CN1189191A - Method for generating and screening novel metabolic pathways - Google Patents

Abstract

The present invention relates to a novel drug discovery system for generating and screening molecular diversity. The system provides methods for mixing and cloning genetic materials from a plurality of species of organisms in combinatorial gene expression libraries to generate novel metabolic pathways and classes of compounds. The system also involves methods for pre-screening or identifying for host organisms containing a library that are capable of generating such novel pathways and compounds. The host organisms may be useful in drug screening for particular diseases, and in commercial production of compounds of interest. The methods of the invention are also useful in preserving the genomes of organisms that are known or prospective sources of drugs.

Description

Produce and screen the method for novel metabolic pathways

1. invention field

The present invention relates to a kind of method of new discovery medicine, specifically, the present invention relates to a kind of system of biological gene group of the good or source likely that saves as medicine; A kind of any mixing from one or more biological genetic material to produce the system of new pathways metabolism; And a kind of prescreen or screen the system of these genetically engineered cells with the compound that produces new biochemical route and the new kind of preparation.This pathways metabolism new or that reconstitute can be used for these compounds of commercial production.

2. background of invention

2.1. medicine forerunner's source

Basic choosing wantonly in the drug discovery is to determine to have required active lead compound, and preferred lead compound is carried out the required requirement of further drug development to satisfy.The usual method of a drug discovery comprises and is created in the bioanalysis and produces the relevant macromole of disease (disease target), the therapeutic activity of wherein testing possible drug candidate, and these molecules can be acceptor, enzyme or transcription factor.

Another kind method comprises the whole cell or the biology of the representative of the pathogenic agent that is provided as disease.These pathogenic agent comprise bacterium and tumor cell line.

Usually, there are two kinds of possible drug candidate sources, a collection of natural product and synthetic compound.Comprise this a collection of discriminating that has realized lead compound of the structure type of wide region as far as possible by any screening.The nearest progress in synthetic combination of compounds storehouse will further increase the number and the kind of the compound that can be used for screening, yet the synthesis method of any synthetic compound library is subjected to the restriction of people's the imagination and synthetic technology.

Screen arbitrarily from for example land endophytic bacteria, fungi, the natural product of invertebrates and plant origin has caused having found many important medicines (Franco etc., 1991, Critical RevBiotechnol 11:193-276; Goodfellow etc., 1989, at " microbial product: novel method ", Cambridge University Press, pp.343-383; Berdy 1974, senior applied microbiology 18:309-406; Suffness etc., 1988, in the importance of halobiontic bio-pharmaceutical, D.G.Fautin, California Academy of Sciences, p.151-157).More than these natural product biologically actives of 10,000 kinds, at least 100 kinds in these are used as microbiotic, agrochemical and antineoplastic agent at present.What of the compound that enters in the screening procedure success of the method for this discovery medicine greatly depend on.Usually, pharmaceutical companies screening contains the compound group of tens thousand of kinds of natural and synthetic compounds.Yet new compound reduces along with the time with the ratio of the compound of finding in the past.In the screening anticancer agent, for example, the microbe species of most of biologically actives can produce certified compound.A part is owing to consistently and sufficiently find, the difficulty that produces again and new natural product sample is provided.Because species diversity largely is because the potential molecular diversity does not have enough species diversities in the biology that is selected for any screening at present, this has just reduced the possibility of separating new compound.

New biological activity is found in various natural origins consistently.Referring to for example, Cragg etc., 1994. (at " Enthnobotany and new drug are looked for " Wiley, Chichester.p178-196).Minority source is by system with test all sidedly and be used for the novel drugs forerunner.For example, estimate that only 5000 kind of plant kinds are used for possible medicinal use by research at large.This is 250 of estimation, 000-3, and the sub-fraction that 000,000 kind is whole, their great majority are grown in the torrid zone (Abelson 1990, science 247:513).And in the millions of kinds of marine microorganisms of estimating, only minority is characterized.Really, has the huge species diversity of being excavated not yet to the lead compound source.

The Lu Sheng microorganism, fungi, invertebrates and plant always are used as the source of natural product.Yet, except some by outside the biological group of careful research, actinomyces for example, it has been developed and has been used for outside drug screening and the commercial production, but duplication of production and production problem still exist.For example, antineoplastic agent, taxol be the part of the bark of ripe Pacific Ocean yew tree, and it has caused paying close attention to about the destructive to the local ecosystem as clinical preparation.Taxol contains 11 chiral centres, has 2048 possibility diastereomeric form, and its de novo synthesis on commercial size is impossible (Phillipson, 1994, imperial tropical medicine and hygiology transactions 88 Supp 1:17-19) like this.

Marine invertebrate is the source of a new compound likely, but the deficiencies of great majority in the technology that is used for carrying out drug screening and provides again on a large scale are provided.For example, marine invertebrate is difficult to collect again, and aspect the content of natural product, many mutabilities with season.

Marine microorganism is a kind of source of new compound likely, but also exists great majority in the deficiency that is used for carrying out drug screening and carries out the technology of industrial fermentation with marine microorganism.For example, marine microorganism is difficult to collect, and sets up and keeps in culture, and manyly have a special nutritional needs.The unpolluted seawater of reliable sources is essential for fermentation usually.The marine bacteria kind of estimation at least 99% can not be survived in the laboratory culture base.And the commercial fermentation unit of purchasing is not suitable for being used for saltwater environment, or under the high pressure.

And some compounds just occur at occurring in nature during only when specific biological interaction with environmental activity.Pathogenic agent may change gene expression in plants and bring out the synthetic of compound, and for example phytoalexin impels the plant anti-erosion.For example, Nicotiana gossei plant (Nicotianasylvestris) increases then that it is alkaloidal synthetic when the erosion that is subjected to from maduca sexta (Manduca sexta) larva.Same fungi can or prevent that their accumulation from producing reaction to phytoalexin by detoxification.By traditional high throughput (throughput) screening, these metabolites are lost, and this method is estimated fungi and its plant host not together.Physical environment passes through the poisonous guided missile frog as can be seen to a tangible example of the influence of biology.Though top that can be by the guided missile of will drying scrapes the sodium channel antagonist alkaloid of collecting lethal dose along the back of the gland of field kind, batrachotoxin, but in the frog that raise on s-generation land, can not detect batrachotoxin (Daly, 1995, the periodical 92:9-13 of institute of NAS).As long as the drug screening technology that utilization is traditional, the possible valuable molecule that resembles these may be found never.

And the lead compound by random screening seldom becomes medicine, because its effectiveness, selectivity, bioavailability or stability may be not enough.Usually, need a certain amount of lead compound, it can be by structural modification to improve its initial activity like this.Yet the method relative efficiency from natural origin especially phytosynthesis and exploitation lead compound is low at present.Have the tangible obstacle relevant with each stage of drug development, for example collect again, produce the growth of the biology of medicine, remove to duplicate (dereplication), bacterial strain improves, and substratum improves and enlarges and produces.These problems have postponed the clinical trial of new compound, and the medicine forerunner's in these new sources economy is used in influence.

At present, to the above-mentioned ocean of natural product, the plant and animal source is under-utilized.For the source of these underexploitations, the method for existing preparation and screening lead compound can not be used effectively at present.Do not resemble some land endophytic bacteria and fungies, these biologies that produce medicine are not easy to be adapted to industrial fermentation technology.Simultaneously, find that the pressure in the new source of medicine is by the triage techniques aggravation of new efficient and high throughput.Therefore, generally need to utilize as the genetic resources in still untapped compound source and the multifarious method of compound to be used to find the purpose of medicine.2.2. expression library

The discovery screening based on mechanism has been transferred in drug discovery plan recently.In case determine a molecule target (for example participating in regulating the hormone receptor of disease), then analysis be designed to discern and/or synthesize at molecular level and the interactional therapeutical agent of target.

The genetic expression storehouse is used to identification, test and preparation target molecule.When not knowing the protein physical properties, cloning by expression has become the ordinary method that obtains the single proteinic target gene of coding.

