CN104593440A - Biosynthesis method for side chain introducing amino acid source to polyketone skeleton and related genes - Google Patents

Abstract

The invention relates to a biosynthesis method for a side chain introducing amino acid source to a polyketone skeleton. The method is based on the biosynthetic pathway of an aroma extending unit of benzyl malonyl mono-acyl coenzyme A; the related genes of the biosynthesis and recognition of the extending unit come from the biosynthetic pathway of splenocin (SPN) and enterocin (ENC) in marine streptomyces Streptomycessp.CNQ431, and comprise genes enccP, enccH, spnE and spnD. The pathway first conducts deamination reduction carboxylation on phenylalanine to transform the phenylalanine into the benzyl malonyl mono-acyl coenzyme A extending unit, and then conducts selective acyltransferase recognition on the aroma and lards type extending units and uploads the extending units on the polyketone skeleton. The strategy and biosynthetic pathway directly transform amino acid into CoA polyketone extending unit and insert the extending unit to the polyketone carbon skeleton, greatly enrich diversified means and structural types of polyketone structure, and generate a positive promotion function on research and development of polyketone medicine.

Description

A kind of biosynthetic means and genes involved introducing the side chain of origin of amino acid to polyketone skeleton

Technical field

The invention belongs to microbiological genetic engineering field, be specifically related in polyketone synthetic system, the gene that biosynthetic means and biosynthesizing to the side chain of polyketone skeleton introducing origin of amino acid are correlated with.

Background technology

Polyketide (polyketides, PKs) is the important natural product of a large class, extensively derives from bacterium, fungi, plant etc.Comprise Macrolide, anthracycline, polyethers, tetracyclines.Polyketide is various structures not only, and has good biology and pharmaceutical activity (Angew Chem Int Ed Engl, 2009,48 (26): 4688-4716.).If antibacterium medicine is (as tsiklomitsin, rifomycin), the medicine in the polyketone such as antifungal drug (as grisovin, amphotericin), antiparasitic (as avilamycin, Nemadectin), anticarcinogen (as Dx, enediyne), anti-cholesterol drug (as lovastatin) source is almost for treatment (the Nat Prod Rep of all important diseases, 2001,18 (4): 380-416.).

Although natural polyketide is of a great variety; its biosynthesizing mechanism is but relatively conservative; say from essence; formed the polyketone carbon skeleton of certain length by the precursor (comprising start element and extension apparatus) that acyl-CoA (acyl-CoA) activates decarboxylation one condensation reaction carried out under the catalysis of polyketide synthase (PKS) repeatedly, and then through cyclisation or aromatize, methylate; glycosylation; redox, the modification such as dehydration, form various baroque polyketides.According to structure and other character of polyketide synthase, polyketide synthase is divided into I-III type PKS (Curr Opin Chem Biol, 2003,7 (2): 285-295.), wherein I type PKS, modularization PKS and repeat type PKS can be divided into, namely modularization forms multifunctional enzyme in modular form, wherein the required structural domain (domain) of a whole set of polyketone synthesis compound carbon skeleton condenses together composition module (module), each structural domain only participate in whole polyketone carbochain build in a step biochemical reaction (non-interative).Repeat type I type PKS and modularization I type PKS Core domain similar, difference is that repeat type PKS can reuse certain structural domain to synthesize object product.The Core domain of polyketone synthesis carbochain comprises acyltransferase (acyltransferase, AT), acyl carrier protein (acyl carrier protein, and beta-keto acyl base synthetase (ketosynthase ACP), KS) form, first by as " entrance guard " (gate keeper) the identification of AT structural domain and optionally start element and extension apparatus are uploaded to ACP come from the phosphopan tetheine ethamine sulfydryl arm of coenzyme A (CoA), by KS, different unit is assembled into carbon skeleton through a series of decarboxylation condensation reaction (claisen condesation) again.I type PKS mainly synthesizes macrolide, polyethers, polyalkenes compound.II type PKS, also claims iterative type (iterative) or fragrant PKS, its main elbs reaction lopps, the aromatics such as tetracyclines.II type PKS is by reusing a set of structural domain, and repeatedly the decarboxylation condensation of the identical extension apparatus of catalysis makes polyketone carbon skeleton be extended, and defines the intermediate of chain, makes it to form aromatic nucleus subsequently by cyclase and aromatase catalysis.III type PKS, belongs to chalcone synthase (chalconesynthase, CHS) class, the biosynthesizing of primary responsibility monocycle or Bicyclic class polyketide.

The sales volume that the medicine that polyketone derives relates to every year reaches hundreds billion of dollar, is an important directions in current new drug development field to the exploitation of polyketone natural product, so obtain the most important condition that diversified polyketone analog is new drug development.Determine that the multifarious link of polyketone natural product is substantially identical, be mainly: 1) start element, 2) extension apparatus and the mode of modification, 3) skeleton dissociate and 4) afterwards modify (Drug Discov Today, 2009,14 (21-22): 1011-1020.).Wherein, start element and extension apparatus constitute main chain and the side chain of polyketone carbon skeleton as building block; The mode of dissociating determines cyclization or the linearizing of carbon skeleton; The modification of extension apparatus and modifying normally to the modification of some reactive groups such as hydroxyl, carbonyl on carbon skeleton afterwards.The structural changes produced is modified for rear, usually can be complementary preferably by chemical process, but for the carbon skeleton generated by start element and extension apparatus condensation, selective chemical transformation cannot realize substantially.But, by synthetic biology means, the AT module that condensation unit responsible in PKS is uploaded is replaced to the AT identifying different units, or the selectivity of AT structural domain is changed by the method for rite-directed mutagenesis, be equipped with the biosynthetic pathway of new condensation unit again, the carbon skeleton that can be implemented in polyketone optionally introduces different side chains, obtains a series of polyketone analogue.This AT module (AT swapping) method of replacing has become creates carbon skeleton multifarious the most effective means (Nat Rev Micro, 2005. 3 (12): 925-36 at present; Curr Opin Microbiol, 2007. 10 (3): 238-45; Curr. Opin. Struct. Biol. 2013,23,603-612).Although in theory by this method, can revise arbitrarily polyketone carbon skeleton, in fact, the derivative obtained by change extension apparatus is little.Its major cause is 1) natural extension apparatus structure type is single; 2) polyketone catalytic module, particularly identifies that the substrate selective of the acyl group transfer protein-AT of extension apparatus is higher.

There are two class polyketone extension apparatus in occurring in nature, the extension apparatus (Fig. 1) of coenzyme A (CoA) carrying and ACP carrying.The extension apparatus application of CoA carrying is the most general; general α carboxylation (the Nat. Prod. Rep. 2009 by acyl-coenzyme; 26; 90-114), propanedioic acid is connected (Eur. J. Biochem. 1998 with CoA; 257; 395-402), reduction carboxylation (the Nat. Prod. Rep. 2012,29,72-86 of alpha-beta unsaturated fatty acids-CoA; J. Am. Chem. Soc. 2009,131,10376-10377; J. Am. Chem. Soc. 2010,133,976-985; J. Am. Chem. Soc. 2011,133,1971-1977; Angew. Chem. Int. Ed. 2011,50,4667-4670) and synthesize.The extension apparatus of ACP carrying, range of application is narrower, is only present in PKS system (the Proc. Natl. Acad. Sci. USA 2006,103,14349-14354 of only a few; Chem. Biol. 2006,13,575-585; J. Am. Chem. Soc. 2013,135,18032-18035).The extension apparatus of CoA carrying is mainly derived from fatty acid metabolism, and therefore, their C-α side chain belongs to simple aliphatic compound mostly.Correspondingly, the specificity of AT structural domain and extension apparatus strict conformance, be only limitted to simple chemical structure.Due to the selection specificity of limited extension apparatus and AT structural domain, at present, the chemical space scope can introducing extension apparatus is limited to simple fatty compounds more.Although the external synthesis of extension apparatus (J. Am. Chem. Soc. 2001; 123; 5822-5823) and with acyl group-NAC thioesters as extension apparatus substitute (Nat. Prod. Rep. 2008; 25; 25-34) this predicament can be alleviated a little, but high preparation cost and extremely low doping efficiency still limit their application.

