f: the structure of characteristic fragment and quality; The chemical structure of D, T6S product.
The LC-EI-Q-TOF of Figure 15, T6C and MS/MS mass spectroscopy figure; Wherein, the LC-EI-Q-TOF of A, three ring thiazolidomycin analogues analyzes and isotopic peak; The relative content that B, isotropic substance peak height characterize; C, bivalent molecule ionic MS/MS fragment are analyzed a-d: the structure of characteristic fragment and quality; The chemical structure of two kinds of ring thiazolidomycin analogues that D, T6C produced.
The LC-EI-Q-TOF of Figure 16, P9A and MS/MS mass spectroscopy figure; Wherein, the LC-EI-Q-TOF of A, four ring thiazolidomycin analogues analyzes and isotopic peak; The relative content that B, isotropic substance peak height characterize; C, bivalent molecule ionic MS/MS fragment are analyzed a-d: the structure of characteristic fragment and quality; The chemical structure of two kinds of ring thiazolidomycin analogues that D, P9A produced.
The biological activity test of Figure 17, ring thiazolidomycin analogue is figure as a result.
Embodiment
Below be to combine accompanying drawing and specific embodiment that embodiment of the present invention is done to specify.Present embodiment is being that prerequisite is implemented with technical scheme of the present invention, provide detailed embodiment and process, but protection scope of the present invention is not limited to present embodiment.
1. point mutation ring thiazolidomycin precursor peptide obtains the method structure of ring thiazolidomycin analogue
As shown in Figure 1, we select the pMD-18T carrier for use, and the 3kb fragment of coming out to contain the part ring thiazolidomycin gene cluster of ring thiazolidomycin precursor-gene cltA with pcr amplification is connected.Restriction enzyme SnaBI and SacI restriction enzyme site are contained in the fragment two ends that pcr amplification produces, and can be used for downcutting being connected with pJTU4892 again.Then, we just can add the restriction enzyme site of restriction enzyme BglII and SpeI on recombinant plasmid pMD-18T-3kb basis.We have designed a pair of primer aSpeIf (5 '-CTC GAC CGG TAC CCC GAC TAG TTG CTG CT-3 ') and aBglIIr (5 '-GTG CAG TTG GAC GCG CAG ATC TCG ACC-3 '), and the method through inverse PCR amplification recombinant plasmid pMD-18T-3kb successfully is added on the restriction enzyme site of restriction enzyme BglII and SpeI on the recombinant plasmid pMD-18T-3kb.Though can cause aminoacid sequence to change behind the introducing restriction enzyme site, can aminoacid sequence be reverted back to wild-type through the primer of wild-type at subsequent process.Owing among the plasmid pJTU4892 1 restriction enzyme SnaBI recognition site and 3 restriction enzyme SacI recognition sites are arranged; Except the SacI recognition site of our needs, other 2 restriction enzyme SacI recognition sites are at the apramycin resistant gene two ends of aac (3) IV.In order to make the SacI restriction enzyme site become the single endonuclease digestion site of plasmid pJTU4892, just must remove these two the restriction enzyme SacI recognition sites that are present in the apramycin resistant gene two ends of aac (3) IV.Because do not contain restriction enzyme SacI recognition site in the kalamycin resistance gene,, thereby reach the purpose of removing 2 restriction enzyme SacI recognition sites so we utilize kalamycin resistance gene to remove to replace the apramycin resistant gene.We utilize one couple of PCR-Targeting primer kanf (5 '-ACT ATT TGC AAC AGT GCC GTT GAT CGT GCT ATG ATC GAC AGC TAT TCC AGA AGT AGT GA-3 ') and kanr (5 '-CAG CAA TCA GCG CGA CCT TGC CCC TCC AAC GTC ATC TCG CTG GAT