By screening-gene expression library identification, be possible disease target from the numerous protein of people and mammalian tissues preparation, for example, acceptor (Simonsen etc., 1994, the dynamic 15:437-441 of pharmaceutical science; Nakayama etc., 1992, current biotechnology comment 3:497-505; Aruffo, 1991, current biotechnology comment 2:735-74l), signal transducer (Margolis etc., US5,434,064).Referring to Seed etc., 1987, the periodical 84:3365-3369 of institute of NAS; Yamasaki etc., 1988, science 241:825-828; With Lin etc., 1992, cell 68:775-785, (type-iii TGF-beta receptor) is the proteinic example by the clone of the functional expression in mammalian cell identification.

In case the target disease is determined, the host cell by genetic design of target protein or expression target protein is used to bioanalysis with screening lead compound (Luyten etc., 1993, the dynamic 11:247-54 of biotechnology).Therefore, in the scheme of drug discovery, the use of gene expression library greatly is confined to the identification and the generation of possible protein disease target.Be protein or little peptide at medicine only, for example, under those situations of antibody, expression library just is produced with generation and screening has required bioactive molecule (Huse etc., 1991, Ciba Foundation Symp159:91-102).

Yet the gene expression library relevant with drug discovery has other purposes.For the purpose of the gene of discerning the biosynthetic pathway that participate in to produce metabolite with pharmaceutical activity and special chemical substance, prepared the gene library of microorganism.These approach need multiple proteins (specifically, enzyme), and are inevitable with than the bigger complicacy of single protein as the medicine target.For example, by screening gene (Malpartida etc., 1984, nature, the 309:462 that this biological gene library has been determined coding bacterium polyketide synthase (PKSs) approach; Donadio etc., 1991, science 252:675-679).PKSs catalysis is biosynthetic a plurality of steps of the polyketide class of important class treatment compound, and the structure diversity of the polyketide of control generation.Permission clone's the PKS gene orthomutation and the host carrier system in streptomyces (McDaniel etc., 1993, the science 262:1546-1550 of expression have been developed; Kao etc., 1994, science 265:509-512).This specific host carrier system has been used to develop the approach of more efficiently generation polyketide class, and be used to reasonably develop new polyketide class (Khosla etc., WO95/08548).

Another example is to prepare dye for fabrics by fermentation in escherichia coli host, and is indigo.Contain coding multienzyme biosynthetic pathway gene two operons by genetic manipulation to improve indigo generation (Ensley etc., 1983, the science 222:167-169 of external source escherichia coli host; Murdock etc., 1993, biology/technology 11:381-386).In a word, the conventional study of the heterogenous expression of the gene of encoding metabolic pathway comprises directed cloning, sequential analysis, the rearrangement of the proteinic specific gene of the pathways metabolism that the design sudden change and the known participation of encoding characterized in the past.

In view of the huge advance made of the understanding that pathogenic mechanism and medicine target are determined, need to improve rapid identification lead compound day by day and their are introduced the strategy and the method for clinical trial.

3. summary of the invention

The invention provides a kind of drug discovery system that produces and screen the molecular diversity that is used for the drug discovery purpose.Method of the present invention will be known as/or be hopeful to catch and be kept in the blended gene expression library for the genetic material of the biology in medicine forerunner source.

In one embodiment, the present invention includes from the biological gene expression library that mixes natural approach that makes up of one or more donors, wherein the donor biology comprises microorganism, plant and animal, especially can not reclaim in a large number at nature, or can not in the laboratory, cultivate those.According to they known biological natures, can select the donor biology in the storehouse, or they can be the mixture of the biology of unknown kind known and/or that collect from nature.Clone and express any fragment of donor biological gene group in host living beings, some in them contain all and the part biochemical route.

According to the present invention, the subgroup of the gene product of the DNA of transfer (subset) can work in host living beings.In host living beings, the biological abiogenous approach of donor can be reconstituted thus.Donor gene can expose other reticent pathways metabolism at xenogenesis host's not similar physiology and the expression of regulating in the environment.The pathways metabolism of donor biology also can interact to produce the compound that new compound or host living beings generally do not produce with the pathways metabolism that is present in the host living beings.

And, owing to the time in office, in host living beings, only express the gene of the donor biology of the subgroup of determining, this system can make and be easier to detect pathways metabolism and compound under the biochemistry that the has characterized/cell background of host living beings.Be in essence, the genetic resources of these donor biologies is captured and is kept in the gene expression library, and this gene expression library can be replicated in different drug discovery steps and use repeatedly.

In another embodiment, the present invention includes the structure of the chimeric approach expression library of bonded, wherein arbitrarily combined in host living beings from the genetic material of one or more donor biologies, clone and expression.These libraries are from a plurality of approach and the biological arbitrary combination that produces gene, before having produced like this at non-existent pathways metabolism of nature and genome independently.Term " independently genome " refers to from any combination of the two or more genes that obtain from the connection of one or more approach the combination gene expression library or biological gene.The several genes product can work in host living beings, and wherein they interact to form the new chimeric pathways metabolism of the compound that produces new classification.Therefore, increased the diversity of the molecular structure that can be used for drug screening by existing approach in the mixed assemblage mosaic gene expression library and biological genetic material.

Though can use the standard method of screening-gene expression library, this library can further be modified satisfies the needs that the clone of required approach and meta-bolites is being expressed in identification to mix a report system.In a specific embodiment, host living beings is comprised coding manipulative capability ground and chemical gene of replying the relevant reporter protein matter of promotor by genetic modification, and this chemistry is replied promotor and replied with the metabolite generation of the required kind that will detect in expression library.

In another embodiment, host living beings can be exposed in the physiology probe for the reporter molecule precursor, and this precursor directly or indirectly is converted into reporter molecule by the compound that produces in the approach that is found.The conversion of activation that reporter molecule characterizes or report precursor has produced and has allowed identification and the signal that separates required clone.

In another embodiment of the invention, host living beings in the library can or contain sample or this other indicator cells type as the target of required compound with the report system, for example anti-infection agent at pathogenic agent or antineoplastic agent at tumour cell be embedded in the semisolid matrix together.Can use the screening method of high throughput, for example, big drip sorting, fluorescence-activated cell sorting or the sorting of magnetic activating cells are to discern in the combination gene expression library and to separate required biology.

Can further analyze the new compound that positive colony produces.By characterize importing genetic material in the separating clone genetics and the biological chemistry of the pathways metabolism that causes producing new compound can be described.

The invention still further relates to the dna vector that is used to make up the combination gene expression library, specific combination gene expression library, the host living beings that contains the report system of particular type, modified so that produce the host living beings of other toxic chemical, with comprise host living beings, indicator cells and/or the report system composition.3.1. definition

Adopt as this paper, below term have specified implication.

" make up natural approach expression library " and be the library of expressing from from the construction of the genetic material preparation of multiple donor biology, the gene operability ground that wherein is present in the genetic material causes that the regulatory region of genetic expression interrelates in suitable host living beings.The combination expression library uses the host living beings of the functioning gene product that can produce the donor biology.Genetic material coding in each host living beings is from the abiogenous biochemical route of a donor biology or its part.

" make up chimeric approach expression library " and be the library of expressing from from the construction of the genetic material preparation of any concatermerization of one or more donor biologies, wherein be present in the genetic material gene operability ground with in suitable host living beings, cause that the regulatory region of genetic expression interrelates.The host living beings that uses can produce the functioning gene product of donor biology.

" the combination gene expression library of bias " is the library of expressing from from the construction of the genetic material preparation of one or more donor biologies, and it has been carried out specific character selects in advance.The genetic material of Xuan Zeing can be used to preparation natural approach of combination or chimeric library in advance.

Adopt as this paper, term " library " refers to expression constructs or contains the host living beings of expression constructs.