The same with lipid acid, amino acid is often widely used as poly-peptide and alkaloidal biosynthesizing assembly unit, is the carbon source of another large class organism promote coagulate tube.But, in the biosynthetic process of polyketone, but Serine and proline(Pro) is only had to be used to synthesizing amino malonyl-list acyl coenzyme A(aminomalonyl-ACP) (Fig. 1 (1)) and dichloro pyrroles propyl group malonyl-list acyl-ACP(dichloropyrrolypropylmalonyl-ACP) (Fig. 1 (2)) take part in two PKS synthetic systems (Proc. Natl. Acad. Sci. USA 2006,103,14349-14354; J. Am. Chem. Soc. 2013,135,18032-18035.).Different with lipid acid, amino acid contains abundant functional group, as amino and mercaptan, can be further used as the target spot of molecular design.In addition, by existing biosynthesizing mechanism, as Phenylalanine hydroxylase (Nature 2010,468,461-464) and tryptophane chlB4 (Mol. BioSyst. 2011,7,852-861) increase further the diversity of amino acid aromatic group, thus increase drug molecule lipotropy and and target protein between pi-pi accumulation power, improve bioavailability and biological activity (the Expert Opin. Drug Discov. 2014 of drug molecule, 9,995-1003).By amino acid converting be extension apparatus, chemistry and the structure diversity of polyketone can be expanded largely.But, at present, the polyketone extension apparatus of origin of amino acid is few, only has two kinds that above-mentioned Serine and proline(Pro) are originated, owing to being both the extension apparatus of ACP carrying, polyketone synthetic system can not be widely used in, in addition, its biosynthetic pathway is comparatively complicated, is difficult to use in transformation polyketone skeleton, and can not generally be applicable to other amino acid to introduce polyketone skeleton, so one is found to be current problem demanding prompt solution by the amino acid converting universal method for polyketone extension apparatus.

Summary of the invention

For above-mentioned situation, the object of this invention is to provide a kind of biosynthetic means introducing origin of amino acid side chain in polyketone skeleton, it comprises the steps:

1, amino acid is formed α, β-undersaturated carboxylic acid through amino acid-cleaving enzymes catalysis;

2, CoA ligase catalysis α, β-undersaturated carboxylic acid, forms α, β-undersaturated carboxylic acid coenzyme A;

3, by crotonyl-CoA reduction carboxylases catalyse α, β-undersaturated carboxylic acid coenzyme A forms the malonyl-list acyl coenzyme A replaced;

4, the AT model choice in polyketide synthases the malonyl-list acyl coenzyme A uploading replacement enters polyketone building-up process, realizes the side chain of origin of amino acid to be introduced in the carbon skeleton of polyketide.

Further, the invention provides a kind of biosynthetic means introducing phenylalanine source side chain fragrant benzyl in polyketone skeleton, it comprises the steps:

1, phenylalanine lyase Enc _cthere is reduction deamination and generate styracin in P catalysis phenylalanine;

2, styracin CoA ligase Enc _cstyracin coenzyme A is generated under the catalysis of H;

3, crotonyl-CoA reduction carboxylase SpnE catalytic reaction former carboxylation reaction of surviving generates benzyl malonyl-list acyl coenzyme A;

4, the special AT structural domain identification benzyl malonyl-list acyl coenzyme A in polyketide synthases SpnD is also uploaded to polyketone skeleton.

In described approach, genes involved derives from the splenocin(SPNs in marine streptomyces Streptomyces sp. CNQ431) and enterocin (ENC) biological synthesis gene cluster, comprise gene enc _c p, enc _c h, spnE, spnD.

Antimycin (antimycin ANTs) is that the polyketone that a class has an allergic asthma activity that excellent suppression is caused by anaphylactogen gathers peptide Hybrid compounds, its parent nucleus is nine yuan of dilactones, and replace on 3 hydroxyls of parent nucleus and have 3-formamido-Whitfield's ointment, 4 and 9 are respectively by methyl substituted.Parent nucleus C-7 position is replaced by the R1 alkyl of straight or branched, by R2 acylations (Fig. 2) on the hydroxyl of C-8 position.Its biosynthetic pathway was illustrated (Org. Lett. 2012,14,4142-4145 in 2013, ACS Chem. Biol. 2012, 7, 1956-1961.), first tryptophane is through a series of oxidation open loop, upload, epoxidation, reset to wait and generate FSA(3-formamidosalicylic acid), lipid acid through the various forms of short chain fatty acids of β-oxidation and formation at crotonyl-CoA reductase enzyme (crotonyl-CoA reductase, AntE) be cofactor with NADPH under effect, carboxylation forms corresponding extension apparatus, and Threonine then, malonyl CoA assembles the skeleton of chain formation ANTs together through PKS and NRPS, finally discharge macrolide precursor by TE catalysis from polyketone chain.Multiple acry radical donor is transferred to the C-8 position of mother nucleus structure by acyltransferase AntB, makes ANTs finally ripe.

Splenocins (SPNs) and antimycin (antimycin ANTs) structure height similar (Fig. 2), from marine streptomyces s. sp. be separated in the fermented liquid of CNQ431 and obtain (J. Med. Chem. 2009,52,2317-2327), maximum difference is that the C-7 position side chain of SPNs is fragrant benzyl, and the C-7 position of ANTs is the fat hydrocarbon chain of straight or branched.The same with ANTs, the C-8 position hydroxyl of SPNs can replace by multiple acry radical donor or be not substituted, in the present invention, by C-8 position R ₁=COCH(CH ₃) C ₂h ₅sPNs called after SPN-C, and C-8 position hydroxyl is not SPN-J by the SPNs Compound nomenclature of i.e. R1=H.This C-7 be the compound of aromatic structure in ANTs producing bacterial strain, as s.sp.nRRL2288, s. albusj1074, s.ambofaciensall do not detect in ATCC 23877 (Org. Lett. 2012,14,4142-4145).According to the biosynthetic pathway of ANTs, the present inventor infers that the fragrant benzyl of C-7 position very likely derives from polyketone extension apparatus benzyl malonyl-list acyl coenzyme A(benzymalonyl-CoA of the present invention) (Fig. 1 (3)).

The invention provides splenocin biological synthesis gene cluster, as SEQ ID:1, this sequence contains 21 genes or complementary sequence altogether, contain 6 independent basises because of: orf-1, orf-2, orf-3, orf-4, orf-5, orf-6; 1 regulatory gene: spnA; 9 responsible start element synthetic genes: spnF, spnG, spnH, spnI, spnJ, spnK, spnL, spnN, spnO; The gene of 4 responsible skeleton synthesis spnB, spnC, spnD, spnM; A crotonyl-CoA reduction carboxylase gene: spnE.

Described spnEgene, nucleotide sequence is positioned at 21443-22663 the base place of SEQ ID:1, and length is 1221 base pairs, and coding crotonyl-CoA reduction carboxylase, length is 406 amino acid.

Described spnDgene; its nucleotide sequence is positioned at 17566-21441 the base place of SEQ ID:1, and length is 3822 base pairs, coding polyketide synthases; length is 1273 amino acid, and acyltransferase AT structural domain wherein can specific recognition and loading benzyl malonyl-list acyl coenzyme A.

The invention provides gene spnEcoded aminoacid sequence, be crotonyl-CoA reduction carboxylase, have 406 amino acid, aminoacid sequence is as shown in SEQ ID:3.

The invention provides gene spnDcoded aminoacid sequence, be polyketide synthases, have 1273 amino acid, aminoacid sequence is as shown in SEQ ID:4.

After bioinformatic analysis is carried out to SPNs biological synthesis gene cluster, fail to find the genes involved synthesizing extension apparatus benzyl malonyl-list acyl coenzyme A.When this gene cluster is passed through genetic operating system, be integrated into non-SPNs producing strains s.eolicolor.M145 genome, also fails to ferment SPNs, and illustrate, the genes involved of synthesize benzyl malonyl-list acyl coenzyme A is not in SPNs biological synthesis gene cluster.The present inventor couple s.sp.cNQ431 genome checks order entirely, have found one with the gene cluster of enterocin biological synthesis gene cluster very high homology.Enterocin biological synthesis gene cluster and approach are at its producing strains s.maritimus illustrated in (Chem. Biol. 2000,7,943-955), wherein had two genes encPwith encHbe respectively phenylalanine lyase gene and styracin CoA ligase gene.

s.sp.enterocin biological synthesis gene cluster in CNQ431, as SEQ ID:2, this gene cluster contains 20 genes or complementary sequence altogether, called after: enc _c a, enc _c b, enc _c c, enc _c d, enc _c e, enc _c f, enc _c g, enc _c h, enc _c i, enc _c j, enc _c k, enc _c l, enc _c m, enc _c n, enc _c o, enc _c p, enc _c q, enc _c r, enc _c s, enc _c t.Wherein enc _c pwith enc _c hrespectively with encPwith encHvery high homology.

The invention provides enc _c pthe nucleotide sequence of gene, is positioned at 15727-17298 the base place of SEQ ID:2, and length is 1572 base pairs, encoding phenylalanine lyase.

The invention provides enc _c hgene nucleotide series, is positioned at 2798-4405 the base place of SEQ ID:2, and length is 1608 base pairs, encoding cinnamate CoA ligase.

The invention provides gene enc _c pthe aminoacid sequence of coding, be phenylalanine lyase, have 523 amino acid, aminoacid sequence is as shown in SEQ ID:5.