GCC GAC GGA TTT GC-3 ') successfully aac (3) the IV apramycin resistant gene of plasmid pJTU4892 to be replaced to the neo kalamycin resistance gene through the method for PCR-Targeting; Obtain plasmid pJTU4895, restriction enzyme SacI recognition site has just become the single endonuclease digestion site in the plasmid pJTU4895 like this.Then; We downcut the fragment of 3kb from the recombinant plasmid pMD-18T-3kb that contains BglII and SpeI recognition site with restriction enzyme SnaBI and SacI; With cut the carrier pJTU4895 that handled with restriction enzyme SnaBI with the SacI enzyme and be connected; Transformed into escherichia coli DH10B, acquisition also has the plasmid pJTU4896 of restriction enzyme BglII and SpeI recognition site.Stride the resistant gene that the intestinal bacteria ET12567 that belongs to conjugal transfer contains kantlex owing to being two parents of intestinal bacteria to streptomycete; PJTU4896 also contains the resistant gene of kantlex; Can not directly pJTU4896 be transformed into intestinal bacteria ET12567; So we have made up with XbaI and EcoRI enzyme and have cut the fragment of handling pJTU4896 and be connected the plasmid pJTU4897 that structure forms with XbaI with the fragment that the EcoRI enzyme is cut processing pSET152, thereby have replaced the neo kalamycin resistance gene with aac (3) IV apramycin resistant gene.Plasmid pJTU4897 contains aac (3) IV apramycin resistant gene like this; And contain restriction enzyme BglII and SpeI recognition site are arranged; Thereby the construction process that just can utilize mutant primer to be connected with the double digestion product is realized former site mutation ring thiazolidomycin precursor-gene cltA; Utilize intestinal bacteria to stride then and belong to conjugal transfer and just can in muta lead mycillin S.lividans 1326, realize heterogenous expression, thereby obtain to encircle the thiazolidomycin analogue to two parents of streptomycete.
2. the ring thiazolidomycin analogue that obtains
Ring thiazolidomycin precursor peptide 18 amino acid residue sequences are SNCTSTGTPASCCSCCCC, the construction process of introducing with the preceding text amino acid between the S1 to P9 that can suddenly change.Amino-acid residue S1 participates in the formation of pyridine ring in the ring thiazolidomycin structure, so can influencing whole ring thiazolidomycin structure, the S1 that possibly suddenly change can not form, so our amino acid between the C3 to P9 of selecting to suddenly change.We select sudden change conservatively, and the structure that has carried out 21 sudden changes altogether is as shown in table 1.
We are directed into the disappearance bacterium E.coliET12567/pUZ8002 that methylates respectively with 21 mutant plasmids and wild-type pJTU4892, distinguish conjugal transfer streptomycete type strain S.lividans1326 then, screen positive transformant with apramycin.The positive transformant that screens extracts the total DNA of streptomycete, on the one hand then with TSBY liquid culture 48 hours; The total DNA of streptomycete that extracts is transformed into E.coli DH10B, screens positive transformant, cultivated 12 hours for 37 ℃ with apramycin; If plasmid is recombinated on the S.lividans1326 karyomit(e); Positive transformant will appear, otherwise, with will not growing bacterium colony on the flat board; On the other hand, this carries out the checking of PCR to primer with Testcltf and Testcltr to extract the total DNA of streptomycete, with total DNA of S.lividans1326 extraction as negative control.The checking result of two aspects shows that all plasmids have been recombinated respectively on the S.lividans1326 karyomit(e) really.