Term " biochemical route ", " natural approach " and " pathways metabolism " comprise the relevant biochemical reaction that is undertaken by biology of all series.This approach can include, but are not limited to biosynthesizing or biodegradation pathway, or energy produces or path for transformation.

" compound " is any molecule for the result of biochemical route or by product, and the interactional product of several genes product normally.

" activity " is that host living beings carries out biochemical reaction or a series of ability that causes producing the biochemical reaction of compound of interest.

As adopting among the present invention, abbreviation expression below: eq (equivalent); M (volumetric molar concentration); MM (millimolar concentration); μ M (micro-molar concentration); N (normally); Mol (mole); Mmol (mmole); μ mol (micromole): nmol (nmole): kg (kilogram); Gm (gram); Mg (milligram); μ g (microgram); Ng (nanogram); L (liter); ML (milliliter); μ l (microlitre); Vol (volume); S (second); With ℃ (centigradetemperature).

In addition, adopt following abbreviation: Cfu: clonogenic unit; The LB:Luria fermented liquid; DdH ₂O: two distillations, reverse osmosis purified water; Seawater: filtering Pacific Ocean seawater; SSW: synthetic sea water; FACS: fluorescence-activated cell sorting; GFP: Victoria's multitube jellyfish (Aequorea victoria) green fluorescent protein; Kbp: kilobase is right; G: gravity; Rpm: per minute rotation; CIAP: calf intestine alkaline phosphatase; EDTA: oxalic acid triethylammonium tetrakis; TE:10mM Tris/1.5mM EDTA pH 7.4; PEG: polyoxyethylene glycol; E.coli: intestinal bacteria; CHO: Chinese hamster ovary; S.cerevisiae: Saccharomyces cerevisiae; A.nidulans: Aspergillus nidulans; S.pombe: schizosaccharomyces pombe; S.lividans: paleness streptomycete; S.aureus: streptococcus aureus; S.coelicolor: streptomyces coelicolor; B.subtilis: subtilis; BAC: bacterial artificial chromosome; YAC: yeast artificial chromosome; PCR: polymerase chain reaction; CaMV: cauliflower mosaic virus; AcNPV: autographa california nuclear polyhedrosis virus; EBV:Epstein-Barr virus; SDS: sodium laurylsulfonate; CsCl: cesium chloride.

4. description of drawings

Fig. 1: the expression constructs that makes up natural approach expression library.This expression constructs contains carrier DNA and comprises encoding metabolic pathway and the donor dna fragment of the natural regulatory region that interrelates.

Fig. 2: the expression constructs that makes up chimeric approach expression library.This expression constructs contains carrier DNA and respectively comprises the box gene of five concatermerizations of donor dna and regulatory region.

Fig. 3: the cloning process that makes up natural approach expression library.The cloned DNA (B) of will be from donor biology (A) but extracting partly digests with product the encode biochemical route of natural generation or the fragment of its a part of genomic dna (C) with Restriction Enzyme.To be connected on the fragment of genomic dna to form expression constructs (F) by the dna vector (D) that has a carrier of compatible end (E) with generation with Restriction Enzyme digestion.

Fig. 4 A-4C: the assembling of box gene.Fig. 4 A has described and has contained toughness BamHI site and be the annealed of flush end with respect to the part in SamI site, the lac promoter fragment of phosphorylation.Fig. 4 B has described to contain and has been that flank is positioned at the promotor dimer in the BamHI site of lac promotor side.Fig. 4 C has described the promoter fragment of concatermerization

Fig. 5 A-5F: the cloning process that makes up chimeric approach expression library.Fig. 5 A shows that preparation is used for promotor and the segmental step of terminator that cDNA and genomic dna insert segmental directed cloning.Fig. 5 B shows that preparation is used for inserting promotor and the segmental step of terminator that fragment links to each other with genomic dna.Fig. 5 C shows that preparation is used for directed cloning, the assembling of box gene, and insert segmental step with the cDNA that is connected of solid support.Fig. 5 D shows that preparation is used for the clone, the assembling of box gene and insert segmental step with the genomic dna that is connected of solid support.Fig. 5 E and 5F show that the series of box gene is connected and goes protection to form concatermer, and concatermer is connected with fission yeast/shuttle vehicle (pDblet's); Expression constructs from the solid carrier release and the cyclisation of expression constructs.

Fig. 6 A-6B: the carrier that is used to prepare the combination gene expression library.Fig. 6 A shows the Streptocos collection of illustrative plates.Cosmid vector Streptocos contains unique the BamHI site that is positioned at T3 and T7 promotor side in a plurality of cloning sites, replication orgin and from the thiostrepton resistant gene of pIJ 699, ColE1 starting point (ori), penbritin gene (Amp) and two cos sites.Fig. 6 B shows the pDblet collection of illustrative plates of modification.Plasmid pDblet is modified at a plurality of cloning sites (MCS), and contains the ColE1 starting point of duplicating, penbritin gene (ApR), the autonomously replicating sequence of two copies (ARS), a ura ⁴Mark, and beta-galactosidase gene (LacZ).A：AatII；B：BamHI；N：NdeI。Fig. 6 C shows and contains the BstXI sequence that changes and the oligomer in NcoI site that it is by the pDblet of the formation modification that excessively is connected with the pDblet of SacI/NotI cutting.

Fig. 7 shows that chemistry replys construction pERD-20-GFP, and it comprises the acceptor gene of an encoding green fluorescent protein (GFP), and chemistry is replied promotor (Pm) regulon (Xyls) relevant with it.

Fig. 8 shows that comprise can penetrating matrix and contain the big thick stream of the indicator cells of receptor area, and the clone from the combination gene expression library in matrix is wrapped.

Fig. 9 A and 9B provide existing and not existing under the situation that comprises marine bacteria expression of gene construction, the Bacillus coli cells storehouse are carried out the example of FACS sorting.Cosmid library with the marine bacteria gene infects the coli strain XLl MR that chemistry is replied construction pERD-20-GFP that contains that is called XLl-GFP.Under 30 ℃, will have or not have the XLl-GFP cell cultures 12 hours of marine bacteria gene, and carry out the FACS sorting of two-wheeled.Fig. 9 A: XLl-GFP with marine bacteria gene; Fig. 9 B: contrast XLl-GFP cell.

Figure 10 shows the arrangement of aminoacid sequence of the actinorhodine dehydratase of streptomyces coelicolor, and from the partial amino-acid series of the prediction of CXC-AMN 20.Barren illustrates that partly sequence is identical, hypographous part explanation conserved sequence homology.

Figure 11: the PCR of clone CXC-AMN 20 sequences in the genomic dna storehouse of marine bacteria detects.This figure shows the painted sepharose that contains from pcr amplification of marine bacteria genomic dna.M: molecular weight marker, size is bp.-: negative control.+: the positive control of amplicon and ribosome-RNA(rRNA).Swimming lane contains the amplicon from T: from the gene DNA of all 37 kinds of marine bacterias; 1,2,3,4: the genomic dna storehouse of marine bacteria.

Figure 12 A-C: the PCR of clone CXC-AMN 20 sequences in the genomic dna of marine bacteria kind detects.This figure shows the painted sepharose that contains from pcr amplification of the various types of genomic dna of marine bacteria.M: molecular weight marker, size is bp.-: negative control.+: the positive control of amplicon and ribosome-RNA(rRNA).Swimming lane contains the amplicon from the marine bacteria genomic dna: be respectively kind #1-10, #12-20 and the #21-35 in the storehouse 1,2,3.

5. detailed Description Of The Invention

The present invention relates to a kind of drug discovery system, this system is provided for catching and preserving nature Multifarious genetic resources, and for catching with the resource translation and expanding to multifarious chemistry The method and composition of structure. The present invention also is convenient to screen required activity and compound.

More particularly, the invention provides for the side that makes up and screen the combination gene expression library Method. These libraries comprise the gene outcome of the multiple kind of any kind, in some cases, and it Can in expressive host, interact, and cause in some cases forming new biochemical route and/or Produce the compound of newtype. And library of the present invention provides and effectively obtains other non-availability The approach in the molecular diversity source of arriving.