The invention provides gene enc _c hthe aminoacid sequence of coding, be styracin CoA ligase, have 535 amino acid, aminoacid sequence is as shown in SEQ ID:6.

The present invention, from SPNs biosynthetic pathway, by body and experiment in vitro, illustrates biosynthesizing and the loading path of fragrant extension apparatus benzyl malonyl-list acyl coenzyme A, and its route of synthesis have employed a unprecedented deamination reduction.Due to a lot of amino acid, as: tyrosine (tyrosine), Histidine (histidine); aspartic acid (aspartic acid) etc. has corresponding amino lyase; the gene of encoding such enzymes is all known (Biochemistry 2011,50,6053-6062; FEBS Lett. 2002,512,240-244; Biochemistry 1999,38,5355-5361; Curr. Opin. Chem. Biol. 2011,15,234-240; Nat. Chem. 2012,4,478-484.).In addition, the wide in range property of substrate of some CoA ligases is very large, CoA ligase in such as Fatty acid degradation approach, can identify various α, β-undersaturated carboxylic acid substrate, change into corresponding carboxylic acid coenzyme A (Angew. Chem. Int. Ed. 2013,52,12308-12312.J. Bacteriol. 2003,185,20-27.Nat. Chem. Biol. 2014, doi:10.1038/nchembio.1718.).The gene information of these CoA ligases same is also known.Crotonyl-CoA reduction carboxylase (SpnE) that the present invention discloses, SpnD-AT module and accordingly some homologous proteins have wide in range substrate identity, various substrate (Angew. Chem. Int. Ed. 2013 can be identified, 52,12308-12312, Nat. Chem. Biol. 2012,8,117-124).Therefore, according to the approach that the present invention discloses, by various amino acid whose lyase and the CoA ligase with wide in range substrate recognition capability, crotonyl-CoA reduction carboxylase and AT block combiner, just can construct the new way other amino acid being introduced polyketone carbon skeleton.For realizing the diversity of polyketone structure, open brand-new situation.

Accompanying drawing explanation

Fig. 1 is occurring in nature known polyketone extension apparatus structure;

Fig. 2 is the mother nucleus structure of SPNs and ANTs;

R in figure ₁and R ₃hydrogen or acyl group, R ₂for alkyl, the involved SPN-C:R of experiment in the present invention ₁=COCH (CH ₃) C ₂h ₅, SPN-J:R ₁=H

Fig. 3 is the route of synthesis of SPNs biological synthesis gene cluster and supposition.

(A) SPNs and ANTs (NRRL2288) biological synthesis gene cluster contrast in figure, the function color inferred of each gene marks, and sums up in Table 1.(B) SPNs and the ANTs route of synthesis inferred in CNQ431, wherein, ACP: acyl carrier protein; C: condensation functional domain; A: polyadenylation functional domain; PCP: peptide pole carrier proteins; KR: keto reductase functional domain; KS: ketosynthase functional domain; TE: thioesterase functional domain.

Fig. 4 is s. sp.CNQ431 and s. SPN-C and ANTs HPLC analysis chart in coelicolor recombinant bacterial strain;

In figure SPN-C and ANTs use respectively ▼ and ● mark, I) CNQ431 wild strain; II) CNQ431 Δ spnCmutant strain; III) CNQ431 Δ enc _c hmutant strain; IV) CNQ431 Δ enc _c pmutant strain; V) SPNs biological synthesis gene cluster is integrated with s. coelicolor mutant strain; V I) s. coelicolor wild strain.

Fig. 5 is the experiment in vitro of SpnE and SpnD;

In figure: the reaction of (A) SpnE and SpnD catalysis; (B) there is reduction carboxylation and generate benzyl malonyl-list acyl coenzyme A(3 in SpnE catalysis styracin coenzyme A (4)) and the HPLC analysis chart of benzyl propionyl coenzyme A (7); (C): the peptide section (PVLVEIGPGDSLTK) in SpnD before the reaction after high resolution mass spectrum HRMS analytical data; (D): the HPLC that benzyl malonyl-list acyl coenzyme A is consumed under SpnD catalysis analyzes.

Fig. 6 is external biochemical reaction and the kinetic parameter thereof of SpnE;

In figure, (A) (B) (C) is respectively SpnE catalytic substrate styracin coenzyme A (4), crotonyl-CoA (5), reduction carboxylation occurs 5-methyl-2-hexenoyl coenzyme A (6) and reduction reaction HPLC detects figure, and select the reaction of 5-methyl-2-hexenoyl coenzyme A (6), be LC-HRMS to detect, the kinetic parameter of (D) SpnE, substrate is styracin coenzyme A (4), crotonyl-CoA (5).

Fig. 7 is Enc _cthe HPLC analysis chart of P catalyzed reaction;

In figure, I) phenylalanine standard product; II) styracin (8) standard substance; III) Enc _cp catalystic converter system.

Fig. 8 is the HPLC analysis chart of SPN-J tunning in AL2110 recombinant bacterium.

In figure, SPN-J ▼ marks, I) AL2110 wild-type; II) be integrated with ermE ^* - enc _c pthe AL2110 mutant strain of fragment; III) integrate ermE ^* - enc _c p- enc _c hthe AL2110 mutant strain of fragment.

Fig. 9 is Δ spnC, Δ enc _c p, Δ enc _c hmutant strain genotype is verified;

In figure, Δ spnCmutant strain checking primer PCR amplifies 1.25 kb fragments, Δ enc _c pmutant strain checking primer PCR amplifies 0.95 kb, Δ enc _c hmutant strain checking primer PCR amplifies 0.68 kb.

Figure 10 is SpnD, SpnE, Enc _c12 %SDS-PAGE of P albumen detect figure

In figure, (A) SpnD albumen, 133kD; (B) SpnE albumen, 45kD; (A) Enc _cp albumen, 57Kd.

The approach of Figure 11 styracin coenzyme A chemosynthesis

In figure, Oxalyl chloride: careless chloric acid; DMF:N, dinethylformamide; Et ₃n: triethylamine; THF: tetrahydrofuran (THF).

Embodiment

The features and advantages of the invention can be understood further by reference to the accompanying drawings by following detailed description.The embodiment provided is only the explanation to the inventive method, and does not limit the present invention in any way all the other contents of announcement.

The experimental technique of unreceipted actual conditions in following Examples, usual conveniently condition, as " Molecular cloning " a laboratory manual third edition, the condition that " Practical streptomyces genetics " described conditioned disjunction production firm is recommended.

1. the clone of SPNs biological synthesis gene cluster and biosynthesizing thereof

SPNs with ANTs structure is very similar, and parent nucleus is all nine yuan of dilactone structures, and maximum difference is at the side chain of C-7 position, and SPNs is fragrant benzyl, and ANTs is fat hydrocarbon chain.According to s. sp. NRRL2288 ANTs biosynthetic pathway (Org. Lett. 2012,14,4142-4145; ACS Chem. Biol. 2012,7,1956-1961.), we gather enzyme/polyketide synthases gene with non-ribosomal peptides antDas PCR probe, success is built s. sp. the Fosmid gene library of CNQ431 is screened, successfully screen a positive and stick grain, sequencing result is the same with anticipation, and this gene cluster and ANTs gene cluster reach the homology of 95%, bioinformatic analysis shows, this gene cluster encodes 21 open reading frame (table 1).

In SPNs biological synthesis gene cluster except a transposase gene orf3, all the other gene orders all with ANTs biosynthesis gene homology.In conjunction with ANT biosynthetic pathway, we have inferred the function of each gene in SPNs biosynthetic pathway: Spn H I J K L M N O is responsible for catalysis and forms start element 3-formamido-Whitfield's ointment; SpnF and SpnG is respectively CoA ligase and the ACP of ATP dependence, and the start element 3-formamido-Whitfield's ointment generated through a series of enzymic catalytic reaction is uploaded to ACP(SpnG) on; SpnC and SpnD is responsible for the synthesis that take part in SPNs skeleton.SpnC is the NRPS of two modules, and structural domain is respectively C-A-PCP-C-A-KR-PCP in order, and wherein SpnC-M1-AT identifies and uploads Threonine, and SpnC-M2-AT is responsible for identifying and uploads pyruvic acid; SpnD is the PKS of a module; structural domain is respectively KS-AT-PCP-TE in order; AT structural domain is wherein responsible for identifying and is uploaded fragrant extension apparatus benzyl malonyl-list acyl coenzyme A; finally form nonatomic ring dilactone at effect ShiShimonoseki ring of SpnD-TE; multiple acry radical donor is transferred on acyl acceptor by SpnB, makes splenocin finally ripe.SpnA plays regulating and controlling effect at this, SpnE and extension apparatus synthesis relevant (Fig. 3).