Next; The S.lividans1326 heterogenous expression mutant strain that above-mentioned structure is successful carries out doing positive control with the solid fermentation S.lividans1326::pJTU4892 and the S.hygroscopicus var.Yingchengensis 10-22 that criticize, does negative control with S.lividans1326.30 ℃ of cultivations are taken out after 7 days with batch cultivating, cut with a knife broken after, with the methyl alcohol soaked overnight of two volumes; Second day with soak solution with filter paper filtering twice, remove solid matter, anhydrate with SODIUM SULPHATE ANHYDROUS 99PCT; With the rotary evaporation in vacuo appearance liquid is revolved driedly then, with 1～2ml methyl alcohol residuum is dissolved again again, the centrifugal 5min of the sample 12000rpm of elder generation; Disposable filter with 0.2 micron filters, and carries out the HPLC-MS detection and carries out mass spectrometric detection with LC-EI-Q-TOF.Table 2 has been listed the chemical structure and the biological activity of 14 mutant strains and 23 novel ring thiazolidomycin analogues.Ten mutant strains wherein in their the methyl alcohol extract of solid fermentation product, detect through LC-EI-Q-TOF and to have found corresponding ring thiazolidomycin analogue (Fig. 2).Further relative isotopic abundance and MS/MS mass spectroscopy accomplished these a series of new compounds carried out structure supposition and preliminary evaluation (Fig. 2～16).T4D solid fermentation thing methanol extract is analyzed (Fig. 3 A), its bivalent molecule quasi-molecular ions [M+2] through LC-EI-Q-TOF
2+, m/z 1504.3030 (theoretical value m/z 1504.2793), and isotopic characteristic peak type tentative confirmation this compound be ring thiazolidomycin analogue; The chemical structure of relatively encircling thiazolidomycin with and positive ion state (Fig. 3 B), the mass-to-charge ratio of the fragment peak of three characteristics with from ring thiazolidomycin parent ion structural formula, derive the theoretical mass-to-charge ratio consistent (Fig. 3 C) of coming out thus derive the chemical structure of the ring thiazolidomycin analogue that T4D produces.In wild-type strain fermentation product, can find two isomer of ring thiazolidomycin usually, in mutant strain T6D tunning, also find two peak by divalent ion, its [M+2] equally with different chemical displacement
2+Be 744.1352, incapability is exist (Fig. 4 B) that isotopic characteristic peak type or second order ms can both confirm these two isomer.Equally, analyze through LC-EI-Q-TOF, isotopic analysis and MS/MS fragment are analyzed, and we are to T6V (Fig. 5); T6G (Fig. 6), G7A (Fig. 7), G7S (Fig. 8), T8D (Fig. 9); T8S (Figure 10), P9H (Figure 11), T68D (Figure 12), T6S (Figure 13); C3S (Figure 14), T6C (Figure 15), the new ring thiazolidomycin analogue that P9A (Figure 16) produces carry out structure and infer and evaluation.The same with T6D, T8S, T68D, T6S all can produce two isomer (Figure 10 A, 12A, 13A).Yet T6G, G7A, T8D but have three identical [M+2]
2+(Fig. 6 A, 7A 9A), show that they can both produce three isomer at the peak.C3S (Figure 14 A) and P9A (Figure 16 A) all have the characteristic peak of four different ring thiazolidomycin analogues on LC-EI-Q-TOF ion flow graph, show that it can produce 4 kinds of different ring thiazolidomycin analogues.Yet for four compounds that P9A produces, the MS atlas analysis only can be inferred and the wherein chemical structures of two kinds of ring thiazolidomycin analogues.T6C has the characteristic peak (Figure 15 A) of three different ring thiazolidomycin analogues, and the MS/MS fragment is analyzed (Figure 15 C) only can infer the structure (Figure 15 D) that two compounds in these the three kinds ring thiazolidomycin analogues.
3. the bioactive test of ring thiazolidomycin analogue
At first, will encircle the thiazolidomycin analogue with solid medium SFM fermentation seven days.After fermentation finishes, with pocket knife with after the substratum chopping, with the methyl alcohol soaked overnight of two volumes; Second day with soak solution with filter paper filtering twice, remove solid matter, anhydrate with SODIUM SULPHATE ANHYDROUS 99PCT; With the rotary evaporation in vacuo appearance liquid is revolved driedly then, with 1～2ml methyl alcohol residuum is dissolved again again, the centrifugal 5min of the sample 12000rpm of elder generation; Disposable filter with 0.2 micron filters, and detects the output of each analogue with LC-EI-Q-TOF.Simultaneously, the corn stigma was cultivated seven days to ten days at 28 ℃ with the PDA substratum, with cotton swab corn stigma mycelium is scraped afterwards; Put into sterilized water, fully vibrate, make the mycelia physical efficiency fully break up with vibrator; Be tiled on the PDA substratum with liquid-transfering gun sucking-off 1ml then, dry up.Then, put the Oxford cup in media surface, in the cup of Oxford, add the ring thiazolidomycin analogue of equivalent, solvent also need keep equal-volume.At last, flat board is put into 28 ℃ of incubators, cultivate three days observationss.The active testing result shows (Figure 17), in 14 ring thiazolidomycin mutant strains, and T6D, T6S, T6V, growth has restraining effect to corn stigma (Bipolaris maydis) for T6G, T8S and P9A product.Mutant strain T6S wherein, T6V, T6G, the T8S restraining effect is stronger relatively; T6D and P9A then relatively a little less than.Except the said mutation strain, the product of all the other mutant strains knows half-and-half that all the growth of subphylum fungi corn stigma does not have restraining effect.