New biochemical route can be carried out, including, but not limited to the structural modification of material, with chemical based Group adds in the material or the process of decomposed substance.

The compound of new kind can include, but are not limited to metabolin, the secondary metabolism thing, and enzyme, or The structure of organism forms. Interested compound can have one or more potential treatment spies The property, antiviral including, but not limited to antibiotic, antitumor, pharmacology or immunomodulatory properties Or other has the compound of commercial value, such as pigment. Compound can be used as a receptoroid or one The excitant of special receptor or antagonist.

As of the present invention, term " combination gene expression library " comprises the natural approach of combination Expression library makes up chimeric approach expression library and contains the host who expresses the construction library and gives birth to Thing.

" making up natural approach expression library " is from the hereditary thing from one or more donor biologies The expression construction library that matter makes, wherein be present in the inhereditary material gene operably with Cause that the regulatory region that gene is expressed interrelates in suitable host living beings. Energy is used in the combinational expression library Produce the host living beings of donor Biofunctional gene outcome. Inhereditary material in each host living beings is compiled Code is from abiogenous biochemical route or its part of donor biology.

" making up chimeric approach expression library " is from any chimeric something lost from multiple donor biology Pass the expression construction library of material preparation, wherein be present in the gene operability in the inhereditary material Ground interrelates with cause the regulatory region that gene is expressed in suitable host living beings. The host who adopts gives birth to Thing can produce the functioning gene product of donor biology. When in host living beings, expressing, the donor biology Gene outcome can interact and form new chimeric biochemical route.

Usually, the inhereditary material that provides from one or more donor biology is provided method of the present invention. Operate described inhereditary material and described inhereditary material is imported the host by means of clone or expression vector In the biology, one or more genes of donor biology are transferred to and show in host living beings like this Reach. These host living beings that contain the donor inhereditary material are compiled forms the library.

The inhereditary material that shifts comprises the gene of any kind usually, and they are expressed by one or more Functional regulatory region causes and controls. Express construction or carrier some such adjustings can be provided The district. The gene of donor biology is transcribed in host living beings, and translation and processing are to produce itself again Produce the functional protein of interested metabolin.

According to the present invention, comprise the gene of biochemical route or its essence part of complete natural generation Expression library can greatly be convenient to seek and be responsible for producing compound or providing the donor of activity interested many Enzyme system. The gene that participates in specific biochemical route can separate routinely and with single expression construction or The clone characterizes. A typical arrangement of this expression construction is shown among Fig. 1.

In case determined required reactive compound, this suitable feature can be convenient to following widely Drug development work, for example improvement of the improvement of bacterial strain and method. Can under standard conditions, cultivate sun Sex clone is to produce the required compound that is used for further research or uses of q.s. This biochemistry way The gene in footpath can be used for order-checking immediately, sudden change, the screening of expression and more wheels. Clone's biochemical way The footpath is suitable for the traditional and/or genetic manipulation for excessive generation required compound easily.

And, be that silence or undetectable biochemical route can be comparatively in the other situation in the donor biology Easily find according to their functional reconstructions in host living beings. Because the life of host living beings Change feature and know, the result's who expresses as the donor inhereditary material many departing from can be by easily Identification. The extract of host living beings that can be by will containing the donor inhereditary material with a series of given Environmental condition under the known compound profiles that is produced by the contrast host living beings compare to detect Noval chemical compound. When biochemical and cell background is characterized well as the host of host living beings, even can Detect very low-level required activity or compound. Described such as the back chapters and sections, the present invention carries Supplied for detection of with the method for separating the clone who produces required activity or type of compounds.

In a preferred embodiment, these methods are applicable to not returning in a large number at nature Receive or can not be at the donor biology of laboratory cultures. By turning to from these biological inhereditary materials Move in the host living beings, biological metabolic pathway can regenerate, and their product can be had Effect ground detects its any required character. Therefore, these biological genetic diversities have been caught and have preserved.

In another embodiment of the invention, can make up the chimeric approach gene expression of combination library, Wherein before importing host living beings, any from the inhereditary material quilt of one or more donor biologies The ground concatermer. Therefore, each host living beings in the library can contain separately from each donor approach Or biological unique, the arbitrarily assortment of genes. Fig. 2 expresses the chimeric approach gene expression of combination library The expression construction in gene and the arrangement of regulatory region. In most cases, the gene in the library These be combined in nature and do not take place. When expressing, in each host living beings, various donors Approach or biological functioning gene product interact, produce cause new chimeric metabolic pathway and/ Or the combination that produces the biochemical reaction of noval chemical compound. Generally speaking, the something lost of the donor biology in the library The biography resource is translated into can not detectable multiple compounds in each donor biology.

In another aspect of the present invention, can be according to their biological nature, or be complementary to the place The ability of required group of not special biochemical reaction of main biology is selected the kind of donor biology. These Desirable characteristics can include, but are not limited to use some nutriment, under extreme conditions survival, The ability of the chemical constitution of deriving, and the ability of decomposition or some type formation of chemical bond of catalysis. When When the gene of donor biology was expressed in host living beings, the donor gene product can be modified and/or the replacement group Become the function of the host gene product of host's metabolic pathway, thereby produce new hybridization approach. New Activity and/or compound can be by comprising donor and producing from the hybridization approach of host's composition Give birth to. The target metabolic pathway of being modified by the donor gene product may be for originally having for host living beings. On the other hand, the target metabolic pathway can be provided by the heterogenous gene product, and this heterogenous gene is endogenous Or before making up gene expression library or simultaneously by genetic manipulation to each host living beings. Cause This, the present invention also comprises structure and screening-gene expression library, wherein coding donor biological metabolism way The dna fragmentation in footpath is cloned and coexpression in the host living beings that contains the target metabolic pathway.

In another embodiment of the invention, host living beings can have the compound of interest branch Secrete to the enhancing complement of the active medicine discharging system of culture medium, thereby reduce compound the host is given birth to The toxicity of thing. Can during cultivating host living beings, use the water imbibition material, such as resinene, thereby Therefore the separable metabolin that is produced and secreted by host living beings is convenient to reclaim metabolin.

In order to make the method for screening the combination gene expression library more effective, the present invention further provides Detect the method that has those host living beings of activity interested or compound in the library. At this In the bright embodiment, host living beings contains the report system, and this report system will be by activation The from the beginning synthetic of reporter molecule produces reaction to variation and the appearance of introducing, and requiredization for example occur Compound or activity. In one embodiment, host living beings contains the precursor of reporter molecule, or gives birth to Rational probe, because required compound or active appearance, it then is converted into reporter molecule. Positive Reporter molecule among the clone produces the positive colony that can detect in the expression library, and never produces it Other clone in the signal that separates.

In many aspects, this drug discovery system for each step of drug development until clinical examination Testing provides obvious facility and jump. Library of the present invention and the porous footprint shape of having set up The Robotics of formula and high throughput screening is compatible. Host living beings of the present invention is lost for being generally used for Pass the biology of operation and/or method exploitation. The present invention has utilized these host living beings or has produced the host and existed Their biological characteristics and keeping demand side are clearly characterized this fact. By in the future , reduced donor to other more familiar expression system from the transfer of genetic material of donor biology The demand of the condition of culture of biological difficulty. Therefore, can study and more effectively optimize in the present invention The biologically active of any lead compound of finding in the system, pharmacokinetics and toxicity character.

The new metabolic pathway that produces in the positive colony can be come by the standard method in the molecular biology Explanation. Can under standard or the condition of culture of rule of thumb determining, cultivate the host living beings that produces medicine The clone synthesize lead compound, the lead compound of so separable q.s is used for further Analysis and exploitation. Existing highly purified production method is for example for some such standard industry The good manufacturing processing (Good Manufacturing Practice) that host living beings is set up (GMP). Do not resemble the conventional method in screening natural products source, need less effort to make sieve Choosing and production method are adapted to the special demand of the biology of each possible generation medicine.