The functional analysis of table 1 splenocins biological synthesis gene cluster

In order to prove that this gene cluster is really relevant to SPNs biosynthesizing, we have interrupted SpnC gene, and HPLC, LC-HRMS analyze and show Δ spnCmutant strain loses the ability (Fig. 4 II) of synthesis SPN-C and ANTs completely, confirms that this gene cluster is responsible for synthesis SPNs and ANTs really.

2, benzyl malonyl-list acyl coenzyme A optionally identifies as extension apparatus and AT structural domain

In ANTs; C-7 position alkyl comes from the identification of AntD-AT structural domain and the alkyl malonyl-list acyl coenzyme A extension apparatus uploaded, extension apparatus be herein alpha-beta unsaturated fatty acids crotonoyl reduce carboxylase (CCR AntE) catalysis under there is reduction carboxylation and generate.So we infer, and the SpnE of AntE very high homology is responsible for synthesize benzyl malonyl-list acyl coenzyme A, and is identified by the SpnD-AT with AntD-AT homology and upload (Fig. 5 A).We carry out great expression to SpnD and SpnE respectively in large intestine system; then one of the substrate styracin coenzyme A (4) of chemosynthesis SpnE; test SpnD and SpnE enzyme in vitro to live; result: SpnE can catalysis styracin coenzyme A (4), crotonyl-CoA (5) and 5-methyl-2-hexenoyl coenzyme A (6); there is reduction carboxylation reaction and reduction reaction (Fig. 6 A B C) in it, kinetic parameter shows that SpnE is to styracin coenzyme A (4) (k _cat/ K _m=1.42 ± 0.008 min ^-1mM ^-1) selectivity is crotonyl-CoA (5) (k _cat/ K _m0.614 ± 0.041 min ^-1mM ^-1) 2.3 times (Fig. 6 D).

Phosphopantetheinyl transferase (phosphopanttetheinyl transferase PPTase) can the phosphopan tetheine ethamine sulfydryl arm covalency of catalysis coenzyme A (CoA) upload on the serine residue hydroxyl of ACP, ACP structural domain is become the activated holo-ACP form (Biochemistry 1998 that can hang over extension apparatus from the apo-ACP form of non-activity, 37,1585-1595), so we are transferred to the expression plasmid with SpnD containing sfp Phosphopantetheinyl transferase gene e. coliin BAP I host, obtain ACP by the holo-SpnD activated by protein expression and purification.According to the route of synthesis of SPN, benzyl malonyl-list acyl coenzyme A will be responsible for selecting by the AT structural domain in SpnD and be uploaded on the serine residue of ACP functional domain.In order to prove that it is uploaded, the benzyl malonyl-list acyl coenzyme A of the holo-SpnD of purifying and above-mentioned synthesis mixes by we, and under 30 ° of C, react 1h, then SDS-PAGE electrophoresis is by SpnD protein salvage.After this albumen of trypsin hydrolyzing, mass spectrometric detection is present in SpnD-ACP structural domain, by the Serine peptide section (PVLVEIGPGDSLTK) that phosphopantetheine activates, proves whether have benzyl malonyl-list acyl to upload.This peptide section of result has the migration of 176.0516 Da, with benzyl malonyl-list acyl coenzyme A molecular weight consistent (Fig. 5 C).Further, along with the carrying out of this reaction, the amount of benzyl malonyl-list acyl coenzyme A is consumed (Fig. 5 D).Thus confirm that extension apparatus benzyl malonyl-list acyl coenzyme A is obtained by SpnE catalysis styracin coenzyme A, and can be identified by SpnD and upload.

3, the biosynthesizing of benzyl malonyl-list acyl coenzyme A, vivo and vitro experiment confirmation originates from amino acid.

Styracin coenzyme A is precursor substance (the Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989 of the synthetic styrene-acrylic element class extensively existed in higher plant body; 40; 347-369); but in bacterial body; distribution is few; only in minority route of synthesis, find (Chem. Biol. 2000,7,943-955; Chem. Commun. 2003,1358-1359).Plant and bacterium utilize phenylalanine lyase (phenylalanine ammonia-lyase, PAL) and CoA ligase (CoA ligase, CL), phenylalanine are transformed styracin and styracin coenzyme A successively.The present inventor, first by the glutinous grain containing the uncertain gene of the several function of SPNs gene cluster and both sides, by genetic operating system, is integrated into the producing bacterial strain of non-SPNs and non-ANTs s.ceolicolorM145 genome, carries out heterogenous expression.Detect with high performance liquid phase-high resolution mass spectrum coupling, confirm that this recombinant bacterial strain only produces ANTs, and fail to produce SPNs(Fig. 4 V, VI), illustrate that the genes involved of synthesize benzyl malonyl-list acyl coenzyme A is present in the outside of SPNs biological synthesis gene cluster.We are right subsequently s. sp. CNQ431 full-length genome carries out sequencing analysis, found one with s.maritimus middle enterocin(ENC) biological synthesis gene cluster (Chem. Biol. 2000,7,943-955) gene cluster of very high homology, this gene cluster contains 20 genes (table 2), before this in the biosynthetic pathway of enterocin, confirm phenylalanine ammonialyase (encP)with styracin coenzyme A ligase enzyme gene (encH)l-Phe can be changed into successively styracin and styracin coenzyme A (J. Bacteriol. 2005,187,4286-4289).Through bioinformatic analysis, we infer respectively with encPwith encHhomology enc _c pwith enc _c hbe responsible for synthesize benzyl malonyl-list acyl coenzyme A.These two genes are interrupted respectively, wherein CNQ431 Δ enc _c pmutant strain no longer produces SPN-C(Fig. 4 IV), and interrupt enc _c hthe generation of SPN-C is not affected (Fig. 4 III).Explanation enc _c pkeying action is played to the synthesis of styracin coenzyme A, and enc _c hcan be compensated by other CoA ligases in genome.The present inventor subsequently in stench false single (pseudomonas) to Enc _cthe protein induced expression of P, vitro enzyme is lived and is tested (Fig. 7), Enc _cphenylalanine can be converted into styracin (8) by P, and does not have activity to other amino acid.Vivo and vitro experiment confirms benzyl malonyl-list acyl coenzyme A jointly, is connected, reduction carboxylation and generating by phenylalanine through deamination, coenzyme A, and illustrate first, amino acid whose metabolism can generate polyketone extension apparatus further.

Each gene function analysis in table 2 enterocin biological synthesis gene cluster

Gene	Size (a.a)	Protein Homolog and Origin	Identity (%)	Proposed function
					E	137	EncE (AAF81720), S. maritimus	96	Transcriptional regulator
F	283	EncF (AAF81721), S. maritimus	96	Transcriptional regulator
					G	166	EncG (AAF81722), S. maritimus	95	Membrane protein
H	535	EncH (AAF81723), S. maritimus	95	Acyl-CoA ligase
					I	260	EncI (AAF81724), S. maritimus	92	Enoyl-CoA hydratase
J	406	EncJ (AAF81725), S. maritimus	97	β-oxoacyl-CoA thiolase
					K	241	EncK (AAF81726), S. maritimus	98	O-methyl transferase
D	266	EncD (AAF81727), S. maritimus	98	Ketoreductase
					A	398	EncA(AAF81728), S. maritimus	99	keto synthase α
B	407	EncB (AAF81729), S. maritimus	98	keto synthase β
					C	82	EncC (AAF81730) , S. maritimus	99	ACP
L	340	EncL (AAF81731) , S. maritimus	95	Acyl transferase
					M	464	EncM (AAF81732), S. maritimus	98	FAD-dependent oxygenase
N	522	EncN (AAF81733) , S. maritimus	95	Aryl-CoA ligase
					O	130	EncO (AAF81734), S. maritimus	92	Unknown protein
P	523	EncP (AAF81735), S. maritimus	98	Phenylalanine ammonia lyase
					O	97	EncQ (AAF81736), S. maritimus	93	Ferredoxin
R	401	EncR (AAF81737), S. maritimus	96	p450 monooxygenase
					S	215	EncS (AAF81739), S. maritimus	96	Regulatory protein
T	482	EncT (AAF81738), S. maritimus	95	Resistance efflux protein

4, transform ANTs biosynthetic pathway, generate SPNs

Infer according to conclusions, why ANTs producing strains (Org. Lett. 2012,14,4142-4145), can not synthesize SPNs, may be owing to not containing styracin coenzyme A synthesis related gene in body.In order to simplify meta-bolites, the present inventor, will by genetic operating system ermE*- enc _c pwith ermE*- enc _c p- enc _c hfragment is incorporated into NRRL2288-AL2110 respectively, and this bacterial strain is Δ antBmutant strain can only produce deacylated tRNA base ANTs(Angew. Chem. Int. Ed. 2013,52,12308-12312.).Fermentation detects, and is integrated with ermE*- enc _c pwith ermE*- enc _c p- enc _c hthe recombinant bacterial strain of fragment can produce new compound (Fig. 8), consistent with vivo gene interruption result, and gene is described enc _c hfunction can obtain complementation from the gene that in body, other have CoA ligase function.Expand recombinant bacterial strain fermentation system, and partly prepared by purifying to this compound, ¹h-NMR, ¹³c-NMR and high resolution analysis, this compound is SPN-J(Fig. 2), its C-7 position comprises a fragrant benzyl really.Due to acyl transferase gene disappearance, the hydroxyl of its C-8 position is not by acylations.The present inventor, successfully the biosynthesizing of benzyl malonyl-list acyl coenzyme A mechanism is applied to the transformation of polyketide structure, prove in ANTs synthetic system, AntD-AT structural domain also can identify and upload benzyl malonyl-list acyl coenzyme A simultaneously.