4. encircle the rapid extraction of new antibiotic in thiazolidomycin and this research
Get 30 ℃ of solid fermentation things of cultivating 5～7 days, cut with a knife broken after, with the methyl alcohol soaked overnight of two volumes; Second day with soak solution with filter paper filtering twice, remove solid matter, anhydrate with SODIUM SULPHATE ANHYDROUS 99PCT; With the rotary evaporation in vacuo appearance liquid is revolved driedly then, with 1～2ml methyl alcohol residuum is dissolved again again, the centrifugal 5min of the sample 12000rpm of elder generation; Disposable filter with 0.2 micron filters, and waits for detections such as HPLC-MS and QTOF.
5. utilize LC-EI-Q-TOF that new antibiotic in ring thiazolidomycin and this research is carried out the analysis of high precision mass spectrometric detection
What LC-EI-Q-TOF analyzed employing is Agilent 1200 systems of Agilent company, and the pillar specification is 2.1 * 30mmC18 reverse-phase column (Agilent Co.CA), and flow velocity is 0.3ml/min.A pump solution is 10% acetonitrile solution (containing 0.1% formic acid), and B pump solution is the acetonitrile that contains 0.1% formic acid.Condition of gradient elution is the concentration from 0% to 90% of solution B in 0 to 10 minute.Mass spectrometric detection is the 6530Accurate-MassQ-TOF mass spectrometric detection appearance through Agilent company.Second order ms (MS/MS) is to adopt the pattern of specifying target spot to rely on to carry out.
The extraction of embodiment 1, e. coli plasmid dna
The intestinal bacteria overnight culture of getting 1～1.5ml adds the EP pipe, thoroughly removes supernatant after centrifugal 30 seconds of 12000rpm is centrifugal.In the EP pipe, add 150 μ l alkaline bleach liquor cleavage solution I with liquid-transfering gun, break up bacterial sediment on the vibrator, make its suspension; Add 300 μ l alkaline bleach liquor cleavage solution II again, mixing makes the abundant cracking of thalline gently; Add 230 μ l alkaline bleach liquor cleavage solution III again; Add 100 μ l chloroforms (trichloromethane), vibration evenly; Then the EP pipe is put into whizzer, the centrifugal 5min of 12000rpm behind centrifugal the finishing, carefully takes out supernatant to new EP pipe; Add isopyknic Virahol, the centrifuge tube that turns upside down, fully mixing is placed 5min for-20 ℃; The centrifugal 1min of 12000rpm then, supernatant discarded; With 75% washing with alcohol deposition twice, will precipitate at last be dissolved in an amount of TE after the drying or the ultrapure water of the bacterium of going out in ,-20 ℃ of preservations uses.
The extraction of embodiment 2, the total DNA of streptomycete
Get 50～100 μ l mycelium and be placed in the EP centrifuge tube, the centrifugal supernatant that goes is collected the streptomycete mycelium, adds 500 μ l lysozyme solns, 37 ℃ of water-bath to mycelium thickness that becomes, and the general time is 30～60min; Add 500 μ l2%SDS, mix and to vibrate to solution thickness not, more transparent till; Add phenol among the 100 μ l, vibration, the centrifugal 5min of 12000rpm gets supernatant, adds an amount of middle phenol again, and vibration is centrifugal, gets supernatant; Add 1/10 times of volume 3mol/LNaAc (natural pH) and equal-volume Virahol; Put upside down mixing; Place 5min for-20 ℃; Then flocks is gone out washed twice in 75% ethanol with the rifle choicest, discards all supernatants at last, be dissolved in an amount of TE damping fluid after the drying or the ultrapure water of the bacterium of going out in.