The present invention also provides donor biology and/or the cell of the method according to this invention from particular group The specific combination gene expression library of type preparation. Be not biology or cell classes all in a group Type, especially biased sample all need to determine one by one or characterize so that prepare combination gene to express literary composition Kucheng is possible.

Any combination gene expression library of the present invention can increase, and duplicates and stores.Amplification refers to cultivate the initial host living beings that contains donor dna, can produce a plurality of clones of host living beings like this.Duplicate each clone who refers to select and to cultivate in the library.Can be by any method caching and the recovery combination gene expression library of the present invention that is suitable for host living beings known in the art, therefore, library of the present invention is a method of effectively catching and preserving the donor Biological Genetic Resources, and this genetic resources can repeat to obtain in the drug discovery scheme.5.1. the preparation 5.1.1. donor biology of combination gene expression library

Any biology can be used as the donor biology that is used to prepare combination gene expression library purpose of the present invention.The donor biology can be from private or public laboratory culture thing, or culture collection unit, American type culture collection for example, and international fungi institute, or from the environmental sample of cultivating or do not cultivate, obtain.

The donor biology may be medicine forerunner's tradition source, for example land endophytic bacteria, fungi and plant.The donor biology can be the transgenosis that is used to produce and/or prepare medicine, by the bacterial strain of genetic manipulation or heredity selection.

The donor biology can or the micro-biological process mentioned of unavailable present prior art cultivate, the genetic material that for example is used for preparing the library can directly obtain from environmental sample.Because the microorganism that only a small amount of (≤1%) nature is found can be in laboratory culture, thus the major advantage of the present invention employed donor biology that is this paper need not to be and can cultivate (Torsvik etc., 1990, applied environment microbiology, 156:782-787).

The present invention is not limited to and uses microorganism as donor.Plant produces a large amount of compounds, and some have strong activity to animal and microorganism, for example phytoalexin (Abelson 1990, science 247:513).In these compounds some can be induced (Cramer etc., 1985, European molecular biology federation magazine, 4:285-289 by wound or from the extract of the cell walls of phytopathogen; Cramer etc., 1985, science 227:1240-1243; Dron etc., 1988, the periodical 85:6738-6742 of institute of NAS).Bioactive compounds, as taxol, camptothecine, Artemisinin is the example from the natural product of plant, they are just carrying out clinical development as antitumor and antimalarial agent respectively.Any plant, those of pharmaceutical properties that especially have possibility may be required donor biology (Phillipson, 1994, imperial tropical medicine and hygiology transactions, 88 supplement 1:S17-9; Volumes such as Chadwick, 1994, at " ethnobotany and new drug research ", Wiley, Chichester, CibaFoundation Symp 185).

Another source with natural product of the antimicrobial or pharmacology character that comes in handy is invertebrates and vertebrates.Some of these compounds play anti-rival, the effect of pathogenic agent and predator's chemical protection.These compounds also can be used to kill and wound ravin or are used as the various forms (Caporale 1995, the periodical 92:75 82 of institute of NAS) of getting in touch.As a rule, the secondary metabolism product is considered to may produce (Faulkner etc., 1993, Gazzetta Chimica Italiana, 123:301-307 for symbiotic Institute of Micro-biology by relevant; Bewley etc., 1995, teach at " the symbiotic summary in the marine natural product chemistry discussion ", La Laguna university, Canary Islands, 9,16,1995, p26 (summary) at Antonio Gonzalez).

Is the source that cherishes a special interest at nature by the biology of other biological biochemical route of known manipulation, and for example some plants as Cycas, can produce the ecdysone stand-in that disturb some insect to grow.These biologies can be survived in identical ecological niche, and wherein they are as the rival, symbiote, and predator and ravin, or as host and parasite and exist.Therefore, the genetic material that uses comfortable nature that chemically interactive biology takes place mutually may be favourable.

The other abundant source of natural product is marine organisms.For example, marine microorganism produces new molecular structure, wherein many biologically actives, for example hot lactim A (octalactin A), it is the possible antineoplastic agent (Tapiolas etc. that do not have the molecular structure of discovery before having in the endophytic bacteria of land, 1991, american chemical federation magazine, 113:4682-83); And Salinamides (Trischman etc., 1994, american chemical federation magazine, 116:755-758), it has the antiinflammatory property of possibility.Contain bromine from more halobiontic compounds from seawater, because the chemically reactive of the halogen that mixes, make compound have high reactivity, marinone (Pathirana etc. for example, 1992, Tetrahedron Lett 33:7663-7666), mix the product of polyketide and mevalonic acid biosynthetic pathway, it has the optionally antibiotic activity of resisting gram-positive bacteria.Has different salt concn, temperature and pressure, the marine organisms kind of the enormous amount of living in the large-scale habitat from the polar region to the tropical zone.The unique characteristic of these habitats is reflected in the genetics and biological chemistry of uniqueness of these biologies, and many useful medicine forerunners may be provided.Referring to, Fenical etc. for example, 1992, " marine microorganism, new Biological resources ".The marine biotechnology progress, Vol.I, PlenumPress, New York.

Environmental sample can obtain from natural or artificial environment, can comprise protokaryon and Eukaryotic mixture, and virus, some of them may be unidentified, and sample can be collected arbitrarily or collect from the area for ecological emphasis, for example the area of close industrial waste, soil, settling around fresh water or the sea water filter thing, hot spring or showing tremendous enthusiasm outlet and ocean or estuarine deposit thing can be used as the source of donor biology.Sample can be from the seabed, and the marine source between sea and tide is collected.Sample can be from the torrid zone, the subtropics, and collect in temperate zone and other zone.The donor biology can be thermophilic, has a liking for salt, has a liking for acid, has a liking for and presses or methanogen.

Also preferred use faces the biology of extinction possibility, those plants and the microorganism for example found in tropical rain forest.Just suffering that in these habitats under the destructive situation, the kind that may produce useful medicine disappears.

Also can collect biology, comprise algae, lichens, fungi, plant and animal according to they purposes in tradition or the medicinal practice that meets the specification with possibility pharmaceutical properties.

In many aspects, wish to use genetic material to make up the library from the donor biology that is unwell to conventional medicament discovery or development technique usually.These donor biologies can have characteristics below one or more: (i) this biology can not be in laboratory breeding or cultivation; (ii) this biology that can not reclaim q.s from nature is used for further experiment; And/or (iii) this biology needs special conditions to produce the unknown or is suitable for industrialized required compound.The characteristics of back have also been described the biology in the existing culture collection thing, wherein owing to unfavorable culture condition, may detect less than the medicine forerunner in conventional screening method.

For the purpose in construction expression library, the donor biology need not be classified to be identified or biochemical the sign.The complicacy per sample and the needs of drug discovery programs, for example, what need the donor kind removes to duplicate (dereplication), can carry out cultivating kind or from the evaluation or the genetic footprinting of the representative group of environmental sample.

Before extracting their nucleic acid, can with the biological concatermerization of donor in the laboratory or the field cultivate, in order to prepare cDNA, may require special growth conditions or in substratum, exist certain chemical substance to induce or to increase transcribing of active gene product interested in the coding donor biology.If only need genomic dna, can use the standard growth condition to cultivate biology.

Because all the donor biologies in environmental sample can not be with phase same rate breeding, if fully under laboratory condition, some donors are biological may hypertrophy also to be caused losing or the slowly dilution of the microorganism of growth.Therefore, can preferably not experimentize formerly cultivating of chamber and the direct biological preparation of the donor from environmental sample nucleic acid.This be when attempting to obtain invertebrates, for example may be particularly useful during the secondary metabolites of sponge, and wherein metabolite is considered to by relevant symbiosis usually and can not cultured microorganism produces.During 5.1.2 saved below the method for the high-quality nucleic acid of the biological preparation of the donor from environmental sample was provided at.