Below provide enforcement embodiment further, these implement embodiments can help to understand the present invention further, only for illustration of, do not limit scope of the present invention.

[embodiment 1] splenocin producing strains s.sp.CNQ431 the foundation of genetic operating system

1. donor bacterium prepares

To be converted into containing target plasmid e. colieT12567, on flat board, picking list colony inoculation contains corresponding microbiotic to 5mL LB() middle overnight incubation, the bacterium liquid drawing 4 mL is received in 50 mL LB (containing corresponding microbiotic), and be placed in 37 DEG C of shaking tables, 250 rpm are cultured between OD600=0.4-0.6.Centrifugal 4 min of 6000 rpm, thalline 20 mL LB wash twice, and to remove microbiotic, are resuspended in 1 mL LB subsequently, as DNA donor bacterium.

2. recipient bacterium prepares

Take out-80 DEG C, spore suspension (6.7x109/mL) one pipe that 20% glycerine is preserved, glycerine is removed after centrifugal 3 min of 8000 rpm, with 0.5 mL TES damping fluid (0.05 M, pH 8.0) washing twice, spore is with deglycerizin, use 500 μ l TES resuspended subsequently, and in 50 DEG C of heat-shocked 10 min, add 500 μ l TSB mixings afterwards and be placed on 37 DEG C, 220 rpm cultivation 2-3 hr, 8000 rpm, centrifugal 5 min collect spore, with 1 mL LB substratum washing once to remove TES damping fluid, resuspended rear as recipient bacterium with 1.5 mL LB.

3. Conjugative tiansfer

MS(soybean cake powder 2 % is spread evenly across, N.F,USP MANNITOL 2 %, agar powder 2 % after recipient bacterium and donor bacterium respectively being gone 100 μ L to mix) substratum is (containing 10 mM MgCl ₂) on flat board, in addition spore identical for 100 μ l is coated in the identical flat board (MgCl containing 10 mM ₂) on, wherein one piece of covering (see step below) is as negative control.Another block does not cover to observe thalline growing way.Cultivate 12 ~ 16 hours for 30 DEG C.Then on every block flat board, add 3 mL sterilized waters, to touch to phage surface by water uniform fold with spreading rod, blot with rifle afterwards.Add a cover 1 mL sterilized water (microbiotic containing respective concentration and 50 μ g/ mL how pyridine ketone acid), cultivate 3 ~ 5 days for 30 DEG C.

[embodiment 2] s. sp. the structure of CNQ431 gene library and screening

1. enzyme is tested conscientiously in a small amount

By 10 u/ μ L's aluit is working concentration (1 ×) that I restriction enzyme (Takara) is diluted to buffer with 10 × L buffer and aqua sterilisa, enzyme is 15 μ L systems of 0.33 U/ μ L concentration, prepare the reaction system of 250 μ l again (containing CNQ 431 gDNA 40 μ l, 10 × L buffer 25 μ l), then 250 μ l reaction systems are divided into 1 × 50 μ l and 7 × 25 μ l, then get 2 μ l and be diluted to 0.33 u/ μ l's in advance alui adds in the reaction system of the first pipe 50 μ l, mix in rear transferase 12 5 μ l to the second pipe 25 μ l, repeat such transfer step 7, above all operation stepss all will be carried out on ice, after then all systems 37 DEG C of enzymes being cut 15 min, 70 DEG C of inactivation 10 min, 4 DEG C of cold houses, 40 V, 0.5 % agarose gel electrophoresis 12 h, dye in ethidium bromide, observe enzyme under gel imaging instrument and cut quality.

2. a large amount of enzyme is tested conscientiously

According to the determined reaction conditions of preliminary experiment, the amount of the STb gene of 4 times and isocyatic enzyme dosage are prepared the DNA fragmentation built needed for library: system mixed on ice, be equally divided into four parts, 37 DEG C of enzymes are cut, respectively at 12,14,16,18 min, take out immediately and be placed in 70 DEG C of inactivation 10 min, strict timing.Every individual system first takes a morsel 0.5 % agarose gel electrophoresis 10 h in 4 DEG C of cold houses, detects the quality that enzyme is cut after ethidium bromide staining under gel imaging instrument.

3. DNA fragmentation reclaims

According to the situation that enzyme is cut, residue system is run the low melting-point agarose gel of 1 %, and the control DNA maker swimming lane of 40 kb sizes is cut, after ethidium bromide staining, again low melting point splicing together, because DNA fragmentation is larger, in order to avoid damaging it after a series of process DNA, or institute's size of cutting is improper, so respectively cut the band of wide about 3 mm respectively at 48 kb maker correspondence positions and top position thereof, weigh the weight of colloid, make glue with β-gelase and reclaim.Add 1/10 volume (1 mg is equivalent to 1 μ L) β-gelase buffer, temperature bath, to colloid fusing, adds β-gelase, (200 mg add the enzyme of 2 μ L), 60 DEG C of temperature bath 1 h.Place 15 min on ice, 12000 rpm 15 min.Get supernatant (containing DNA), add the NaCl solution of 1/10 volume and the Virahol of 1V, fully-80 DEG C of placement 20 min after mixing.12000 rpm, 15 min, removing supernatant, precipitates 2 times with the ethanol purge of 75 %, is dried to without ethanol taste, with the aqua sterilisa dissolving DNA of 30 μ l, get 1 μ l 0.5 % agarose gel electrophoresis 10 h, detects recovery quality stand-by.

4. the packaging in library

Get 25 μ l packaging proteins to join in the STb gene fragment be connected with carrier of 8 μ l.Remaining 25 μ l packaging proteins are put back into rapidly-80 DEG C of refrigerators stand-by, 30 DEG C of temperature bath 2 h.Remaining 25 μ l packaging proteins are added, 30 DEG C of temperature bath 2 h.Add the PDB buffer of 960 μ l to final volume 1 ml, then add the trichloromethane of 25 μ l, 4 DEG C of one week can be placed on stand-by.

5. transfection

In the LB of single bacterium colony to 3 ml of picking library construction host e. coli EPI-300,37 DEG C of incubated overnight, contain maltose by the LB(that bacterium liquid is forwarded to 50 ml) in, be cultured to OD ₅₀₀=0.8 immediately for transfection, as follows by transfection conditions: 2.5 μ l pack liquid+22.5 μ l PDB+ 25 μ l EPI300 room temperature (25 DEG C) 30 min, after add 200 μ l LB 37 DEG C cultivate 75 min, above-mentioned transfection liquid is coated on little culture plate, incubated overnight, grows about 1500 bacterium colonies.

6. titration and amplification

Random choose 11 single bacterium colonies in the clone grown, are linked in 3 ml LB, incubated overnight respectively, extract plasmid in second day, use bamh do enzyme to cut, through the agarose gel electrophoresis inspection of 0.5 %, randomness is good, carries out a large amount of transfection with same transfection method, cultivates library bacterium colony.Then bacterium colony is chosen in 96 orifice plates, have 34 blocks of plates, and numbering 1-34, put after 37 DEG C of incubators cultivate 28 h, add the glycerine of 40 %, to final glycerol concentration 20 %, be placed on-80 DEG C of refrigerators frozen.

7. the screening in library

Use ANTs producing strains s. sp. gene in ANTs route of synthesis in NRRL 2288 antDgene order design library screening primer 5 '-GCTCGGTGGCCCTGGTCCACGC-3 ' and 5 '-GGCAGCGCCTCGTCGATCTCC-3 ' finally screen and positively stick grain called after pWHU2404.