Preparation and foreign DNA thereof that embodiment 3, intestinal bacteria calcium change the method competent cell transform
With the activation on new LA plate of intestinal bacteria DH10B bacterial strain, the single bacterium colony of the intestinal bacteria on the fresh LA plate of picking inserts in the LB liquid nutrient medium of 5ml, and 37 ℃ of shaking tables were cultivated 12 hours.Inoculum size according to 1/100 is transferred to colibacillary overnight culture in the LB liquid nutrient medium of 100ml (can add 20mmol/L MgCl among the LB
2), 37 ℃ of shaking tables are cultivated and were made thalline OD600 value be about 0.6 in 2.5 hours to 3 hours, rapidly thalline are placed the ice ice bath, and from then on cell should be in 0 ℃ to 4 ℃ the environment always.4 ℃ of centrifugal 6min of 3500rpm collect thalline, supernatant discarded, ice-cold 0.1M CaCl
2Again the thalline that suspends is positioned on the ice bath; 4 ℃ centrifugal, removes supernatant, collects thalline, and every 50ml initial incubation thing is with the ice-cold 0.1M CaCl of 2ml
2Thalline is suspended again; Divide to install in the Ep pipe, (microorganism collection, washing, centrifugal and packing all will be carried out the rifle head of use, Ep pipe, Ep pipe support, centrifuge tube and 0.1mol/L CaCl on ice to every pipe 50 μ l
2All want precooling, and note aseptic technique); ℃ preservation at last-70.Before the conversion, take out the competent cell of preserving, place on ice and thaw from-70 ℃ of refrigerators, treat cell thawing after, add 1～5ul transfer DNA, mixing, behind the ice bath 30min, 42 ℃ of heat shock 90s, ice bath 1～2min once more.Add LB 1ml, after 37 ℃ of shaking tables are cultivated 30min, be coated with out containing on the corresponding antibiotic LA flat board, dry up, cultivate for 37 ℃ and can obtain transformant in 12～16 hours.
The preparation and the foreign DNA thereof of embodiment 4, intestinal bacteria electricity commentaries on classics method competent cell transform
With the activation on new LA plate of intestinal bacteria DH10B bacterial strain, the single bacterium colony of the intestinal bacteria on the fresh LA plate of picking inserts in the LB liquid nutrient medium of 5ml, and 37 ℃ of shaking tables were cultivated 12 hours.Inoculum size according to 1/100 is transferred to colibacillary overnight culture in the LB liquid nutrient medium of 100ml (can add 20mmol/L MgCl among the LB
2), 37 ℃ of shaking tables are cultivated and were made thalline OD600 value be about 0.6 in 2.5 hours to 3 hours, rapidly thalline are placed the ice ice bath, and from then on cell should be in 0 ℃ to 4 ℃ the environment always.The centrifugal 6min of 4 ℃ of 3500rpm collect thalline, and supernatant discarded is washed thalline about 3 times with 10% glycerine of precooling; Remove supernatant, add a little precooling 10% glycerine, thalline is suspended again; Divide to install in the Ep pipe every pipe 50 μ l (microorganism collection, washing, centrifugal and packing all will be carried out on ice, and the rifle head of use, Ep pipe, Ep pipe support, centrifuge tube and 10% glycerine are all wanted precooling, and the attention aseptic technique); ℃ preservation at last-70.Before the conversion, take out the competent cell of preserving, place on ice and thaw from-70 ℃ of refrigerators, treat cell thawing after, add 1～3ul transfer DNA, mixing, ice bath 1～2min adds electricity and transforms cup and (in 50 * 1mm), regulate electric conversion instrument (EasyjecT Plus
, EquiBio company), making parameter be arranged to output voltage is 1800Volts, strength of electric field is 18000V.cm
-1After electricity swashs, place ice bath 1～2min rapidly, add LB 1ml, after 37 ℃ of shaking tables are cultivated 30min, be coated with out containing on the corresponding antibiotic LA flat board, dry up, cultivate for 37 ℃ and can obtain transformant in 12～16 hours.