The donor biology of the present invention's imagination can include, but are not limited to virus; Bacterium; Unicellular eukaryote is as yeast and protozoon; Algae; Fungi; Plant; Tunicate; Moss animal; Worm; Echinoderms; Insect; Mollusk; Fish; Amphibians; Reptilia; Birds; And Mammals.The limiting examples of donor biology is listed in Table I and II.

Table I

(Berdy 1974, practical microorganism progress, 18:309-406 for the example table of bacterium and fungi donor biology; Goodfellow etc., 1989, " microniological proudcts: novel method ", Cambridge University Press 343-383) divides generic bacterium actinomycetales streptomyces, Micremonospora, Norcodia, put the Madura

The line Pseudomonas, actinoplanes, streptosporangium, little two

Spore Pseudomonas, kitasatosporia belong to the solid chlorine Pseudomonas of Eubacteriales, rhizobium, and Achromobacter, enterobacter,

Brucella, micrococcus, lactobacillus genus, gemma bar

Pseudomonas, Clostridium, brevibacterium sp pseudomonas order Pseudomonas, aerobacter, Vibrio, Halobacterium mycoplasmatales mycoplasma fruiting myxobacteria phagocyte bacterium, Myxococcus fungi Myxothallophytes Physarum, flowers-of-tan belongs to algae shape Gammaproteobacteria Mucor, phytophthora, Rhizopus Ascomycetes Aspergillus, Penicillium Basidiomycetes Coprinus, Phanerochaete deuteromycetes Acremonium (Cephalosporium), Trochoderma, long

Child's spore belongs to, Fusarium, and Alternaria belongs to, Myrothecium Yeast sugar yeast belong

Table II

The example classified instance of the donor biology of high form belongs to, compound and characteristic plant algae Digenea simplex (Wnlf.) C. Ag. (kainic acid is controlled anthelminthic)

Narrow leaf sea-tangle (laminine is controlled ypotension) lichens pine sieve (close ear acid, antibiotic; Usnic acid, antitumor) higher plant Vinca (Vinca), Digitalis (cardiac glycoside),

Podophyllum emodi var chinense belongs to (podophyllotoxin), Taxus (taxol),

Cephalotaxus (homoharringtonine), Camptotheca acuminata belongs to (camplotheca acuminata

Alkali), artemisia (artemisin), Coleus (forskolin),

Angle silk desmids belongs to the crafty spider of (potassium channel antagonist) Protozoa dinoflagellate ptychodiscus brevis (the cardiovascular property of brevitoxin) insect and belongs to (" stealing spider " venom), plants ladybug and belongs to (Mo Xi

Brother's Epilachna spp alkali) the thin bud sponge of polyzoan polyzoan (bryostatins, antitumor) mollusk cone shell sponge (ectyonin, antibiotic) Cryptotethya

Cryta (D arbinofuranose glycosides) coral Pseudoterogonia (pseudoteracins, anti-inflammatory),

Erythropodium (erythrolides, anti-inflammatory) worm Annelita rope clam worm (nereistoxin, desinsection) Spinunculida splits Yi and (splits the Yi element, neural activity) tunicate Trididemnum solidum (didemnin, antitumor

With antiviral)

Ecteinascidia turbinata (ecteinascidins, anti-

Tumour) fish hagfish (hagfish toxin, cardiac stimulus), imperial Teng (egg

The white matter toxin brings high blood pressure down, and breathes and the minimizing heart rate) the Amphibians Dendrobates (batrachotoxin, pumiliotoxins,

Histrionicotoxins and other polyamine) Reptilia snake venom toxin birds histrionicotoxins, the carotenoid of modification, class is looked look

Element and steroide (Goodwin 1984 " class Radix Dauci Sativae

Plain biological chemistry " Vol II, Chapman and Hall,

New York, pp 160-168) Mammals duckbill platypus (duckbill platypus venom), the carotenoid of modification,

Retinoids and steroide (Goodwin 1984,

Supra pp.173-185; Devlin 1982 " bioids

Learn textbook " Wiley, New York, p.750) 5.1.2. is from the high-quality nucleic acid of the biological preparation of donor

Depend on the biological type and the source of sample, can be by the whole bag of tricks from donor bioseparation nucleic acid.For the gene expression library of the genetic information that makes up abundant representative donor biology, obtain not incise, the high-quality nucleic acid of strand breach and partially denaturing is very important.In order to prepare high-quality nucleic acid, the gentleness of the donor biology in the method sampling of the present invention, fast and complete molten born of the same parents, and proteinic fast from this biological nuclease and other degradation property, complete deactivation.Initial extraction can be carried out in the field can carrying out in the laboratory until further separating step with the nucleic acid of stablizing in the sample.

Any separate nucleic acid method all requires the donor biology to rupture fully.Can adopt many standard methods, be included under the liquid nitrogen and solidify, when having glass or other cracking agent, grind, and simple and mechanical shearing or enzymic digestion.

For compounding substances, soil for example, or for containing high-load tough and tensile material, for example Mierocrystalline cellulose or chitin (as in filamentous fungus and plant) can adopt lyophilize to make the sample fragility, thereby it is more suitable in breaking.This is frozen the exsiccant material and only preserves tool enzymic activity and high molecular weight material (for example nucleic acid) (Gurney 1984, molecular biology method, Vol.2, p 35-42, John M.Malker volume) for a long time.Sample can be by flash freezing in liquid nitrogen.Loose sample, as soil, can be freezing in fine-structure mesh or nylon mesh.Under vacuum, can carry out 24-72 hour lyophilize to freezing sample.Can be under vacuum-70 ℃ with the freeze drying example dry storage.May need other step to prepare environmental sample, for example the concentrate microbial population (Jacobson etc. 1982, practical environmental microorganism, 58:2458-2462; Zhou etc. 1996, practical environmental microorganism, 62:316-322; Somerville etc. 1989, practical environmental microorganism, 55:548-554).

A basic skills of the present invention, though of course not unique the employing, be to Chirgwin etc. (1979, biological chemistry, 24:5294), Sadler etc. (1992, modern genetics, 21:409-416) and Foster (1991, Ph D dissertation, University of California, Santa Barbara) improvement.This method is used strong chaotropic agent, and guanidinium isothiocyanate and 2 mercapto ethanol make protein denaturation and make the nuclease inactivation, then comes the purification of nucleic acid material with caesium chloride density gradient centrifugation.The method institute difference of method provided herein and Chirgwin is to have extracted DNA and RNA.Method of the present invention has also comprised the high speed centrifugation step and has optionally added the dibenzoyl imide dye.Depend on the donor biology that is adopted, other step can comprise, but be not limited to cetylpyridinium chloride muriate or cetyl trimethylammonium bromide (CTAB) optionally to remove polysaccharide, handle to remove phenol with polyvinylpyrrolidone, and carry out the Mierocrystalline cellulose chromatography to remove starch and other carbohydrate (Murray and Thompson, 1980, nucleic acids research 8:4321-25).

Use ThermoScript II the RNA from the donor bioseparation can be converted into complementary DNA.

Impaired nucleic acid may be difficult to the clone, thereby causes the clone of low number in the loss of the biological DNA of donor and the library.Source DNA repair mechanism in if host living beings allows reorganization and lacks effectively, problem can seriously be changed so.The present invention also provides use, and enzymatic reaction commonly used can be in the impaired DNA of external reparation (Sambrook etc. 1989, " molecular cloning " 2 editions, chapters and sections 8) before the clone between synthesis phase at complementary DNA second chain.For example, can come the DNA plerosis crack and incise by the Klenow fragment and the e. coli dna ligase of archaeal dna polymerase.These enzymatic reactions are known for those skilled in the art.