The structure of [embodiment 3] sudden change or recombinant bacterial strain

1. SpnC gene disruption

Use primer 5'-CG gAATTCgAGCGGCTGACCTCGTTCTG-3' and 5'-C cAAGCTTg

ACGACCGTGCACAGTCCGT-3'( ecoRi and hindiII digestion site underscore mark) with the genome of CNQ431 for template, amplify spnC gene intermediate segment 3.08 kb, be connected to PMD19-T, order-checking is determined correct, uses bamhI- hindiII digestion, because the intermediate segment of spnC has one bamhI restriction enzyme site, so the fragment cut only has 1.1 kb, is then connected to plasmid pYH7's bgliI- hindiII site.By the plasmid called after pWHU2406 built, then by genetic operating system, Conjugative tiansfer is to CNQ431.The zygote grown, rules and to recombinate with induced gene to MS flat board.From the further streak inoculation of the dull and stereotyped picking colony of MS to the MS flat board of the apramycin containing 50 μ g/ mL, 30 DEG C of cultivations, filter out Δ spnCmutant strain called after mWHU2450, genotype checking (Fig. 9 A) verifies that primer is: spnC-gt (5 '-CACCTACAACATTCCCCTGCCCGTC-3 ') and YH7-gt (5 '-GGCA

AGTTGGTACGCGTCGATTATC-3’) 。

2. enc _c pgene disruption

With primer 5 '-G gAATTCgCACTGGTGTGCGCGGGG-3 ' and 5 '-C cA

aGCTTcCCGTTGTGGAACACCTCCGC-3 ' ( ecoRi and hindiII restriction enzyme site underscore mark) with the genome of CNQ431 for template, amplify enc _c pgene intermediate segment 0.57 kb, is connected to PMD19-T, and order-checking is determined correct, bamhI- hindiII digestion is also connected to plasmid pYH7's bgliI -HindiII site, will obtain plasmid called after pWHU2407, Conjugative tiansfer, to CNQ431, obtains Δ enc _c pmutant strain, called after mWHU2451.Genotype checking (Fig. 9 B), checking primer is: enc _cp-gt 5 '-ACGAACGTGTCATCTACGGGGTC-3 ' and YH7-gt PCR.

3. enc _ch gene is interrupted

With primer 5 '-TCTCCTTAGATCTGACGGTCCGGGCCTGTCGGC-3 ' and 5 '-ATTCCT cAAGCTTcATGGGCCTTCCCCGCACGAG-3 ( bgliI and hindiII restriction enzyme site underscore mark) with the genome of CNQ431 for template, amplify enc _c hgene intermediate segment 0.63 kb, is connected to PMD19-T, and order-checking is determined correct, bgliI- hindiII digestion is also connected to plasmid pYH7 same loci, will obtain plasmid called after pWHU2408.Conjugative tiansfer, to CNQ431, obtains Δ enc _c hmutant strain, called after mWHU2452, genotype checking, as (Fig. 9 C), verifies primer: enc _ch-gt (5 '-CCAGACGTTATGCGAACTG-3 ') and YH7-gt PCR.

4. SPNs biological synthesis gene cluster heterogenous expression recombinant bacterium builds

With primer 5 '- aCTATCCCGAGGAGGGCGAGAGTGACAAGTACGCGCTGA

GGTTCATGTGCAGCTCCATC-3 ' and 5 '- cGAGCACCGCCGTGGAGGCCGAG

tCGAAGACGATGTGATgCTATTTACCC GCAGGACTAT-3 ' amplifies one section containing apramycin resistance gene aac (3)-IV from plasmid pSET152, oriTsite, attPsite, the DNA fragmentation of integrator gene.Before and after this fragment respectively containing one section of 39 bp and orf-1the sequence of homology, thus can by the mode of gene targeting this fragment is incorporated into SPNs biological synthesis gene cluster independent basis because of orf-1place, by the recombinant cosmid called after pWHU2405 obtained, and is integrated into pWHU2405 by genetic operating system s. coelicolorm145 genome, by the mutant strain called after mWHU2453 obtained.

5. AL2110 ermE*- enc _c p- enc _c hwith ermE*- enc _c pallos complemented strain builds

Gene fragment enc _c pwith enc _c hrespectively by primer (5 '-G gAATTCgGAGGGAACTTC

ATGACCTTCGTCATAG-3, and 5 '-CC aTCGATgAAGT CCGTAACCGCATGCT-3 '

ecoRi and clai restriction enzyme site underscore marks) and (5 '-CC aTCGAT gGAGGaGCCGACATGACCACCGACC-3 ', 5 '-G gAT

aTCgCTCATCGTCGTCCGCCAGG-3 ', clai and ecoRv restriction enzyme site underscore marks, and RBS site italic marks) obtain from CNQ431 genome amplification, be then cloned into pMD19-T.After order-checking is correct, use respectively ecoRi- clai and clai- ecoRv enzyme is cut. enc _c pfragment is cloned into plasmid pll6215 (J. Bacteriol. 2008,190,251-263) ecoRi- clai site, will obtain plasmid called after pWHU2409, or will enc _c pand enc _ch fragment is connected to plasmid pll6215 together ecoRi- ecoRv site, will obtain plasmid called after pWHU2410.PWHU2409 plasmid xbai- ecoRv fragment is cloned into pSET152 further and is contained ermE*-enc _c pthe heterologous expression vector pWHU2411 of fragment, pWHU2410's xbai- ecoRv fragment is cloned into pSET152 further and is contained ermE*-enc _c p-enc _c hthe heterologous expression vector pWHU2412 of fragment, pWHU2411 and pWHU2412 Plasmids conjugation is transferred to deacylated tRNA base ANTs producing strains AL2110 and obtains recombinant bacterium called after mWHU2454 and mWHU2455 respectively after the same method.

[embodiment 4] s. sp.cNQ431 and s.the fermentation of NRRL2288, product separation Purification and Characterization

1. liquid fermenting

Wild mushroom or targeted mutagenesis bacterial classification is taken out from-80 DEG C of cryopreservation casees, pick spore liquid with cotton swab is coated with evenly on MS solid medium, cultivate 4-6 days for 30 DEG C, growing fine with rifle head to thalline, to have the solid medium of thalline to cut broken by long, the amount of getting half block is inoculated into 50 mL M3 substratum (5 % corn steep liquors, 1 % glucose, 0.15 % CaCO3,0.6 %. (NH ₄) ₂sO ₄pH=7.0) 28 DEG C, 220 rpm, cultivate and collect fermented liquid in four days.

2. solid fermentation

Get 40 μ L spore liquid and be inoculated into 3ml TSB substratum, after 30 DEG C of cultivation 2 d, draw 400-500 μ L and be spread evenly across containing corresponding antibiotic MS substratum, be put in 30 DEG C of incubators, cultivate 5-7 d.

3. the separation and purification of target product

Fermented liquid or flat board are cut broken after, add 150 ml methylene dichloride, soak 5 h after ultrasonic extraction 15 min, separate dichloromethane layer, is spin-dried for.The dehydrated alcohol adding 1 ml redissolves, and after centrifugal 10 min of 12000 rpm, gets the organic phase millipore filtration that supernatant also crosses 0.22 μm

4. the qualification of target product

The HPLC of tunning analyzes and carries out in Shimazdu LC-20AT workstation, LC-MS and LC-HRMS carries out at LCQ (ThermoFisher Scientific Inc.) and ESI-LTQ Orbitrap (ThermoFisher Scientific Inc.) respectively.HPLC analyzes the separation condition that employing C-18 reversed-phase column (Diamonsil C18 250 x 4.6 mm, 5 μm) flow velocity is 1 mL/min, SPNs and ANTs: T=0 min, 60 % B; T=5 min, 65 % B; T=12 min, 82 % B; T=30 min, 85 % B; T=35 min, 100 % B; T=36 min, 60 % B; T=40 min, 60 % B. (A, H ₂o; B, CH ₃cN).

5. SPN-J half preparation:

SPN-J is prepared in (5 μm, Agilent Zorbax SB-C18 post, 250 × 9.4 mm) carry out with the flow velocity of 3 mL/min. obtain white powder after freeze-drying, and carry out 1H-NMR, 13C-NMR and HR-MS qualification: 1H-NMR (300 MHz, CDCl ₃): δ 12.62 (s, 1H), 8.54 (d, J=8.4 Hz, 1H), 8.49 (s, 1H), 7.90 (s, 1H), 7.35 ~ 7.15 (m, 6H), 7.03 (d, J=7.5 Hz, 1H), 6.91 (t, J=7.8 Hz, 1H), 5.60 (m, 1H), 5.24 (t, J=7.9 Hz, 1H), 4.87 (m, 1H), 3.75 (t, J=9.6 Hz, 1H), 3.21 (m, 1H), 3.00 (m, 1H), 2.71 (m, 1H), 1.70 (brs, 1H), 1.49 (d, J=6.2 Hz, 3H), 1.17 (d, J=6.4 Hz, 3H). 13C-NMR (75 MHz, CDCl3): δ 173.4, 170.5, 169.8, 159.3, 151.1, 138.7, 129.2, 128.9, 127.7, 126.0, 125.2, 120.4, 119.3, 112.9, 77.6, 76.9, 71.1, 54.3, 53.9, 35.5, 18.8, 15.1. HRMS (m/z): C24H27N2O8 [M+H] ⁺theoretical value, 471.1762, measured value, 471.1767.