After will having the competent cell of purpose plasmid of orlT through conversion entering intestinal bacteria ET12567/pUZ8002, incubated overnight transformant cell in the LB of 3ml substratum.With 1: 10 inoculum size overnight culture is connected among the fresh LB, cultivated 2～3 hours for 37 ℃; Simultaneously, can do heat shock and preparatory processing of sprouting to the streptomycete spore synchronously: collect the streptomycete spore with the cotton swab of sterilizing, again in suspension and the TES damping fluid; 50 ℃ of heat shock 10min; To be cooled to room temperature, add the preparatory germination medium of isopyknic spore again, cultivated 2～3 hours for 37 ℃.After having cultivated, the centrifugal 6min of 4000rpm collects coli somatic and streptomycete, to the intestinal bacteria washed twice, presses Bacillus coli cells number and spore count 10 with fresh LB substratum
10: 10
8Ratio both are mixed; And coat on the flat board that contains the SFM substratum; Dry up and place 30 degree incubators to cultivate, after 16～18 hours, flat board is covered with the 1ml sterilized water that contains corresponding microbiotic and nalidixic acid; 1～2h dries up flat board, places 30 ℃ of incubators and cultivates conjugal transfer that can see white after 2～3 days.
The title of table 1 sudden change ring thiazolidomycin precursor and concrete aminoacid sequence
Table 2 is summed up compound and the biological activity that mutant strain produced
| Mutant | Compounds Observed | Antibiotic?Activity |
| C3S | 4 | - |
| T4S | - | - |
| T4D | 1 | - |
| S5T | - | - |
| |
2 | ++ |
| T6D | 1 | - |
| |
1 | ++ |
| |
1 | ++ |
| T6C | 3 | - |
| G7A | 1 | - |
| G7S | 1 | - |
| T8D | 1 | - |
| P9G | - | - |
| |
4 | + |
| P9K | - | - |
| P9H | 1 | - |
| P9D | - | - |
| T46D | - | - |
| T48D | - | - |
| T68D | 1 | - |
| |
1 | ++ |
Claims (10)
1. the ring thiazolidomycin analogue that the sudden change of precursor peptide group produces is characterized in that, is to obtain through the method that may further comprise the steps: the restriction enzyme site that in ring thiazolidomycin precursor peptide gene cltA, inserts two BglII and SpeI restriction enzyme; Be connected with the double digestion product to make up with mutant primer and realize former site mutation ring thiazolidomycin precursor peptide gene cltA; Obtain ring thiazolidomycin analogue through the heterogenous expression in the pattern of streptomycete then.
2. the ring thiazolidomycin analogue that precursor peptide group sudden change according to claim 1 produces; It is characterized in that; The said method of in ring thiazolidomycin precursor peptide gene cltA, inserting the restriction enzyme site of two BglII and SpeI restriction enzyme specifically comprises the steps:
(1) selects the pMD-18T carrier for use; The 3kb fragment of coming out to contain the part ring thiazolidomycin gene cluster of ring thiazolidomycin precursor peptide gene cltA with pcr amplification is connected; Restriction enzyme SnaBI and SacI restriction enzyme site are contained in the fragment two ends that pcr amplification produces, and downcut to be connected with pJTU4892 again;
(2) method through PCR-Targeting replaces to the neo kalamycin resistance gene with the aac among the plasmid pJTU4892 (3) IV apramycin resistant gene, obtains plasmid pJTU4895;
The fragment of (3) downcutting 3kb from the recombinant plasmid pMD-18T-3kb that contains BglII and SpeI recognition site with restriction enzyme SnaBI and SacI; With cut the carrier pJTU4895 that handled with restriction enzyme SnaBI with the SacI enzyme and be connected; Transformed into escherichia coli DH10B, acquisition also has the plasmid pJTU4896 of restriction enzyme BglII and SpeI recognition site;
(4) cutting the fragment of handling pJTU4896 with XbaI and EcoRI enzyme is connected structure with the fragment of cutting processing pSET152 with XbaI and EcoRI enzyme and obtains plasmid pJTU4897; This plasmid pJTU4897 contains aac (3) IV apramycin resistant gene, and contains restriction enzyme BglII and SpeI recognition site.