When the DNA that is extracted by environmental sample prepares recombinant expressed library, obtainable DNA amount usually is restricted, and is the problem that will consider in the selection of method of attachment.Described in 5.1.3., if extract or concatermerization after its amount few (＜100 μ g), DNA can be connected in the high-efficient cloning system, for example SuperCos.Insertion fragment among the amplification clone, and discharge from carrier by Restriction Enzyme digestion.Because the character of environment DNA sample, it may comprise protokaryon and eucaryon donor biology, may need to use a plurality of host living beings.If can obtain the initial environment DNA sample of q.s, if or DNA increase, DNA can be connected in a collection of carrier that is suitable for required batch of expression host cell each.Preferably, carrier has the ability of shuttling back and forth between two or more expressive hosts.5.1.3. host living beings and carrier

The term that this paper adopted " host living beings " comprises unicellular organism widely, and for example bacterium, and multicellular organism is as plant and animal.Can use any cell type, be included in vitro culture or by those of genetic modification.Any host-vector system well known in the prior art can be used for the present invention.Use can be replicated and the shuttle vectors of surviving in more than one host living beings is favourable.

Host living beings or host cell can be from private laboratory preservation things, public culture center, for example American type culture collection or obtain from commercial suppliers.These host living beings or cell can further be modified by technology known in the art is used for specific purposes.

According to the present invention, preferred host living beings or host cell have been used to expression of heterologous genes, and have reasonably been carried out biological chemistry well, and physiology and/or heredity characterize.These host living beings may be improved one's methods by the hereditary bacterial strain by routine, cultural method, fermentation process and/or recombinant DNA technology and use.Need to use and to have developed the donor biology that is used for large-scale production process, and become known for growing and produce the condition of secondary metabolites.

Can be in the temperature of standard, soaking time, light intensity and be adapted to the nutrition of expressive host and the condition of the culture media composition of physiological requirements under cultivate host living beings.Yet, be used to keep with the condition that produces the library may be used to express and to screen those of library different.Improved culture condition and substratum also can be used to imitate some nutrition and the physilogical characteristics of donor biology, and so that produce interested metabolite.For example the precursor of compound of interest can be provided in nutritional medium so that modify those precursors.Any technology known in the art can be used to set up optimal conditions.

Host living beings should preferably lack the ability of carrying out homologous recombination and restriction foreign DNA.Host living beings should preferably have the codon that is similar to the donor biology and select.If adopt eucaryon donor biology, preferred host living beings has the ability of suitable processing donor messenger RNA(mRNA), for example cuts intron.

Preferred prokaryotic hosts biology can include, but are not limited to intestinal bacteria, subtilis, paleness streptomycete, streptomyces coelicolor.Also can use yeast specie, for example Saccharomyces cerevisiae (Saccharomyces cerevisiae), grain wine fragmentation sugar yeast (fission yeast), Pichia pastoris and multiform Hansenula anomala (methylotrophic yeast).Also can adopt thread ascomycetes, for example Neurospora crassa and Aspergillus nidulans.The preferred plant cell is for example from those of Nicotiana and Arabidopsis.Preferred mammalian host cell comprises, but be not limited to from the mankind, monkey and rodent, Chinese hamster ovary (CHO) cell for example, NIH/3T3, COS, 293, VERO etc. (seeing Kriegler M. " transgenosis and expression: laboratory manual " New York, Freeman and Co.1990).

Can select as required to modify and the host living beings of the gene product of expression processing with ad hoc fashion.The effect in biochemical route may be very important for protein for these modifications of protein (for example glycosylation) and processing (for example cracking), can select correct modification and the processing with the exogenous protein guaranteeing to express of the clone that suits or host system.At last, if the donor biology is an eukaryote, have the correct and accurately processing of the primary transcript of being used for, the eukaryotic host cell of the organoid of the phosphorylation of glycosylation and gene product may be preferred.

For example, shown that the eucaryon fungi has many identical core element biology, between many kinds of the most common fungal species producer exchange be possible (Gurr etc. 1987, the gene structure of eukaryotic microorganisms, Kinghorn compiles, p.93; Bennet and Lasure 1992, the genetic manipulation in the fungi, Academic Press, NY).The preferred embodiment of eucaryon host biology is fissiparous yeast, grain wine fragmentation sugar yeast.At first the molecular biology of grain wine fragmentation sugar yeast is fully studied, and many main cultivations and purification process and operation are carried out routinely.The second, it is unicellular, therefore can easily be cultivated under laboratory condition, stores and operation.The 3rd, the blended eukaryotic DNA is particularly important being used for expressing, its montage correctly and fungi, plant and the mammiferous gene of expressing other kind.Illustrated that for the research of the montage of allos nRNA (RNA that contains intron) and processing grain wine fragmentation sugar yeast and other fungi and high Orthoptera have the type that is used for the needed small nuclear RNA of montage (snRNA) composition significantly and the similarity of structure.At last, many non-grain wine fragmentation sugar yeast promotors, some of them are from Mammals and plant virus, can moderately cause high-caliber genetic expression (Forsburg, 1993, nucleic acids research, 21:2955).This feature can allow fungal DNA/cDNA library to shuttle back and forth in the mammalian cell expression host, NIH3T3 (inoblast) for example, GT1 7 (neurone), or other cell type.

Cloning vector or expression vector can be used to donor dna is imported the host living beings that is used for expressing.Expression constructs is the expression vector that contains the operability ground donor dna sequence relevant with one or more regulatory regions.Regulatory region can be provided by donor dna or carrier.Can use variety carrier, it includes, but are not limited to plasmid; Clay; The phage plasmid chimera; Artificial chromosome, for example yeast artificial chromosome (YACs), and bacterial artificial chromosome (BACs, Shizuya etc. 1992, the periodical 89:8794-8797 of institute of NAS) or the virus of modifying, but carrier must be compatible with host living beings.The limiting examples of available support is λ gt11, pWE15, SuperCosl (Stratagene), pDblet (Brun etc. 1995, gene, 164:173 177), pBluescript (Stratagene), CDM8, pJB8, pYAC3, pYAC4 (referring to the appendix of the same method in the molecular biology, 1988, editor Ausubel etc., Greene Publish.Assoc. and Wiley Interscience, it at this paper by incorporated by reference).

When the regulatory region of host and donor biology was compatible with transcription factor, the transcriptional domain of donor can be in conjunction with host's factor, and RNA polymerase for example is to influence transcribing in the host living beings.If donor and host living beings are incompatible, the regulatory region compatible with host living beings can link to each other with the donor dna fragment, with the expression of gene that guarantees to be transferred.

At whole operons, comprise the translation initiation codon of itself, rrna land and contiguous sequence are inserted under the suitable clone or the situation in the expression vector, can not need other control signal.Yet, when only the encoding sequence of some gene is inserted into, must provide the control signal of external source, comprise translation initiation codon (being generally ATG) and contiguous sequence.The regulatory region of these external sources and initiator codon can be various sources, natural and synthetic.Composing type and induction type regulatory region can be used to the expression of donor dna.When the product possibility of expression library is poisonous, need to use evoked promoter.Can improve the efficient (referring to Bittner etc. 1987, Enzymology method 153:516-544) of expression by the inclusion of suitable transcriptional enhancer element.

" operability links " refers to a kind of contact, and wherein regulatory region and the dna sequence dna that will express are connected with the mode of final translation and locate to allow to transcribe.The precise nature of the regulatory region that genetic expression is required can be with biological and biological the variation.Usually, needing can be in conjunction with RNA polymerase and start the promotor that nucleotide sequence that operability links is transcribed.These regulatory regions can comprise 5 '-the non-keying sequence participates in transcribing and translate those of beginning, and for example the TATA box adds the cap sequence, CAAT sequence etc.The Transcription Termination of non-coding region 3 ' also can be retained or duplicate it to encoding sequence is regulated sequence, for example terminator and poly adenylylation site.Two sequences of a nucleic acid molecule are said to be " linking to operability " when they expand to second mode in the sequence and get in touch mutually with the rna transcription thing that allows two sequences and be transcribed on the identical rna transcription thing or allow to have begun in a sequence.Can produce the polycistronic transcription thing thus.If transcribing of beginning at the promotor place will produce the rna transcription thing of the sequence that operability links, so two or more sequences, for example promotor and any other nucleotide sequence are that operability links.In order to become " operability links ", two sequences must be not to be close to mutually.