The clone of [embodiment 5] SpnD and SpnE albumen, expression and purification

1. clone

spnDwith spnEgene uses primer spnD-ex(5-T respectively gAATTCCATATGg

ATGACCCGCAGGCCCTG-3 and 5 '-T aAGCTTgTCACGGGGAGTCC

TTCTGGAGC-3 ') ( ecoRi, ndei and hindiII digestion site underscore mark) spnE-ex (5 '-T gAATTCCATATGaCCAAGGACCTCTACGAACTCG-3 ' and 5 '-T aAGCTTcTACTCGGTGGCGTTCACCAGTAC-3 ') (( ecoRi, ndei and hindiII digestion site underscore mark)) carry out PCR, be connected to pMD19-T after PCR primer purifying, after order-checking is correct, will spnDwith spnE's ndei- hindiII fragment is connected to pET28a expression vector respectively, by plasmid called after pWHU2415 and pWHU2416 respectively obtained.

2. express

SpnD and SpnE expression plasmid will be built be converted into respectively e. colibap1 and BL21 (DE3).Then picking individual colonies 3 mL contains corresponding antibiotic LB substratum, 37 DEG C of incubated overnight, cultured thalline is transferred 2 ml to containing corresponding microbiotic containing corresponding antibiotic 500 ml LB() in the shaking flask of 2 L, 37 DEG C of cultivations, when reaching 0.4 to OD600 value, place after 10 min make it cool on ice, add the IPTG 50 μ L of 1M, to final concentration 0.1 mM, 16 DEG C of abduction delivering 20 about h.

3. purifying

By the thalline of having induced, with 500 ml Centrifuge Cups, 6000 rpm, 10 min remove supernatant immediately, collect thalline in clean centrifuge tube, 30 ml lysis buffer(25 mM HEPES pH 7.5,300 mM NaCl, 5 mM imidazole, 10 % glycerol)) resuspended.Ultrasonication 10 min(surpasses 5 s and stops 10 s), and (15000 rpm, 1 h), in transfer supernatant to clean centrifuge tube for high speed centrifugation.Get 2 ml nickel beads and join protein supernatant liquor, 4 DEG C of jogs are in conjunction with 2 h.Rely on gravity imidazole concentration wash-out from small to large, collect target protein, Amicon Ultra-4 evaporating pipe, protein concentrate.Then the good albumen (Figure 10 A B) of purifying is detected with acrylamide SDS-PAGE.The method of Braford, with BSA standard curve determination protein concentration.Finally use liquid nitrogen flash freezer, and be stored in-80 DEG C for subsequent use.

[embodiment 6] Enc _cthe expression and purification of P albumen

We have gone out soluble E nc by aforesaid method successful expression in pET28a _cp albumen, can't detect activity, although we have attempted MBP amalgamation and expression respectively or Nus-tag expresses, be all the same result, finally we have gone out activated Enc in Pseudomonas putida successful expression _cp.In order to express recombinant protein in Pseudomonas putida KT 2440 system of tetracyclin resistance, the present inventor is by tetracycline resistance gene fragment in plasmid pCIBhis (Chem. Biol. 2005,12,445-452) tetR-tetAkalamycin resistance gene is replaced as by the method for gene targeting.Target practice primer is 5 '- gTTCCTGACAACGAGCCTCCTTTTCGCCAATCCATCGACg

CAGTGGGCTTACATGGCG-3 ' and 5 '- cCCGCGACGCAGCGCCGG

cAGGCAGAGCAAGTAGAGGGCin CTCGTGATGGCAGGTTGGG-3 ' primer, homologous fragment underscore marks.Here the expression plasmid called after pCIBKm will obtained.With primer 5 '-T gAATTCcATATGACCTTCGTCATAGAGCTCGACA-3 ' and 5 '-T aAGCTTgTGCTCCATTTTCGGACGAAGTC-3 ' ( ecoRi and hindiII digestion site underscore mark) obtain from CNQ431 genomic PCR amplification en _c cPfragment.Then primer 5 '-TTT is used aAGCTTTTAATTAAgGTTCATGTGCAGCTCCATCAGCAAAAG-3 ' and 5 '-TTT cATATGTTAATTAAtCAGCCAATCGACTGGCGAGCGGCAT-3 ' ( hindiII, paci and ndei restriction enzyme site underscore marks) obtain A Baila resistant gene from pSET152 plasmid clone aac (3)-IV fragment, and by enzyme cut connection method and enc _c pgene fragment connects, and obtains enc _c p-aac (3)-IV. enc _c p-aac (3)-IV fragment use further primer 5 '- gGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGaTGACCTTCGTCATAGAGCTCG-3 ' and 5 '- tTCGAATTCCATGGTACCAG

cTGCAGATCTCGAGCTCGG tTAATTAA(homologous fragment underscore marks TCAGCCAATCGACTGGC-3 ' , Paci restriction enzyme site italic marks) amplification, by the method for gene targeting, be connected to pCIBKm expression vector and obtain pWHU2417.The carrier electricity obtained is forwarded to p. putida, by the pseudomonas putida with expression plasmid, 37 DEG C of cultivations in the LB containing tsiklomitsin and A Baila, after OD600 reaches 0.8-1.0, the IPTG adding 1 mM induces target protein to express.The purifying concentration determination of albumen is the same with aforesaid method with preservation subsequently.12 % acrylamide SDS-PAGE electrophoresis detection of this albumen are as (Figure 10 C).

The chemosynthesis of [embodiment 7] SpnE substrate styracin coenzyme A

The present inventor is according to the synthetic route synthesizing cinnamic acid coenzyme A of such as (Figure 11)

1. S-phenmethyl styracin (9) (S-methylphenyl cinnamate)

DMF (0.46 ml, 5.9 mM) is distilling room temperature reaction 1 h in pure methylene dichloride (9 ml) with oxalyl chloride (1.5 ml, 17.6 mM).After the residual oxalyl chloride of vacuum removing, the light powder of reaction soln simmer down to, resuspended with 1 ml tetrahydrofuran (THF), in advance by styracin (8) (740.8 mg, 5 mM) be dissolved in 9 ml tetrahydrofuran (THF)s, slowly instill in above-mentioned solution with 4.9 ml pyridines, after reaction system being placed in-30 ° of C stirring reaction 2 h, add subsequently and be dissolved in methylthiophenol (621.05 mg, 5 mM) in 10ml methylene dichloride and 7.5 ml triethylamines.The solution obtained at room temperature is continued stirring reaction 2hr.With the solution that dchloromethane obtains, then use the HCl of 2 %, saturated NaHCO ₃, saturated NaCl solution washing.Then anhydrous Na SO is used ₄dry organic layer, after concentrated, finally uses sherwood oil: object product gets off from silicagel column elution by the eluent of ethyl acetate=8:1.Obtain a kind of white powder (9) (1.08 g, yield 85 %).1H-NMR (400 MHz, CDCl3): δ 7.67 (d, J= 16.0 Hz, 1H), 7.57~7.55 (m, 2H), 7.41~7.36 (m, 5H), 7.25 (d, J=2.8 Hz, 1H), 6.78 (d, J= 16.0 Hz, 1H), 2.39 (s, 3H). 13C-NMR (100 MHz, CDCl3): δ 188.5, 141.3, 139.8, 134.6, 134.1, 130.7, 130.1, 129.0, 128.5, 124.2, 124.1, 21.4.

2. styracin coenzyme A (4) cinnamonyl-CoA

By 2 ml PBS damping fluids (phosphate buffered saline buffer, 1 M, PH=8.5) in a nitrogen environment in advance ultrasonic 20 min with remove trace air, add coenzyme A, then add the compound 9(20 mg be dissolved in 2 ml tetrahydrofuran (THF)s, 0.02 mM).Under room temperature, with nitrogen protection, after vigorous stirring reaction 40hr, the removing THF aqueous solution, remove unreacted thioesters with 2 times of volume washed with diethylether.Cinnyl coenzyme A (4) is by semi-preparative high performance liquid chromatography purifying, and lyophilize obtains white powder (16.3 mg, 71% yield).1H NMR (400 MHz, D2O): δ 8.33 (s, 1H), 7.93 (s, 1H), 7.45~7.15 (m, 6H), 6.56 (m, 1H), 5.92 (m, 1H), 4.75 (m, 2H), 4.44 (s, 1H), 4.12 (s, 2H), 3.91 (s, 1H), 3.72 (m, 1H), 3.44 (m, 1H), 3.34 (m, 2H), 3.27 (m, 2H), 2.98 (m, 2H), 2.33 (m, 2H), 0.789 (s, 3H), 0.65 (s, 3H). HRMS (m/z): [M+H] ⁺calcd. For C ₃₀H ₄₃N ₇O ₁₇P ₃S, 898.1643; observed, 898.1641.