3. the ring thiazolidomycin microbiotic analogue that precursor peptide group sudden change according to claim 1 produces; It is characterized in that; Said former site mutation ring thiazolidomycin precursor peptide gene cltA is specially the amino-acid residue in arbitrary site in 7 sites of the ring thiazolidomycin precursor peptide gene that suddenlyd change or two sites, and said 7 sites are C3, T4, S5, T6, G7, T8 and P9 position.
4. the ring thiazolidomycin analogue that precursor peptide group sudden change according to claim 1 produces is characterized in that said heterogenous expression is the heterogenous expression in the type strain muta lead mycillin Streptomyces of streptomycete lividans 1326.
5. the ring thiazolidomycin analogue that precursor peptide group sudden change according to claim 1 produces; It is characterized in that the mutant strain that said ring thiazolidomycin analogue is corresponding is C3S, T4D, T6D, T6V, T6G, T6S, T6C, G7A, G7S, T8D, T8S, P9H, P9A or T68D.
6. the ring thiazolidomycin analogue that precursor peptide group sudden change according to claim 5 produces is characterized in that the mutant strain that said ring thiazolidomycin analogue is corresponding is T6D, T6S, T6V, T6G, T8S or P9A.
7. the ring thiazolidomycin analogue that precursor peptide group sudden change according to claim 6 produces is characterized in that the mutant strain that said ring thiazolidomycin analogue is corresponding is T6S, T6V, T6G or T8S.
8. the ring thiazolidomycin analogue that precursor peptide group sudden change according to claim 7 produces is characterized in that the structural formula of said ring thiazolidomycin analogue is shown in formula (I), formula (II), formula (III), formula (IV) or the formula V,
9. the described ring thiazolidomycin of claim 6 analogue suppresses the purposes in the suppressor factor of corn stigma growth in preparation.
10. the purposes in the suppressor factor of preparation inhibition corn stigma according to claim 9 growth, it is characterized in that: the mutant strain that the ring thiazolidomycin analogue that said suppressor factor adopts is corresponding is T6S, T6V, T6G or T8S.
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| CN110305879A (en) * | 2019-07-10 | 2019-10-08 | 吉林大学 | A kind of southern corn leaf blight ChCDC3 gene and its application |
| CN111175397A (en) * | 2020-01-09 | 2020-05-19 | 大连理工大学 | GC-QTOF construction-based VOCs non-target screening method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007238453A (en) * | 2006-03-03 | 2007-09-20 | Hirosaki Univ | Novel cyclic thiopeptide and method for acquiring the same |
| CN101838642A (en) * | 2009-12-16 | 2010-09-22 | 上海交通大学 | Cyclothiazomycin biosynthesis function gene cluster |
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| JP2007238453A (en) * | 2006-03-03 | 2007-09-20 | Hirosaki Univ | Novel cyclic thiopeptide and method for acquiring the same |
| CN101838642A (en) * | 2009-12-16 | 2010-09-22 | 上海交通大学 | Cyclothiazomycin biosynthesis function gene cluster |
Non-Patent Citations (2)
| Title |
|---|
| AOKI ET AL: "Cyclothiazomycin, a novel polythiazole-containing peptide with renin inhibitory activity. taxonomy,fermentation, isolation and physico-chemical characterization", 《J ANTIBIOT》 * |
| 王绛: "吸水链霉菌10-22中硫肽类抗生素cyclothiazomycin生物合成机制研究", 《中国博士学位论文全文数据库》 * |
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| WO2015191789A3 (en) * | 2014-06-10 | 2016-08-11 | The Board Of Trustees Of The University Of Illinois | Reactivity-based screening for natural product discovery |
| CN110305879A (en) * | 2019-07-10 | 2019-10-08 | 吉林大学 | A kind of southern corn leaf blight ChCDC3 gene and its application |
| CN110305879B (en) * | 2019-07-10 | 2022-03-29 | 吉林大学 | Maize small leaf spot pathogen ChCDC3 gene and application thereof |
| CN111175397A (en) * | 2020-01-09 | 2020-05-19 | 大连理工大学 | GC-QTOF construction-based VOCs non-target screening method |
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