[embodiment 8] Enc _cp, SpnE and SpnD vitro enzyme is lived and is tested

1. Enc _cthe enzyme activity assay of P

Enc _cthe 100 μ L reaction systems of P are as follows: 100 mM Tris buffer(50 mM Tris, 50 mM NaCl, pH=8.0), 1 mM TCEP, 1-5 μM of enzyme, 200 mM amino acid substrate, comprise phenylalanine, tyrosine, tryptophane, Histidine.30 DEG C of water-baths, add 10 μ L 100 % trichoroacetic acid(TCA) (TCA), HPLC or LC-HRMS analyzes and carry out in Inertsil ODS-3 chromatographic column (5 μm, 4.6 × 250 mm, GL Science Inc).HPLC analyze with the flow velocity of 1 ml/min under 260 nm wavelength: T=0 min, 5 % B; T=10 min, 20 % B; T=15 min, 50 % B; T=20 min, 70 % B; T=25 min, 75 % B; T=26 min, 5 % B; T=30 min, 5 % B (A=H ₂o, B=CH ₃cN).HPLC-ESI-MS analysis condition is the same with HPLC condition.

2. SpnE enzyme activity assay

The test alive of SpnE enzyme has been carried out: 100 mM HEPES buffer pH=7.0 by following system; 0.5 mM NADPH; 60 mM NaHCO3; 10 μMs of SpnE; 0.5 mM substrate; comprise styracin coenzyme A (4), crotonyl-CoA (5) and 5-methyl-2-hexenoyl coenzyme A (6); 30 DEG C of reaction 2 h; add 1 % TCA(trichoroacetic acid(TCA)) termination reaction; after 12000 rpm are centrifugal; HPLC and LC-HRMS detects, and result display SpnE can three kinds of substrates (Fig. 6 A, B, C) all can catalyzed reaction above.Kinetic parameter test is carried out to styracin coenzyme A (4) and crotonyl-CoA (5), the concentration of NADPH is remained on 1mM, and the concentration of substrate 4 and 5 changes at 0.2-2 mM, react to add 10 μMs of SpnE and start.Under 360 nm wavelength, the reduction of NADPH is as speed calculating parameter, wherein using NADPH standard equation as the quantitative criterion of NADPH.Each data point at least repeats 3 times.With originlab originpro 9 software matching Michaelis-Menten equation, obtain the k of styracin coenzyme A (4) and crotonyl-CoA (5) _cat/ K _mvalue (Fig. 6 D).

3. SpnD vitro enzyme is lived and is tested

Add 10 μ L holo-SpnD albumen, in above-mentioned 100 μ L SpnE reaction systems, 30 DEG C are continued reaction 2 h, add isopyknic chloroform termination reaction.Afterwards albumen is made lyophilized powder, be sent to national protein science center, do tryptic digestion and high resolution mass spectrum.

Claims (13)

1. in polyketone skeleton, introduce a biosynthetic means for origin of amino acid side chain, it is characterized in that: comprise the steps:

(1), amino acid is formed α, β-undersaturated carboxylic acid through amino acid-cleaving enzymes catalysis;

(2), CoA ligase catalysis α, β-undersaturated carboxylic acid, formed α, β-undersaturated carboxylic acid coenzyme A;

(3), by crotonyl-CoA reduction carboxylases catalyse α, β-undersaturated carboxylic acid coenzyme A forms the malonyl-list acyl coenzyme A replaced;

(4) the AT model choice, in polyketide synthases the malonyl-list acyl coenzyme A uploading replacement enters polyketone building-up process, realizes the side chain of origin of amino acid to be introduced in the carbon skeleton of polyketide.

2. the biosynthetic means introducing origin of amino acid side chain in polyketone skeleton according to claim 1, is characterized in that: described amino acid is phenylalanine, and described side chain is fragrant benzyl, comprises the steps:

(1) phenylalanine lyase Enc _cthere is reduction deamination and generate styracin in P catalysis phenylalanine; (2) styracin is at styracin CoA ligase Enc _cstyracin coenzyme A is generated under the catalysis of H; (3) crotonyl-CoA reduction carboxylase SpnE catalytic reaction former carboxylation reaction of surviving generates benzyl malonyl-list acyl coenzyme A;

(4) the AT structural domain identification benzyl malonyl-list acyl coenzyme A in polyketide synthases SpnD is also uploaded to polyketone skeleton.

3. the biosynthetic means introducing origin of amino acid side chain in polyketone skeleton according to claim 2, is characterized in that: described phenylalanine lyase Enc _cp, styracin CoA ligase Enc _ch, crotonyl-CoA reduction carboxylase SpnE, polyketide synthases SpnD derive from the splenocin(SPNs in marine streptomyces Streptomyces sp. CNQ431) and enterocin (ENC) biological synthesis gene cluster, be gene respectively enc _c p, enc _c h, spnE, spnDcoded protein.

4. the splenocin(SPNs derived from marine streptomyces Streptomyces sp. CNQ431 according to claim 3) biological synthesis gene cluster, it is characterized in that: its nucleotide sequence is as SEQ ID NO:1, this sequence totally 21 genes or complementary sequence, contain 6 independent basises because of: orf-1, orf-2, orf-3, orf-4, orf-5, orf-6; 1 regulatory gene: spnA; 9 responsible start element synthetic genes: spnF, spnG, spnH, spnI, spnJ, spnK, spnL, spnN, spnO; The gene of 4 responsible skeleton synthesis spnB, spnC, spnD, spnM; A crotonyl-CoA reduction carboxylase gene: spnE.

5. splenocin(SPNs according to claim 4) biological synthesis gene cluster, it is characterized in that: described spnEgene, nucleotide sequence is positioned at the 21443-22663 base place of SEQ ID NO:1, and length is 1221bp, coding crotonyl-CoA reduction carboxylase.

6. splenocin(SPNs according to claim 5) biological synthesis gene cluster, it is characterized in that: gene spnEcoded aminoacid sequence, be crotonyl-CoA reduction carboxylase, have 406 amino acid, aminoacid sequence is as shown in SEQ ID NO:3.

7. splenocin(SPNs according to claim 4) biological synthesis gene cluster, it is characterized in that: described spnDgene, nucleotide sequence is positioned at the 17566-21441 base place of SEQ ID NO:1, and length is 3822bp, coding polyketide synthases, and acyltransferase AT structural domain wherein can specific recognition and loading benzyl malonyl-list acyl coenzyme A.

8. splenocin(SPNs according to claim 7) biological synthesis gene cluster, it is characterized in that: gene spnDcoded aminoacid sequence is polyketide synthases, and have 1273 amino acid, aminoacid sequence is as shown in SEQ ID NO:4.

9. enterocin (ENC) biological synthesis gene cluster derived from marine streptomyces Streptomyces sp. CNQ431 according to claim 3, it is characterized in that: its nucleotide sequence is as SEQ ID NO:2, this gene cluster contains 20 genes or complementary sequence altogether, called after: enc _c a, enc _c b, enc _c c, enc _c d, enc _c e, enc _c f, enc _c g, enc _c h, enc _c i, enc _c j, enc _c k, enc _c l, enc _c m, enc _c n, enc _c o, enc _c p, enc _c q, enc _c r, enc _c s, enc _c t.

10. enterocin (ENC) biological synthesis gene cluster derived from marine streptomyces Streptomyces sp. CNQ431 according to claim 9, is characterized in that: described enc _c pthe nucleotide sequence of gene, is positioned at the 15727-17298 place of SEQ ID NO:2, and length is 1572bp, encoding phenylalanine lyase.

11. enterocin (ENC) biological synthesis gene clusters derived from marine streptomyces Streptomyces sp. CNQ431 according to claim 10, is characterized in that: described enc _c pthe aminoacid sequence of genes encoding is phenylalanine lyase, has 523 amino acid, and aminoacid sequence is as shown in SEQ ID NO:5.

12. enterocin (ENC) biological synthesis gene clusters derived from marine streptomyces Streptomyces sp. CNQ431 according to claim 9, is characterized in that: described enc _c hgene nucleotide series, is positioned at the 2798-4405 place of SEQ ID NO:2, and length is 1608bp, encoding cinnamate CoA ligase.

13. enterocin (ENC) biological synthesis gene clusters derived from marine streptomyces Streptomyces sp. CNQ431 according to claim 12, is characterized in that: described gene enc _c hthe aminoacid sequence of coding, be styracin CoA ligase, have 535 amino acid, aminoacid sequence is as shown in SEQ ID NO:6.