In addition, expression vector can contain and is useful on initially-separate, identifies or follow the trail of the marker gene that maybe can screen selected of the host living beings that comprises donor dna.Expression vector also can provide restriction site unique or that conveniently be positioned to insert fragment to allow the DNA that preserves and/or reset in the part expression constructs.

Expression vector can contain and allows carrier to exist in one or more host living beings and/or duplicate, or the sequence of vector integration to the host chromosome.These sequences can include, but are not limited to, duplicate field, automatic replication sequence (ARS), centromere DNA and telomeric dna.As a result, in host living beings, can produce and preserve one or more copy expression libraries.Expression constructs can be incorporated in the host genome or keep free in the host living beings.

Usually, it may be favourable using shuttle vectors, and this shuttle vectors can duplicate at least two kinds of host living beings and survive, for example, as bacterium and mammalian cell, bacterium and yeast, bacterium and vegetable cell or Gram-positive and gram negative bacterium.Shuttle vectors can contain wide host range replication orgin, for example from IncP, and those of IncQ plasmid, or two or more at least duplicate field.Shuttle vectors also can comprise the sequence from the plasmid of natural generation, and it can be used to by means of conjugal transfer (Hayman etc., 1993, plasmid 30:251-257) in the various compatible host living beings be moved in the library.By using shuttle vectors to make up the library, the dna sequence dna of donor biology can easily be replicated and transfer to another host living beings from a host living beings.

For example, a preferred typical expression vector-host living beings be combined into clay, SuperCos 1 and coli strain, XLl-Blue MR, they all can available from Stratagene (La Jolla, CA).Carrier is accepted the DNA insertion fragment that the size variation scope is 30-42 kbp by the BamHI cloning site, and the SV40 promotor that has neomycin resistance mark (neoR) and be used for expressing at mammalian cell.Carrier also contains and be used to the ampicillin resistance gene selected in prokaryotic cell prokaryocyte.Biological some restriction system (hsdR, mcrA, mcrCB and mrr) that lacks of escherichia coli host, endonuclease (endA1) defective, and be defective recombinant (recA).Host living beings can not enzyme be cut and is had halfcystine and/or the methylated insertion fragment of VITAMIN B4, and this insertion fragment is present in eukaryotic DNA usually and uses among the methyl dNTP analogue synthetic cDNA.

The advantage of this system is included in and externally efficient λ packaging system is used to be initially at sex-limited negative, produces the library in the negative escherichia coli host of recA.Transform required amount because the character in source, genomic dna may be less than naked DNA, the genomic dna of packing inserts fragment can protectedly avoid degraded.In case in e. coli host cell, impaired insertion fragment can be repaired by the host cell DNA repair mechanism.This system only needs a small amount of initial gene group DNA (5-10 μ g), and because packaging system is only accepted the insertion fragment of a certain magnitude range, so may not need size to select.Initial library in the intestinal bacteria can be amplified producing the superhelix cosmid DNA, and this cosmid DNA can be used for importing in the efficient method for transformation in other expressive host biology.

Can further modify to be used for clone the SuperCos1 carrier by SV40 starting point and neoR gene that displacement is duplicated with replication orgin (for example from plasmid pIJ 101 or pIJ 922) and the displacement of thiostrepton resistant gene of streptomycete the streptomycete host.This shuttle vectors is called Streptocos (seeing Fig. 6 A), it is by digesting with KpnI and HindIII, from containing the pIJ 699 (Hopwood etc. of pIJ 101 starting points and thiostrepton resistant gene, 1985, the genetic manipulation of streptomycete, laboratory manual, The John Innes Foundation) separates 4.0 kb fragments and make up.This fragment by concordantization of end, and is cloned in (referring to Bierman 1992, related example is seen Denis1992) among the SuperCos at the SamI-HindIII restriction site in the KpnI site.In addition, can import be used to the to shuttle back and forth migration (Bierman etc. 1992, gene 416:43-49) of clay of sequence composition by means of conjugal transfer.The different Streptocos form that contains the streptomycete specificity promoter can be imported in the carrier at contiguous BamHI cloning site place.By using PCR, can produce the streptomycete promoter fragment in the NotI/EcoRI site that can directly be cloned into SuperCOS 1.Can use multiple known streptomycete promotor, comprise ermE, Pptr (1995, molecular biology, 17:989) and hrdB (Buttner, M.J.1989, molecular biology, Vol.3, pp.1653-1659).This method can be used for large-scale host-vector system usually.Therefore, if desired, can be by importing host's replication orgin, selectable marker gene and homologous promoter are modified SuperCos 1.

For the combination gene expression library that uses vegetable cell as the host, the expression of donor encoding sequence can be caused by any of multiple promotor.For example, preferred bacterial strain is described in Principles of gene manipulation 1985, R.W.OLD and S.B.Primrose 3rd editor Blackwell ScientificPub; Carrier: the research 1988 of molecular cloning vector and their purposes, R.L.Rodriquez, D.T.Denhardt, Butterworths Pub.; Molecular cloning practical guide 1988, B.Perbal, John Wiley and Sons, viral promotors, for example the 35S RNA of CaMV and 19S RNA promotor (Brisson etc. 1984, natural 310:511-514) maybe can adopt the coat protein promotor of TMV; On the other hand, can use plant promoter, for example the small subunit of RuBISCo (Coruzzi etc., 1984, EMBO is J.3:1671-1680; Broglie etc. 1984, science 224:838-843); Or heat-shocked promotor, for example soybean hsp 17.5-E hsp 17.3-B (Gurley etc. 1986, molecular cytobiology 6:559-565).

Vegetable cell and protoplastis all can be used as host cell.Plant host can include, but are not limited to, corn, wheat, rice, soybean, tomato, tobacco, Radix Dauci Sativae, peanut, potato, beet, Sunflower Receptacle, potato, Arabidopis thaliana, the cell of rape and petunia.Since do not have cell walls and they may be along with cell culture hyperplasia, and be regenerated as plant, so the preferred plant protoplastis.

In addition, the recombination to construct thing can comprise the effable marker gene that maybe can screen selected of plant, it comprises, make it have antibiotics resistance (for example kantlex or hygromycin resistance) or Herbicid resistant (for example anti-sulfonylurea but be not limited to, phosphinothricin, or glyphosate) gene.The mark that can screen comprises, but be not limited to coding beta-Glucuronidase (Jefferson, 1987, molecular biology of plants, Rep5:387-405), luciferase (Ow etc. 1986, science 234:856-859), gene with the B albumen (Goff etc. 1990, and EMBOJ9:2517 2522) of regulating the generation of anthocyanin pigment.

For the biological DNA of donor is imported vegetable cell, can adopt the Agrobacterium tumefaciens system that is used to transform plant.The appropriate design of this conversion carrier based on T-DNA and making up is known for those of ordinary skills.These transform selects to use binary edaphic bacillus T-DNA carrier (Bevan, 1984, nucleic acids research 12:8711-8721), and co-culture method (Horsch etc. 1985, science 227:1229-1231).Usually, the edaphic bacillus conversion system is used to genetic modification dicotyledons (Bevan etc., 1982, Ann.Rev.Genet 16:357-384, Rogers etc. 1986, Enzymology method 118:627-641), but it also can be used to transform and DNA is transferred in monocotyledons and the vegetable cell (referring to Hernalsteen etc. 1984, EMBOJ3:3039-3041; Hooykassvan Slogteren etc. 1984, natural 311:763-764; Grimsley etc. 1987, natural 325:1677-1679; Boulton etc. 1989, molecular biology of plants 12:31-40; Gould etc. 1991, plant physiology 95:426-434).

In another embodiment, also can use various other methods that are used for the recombinant nucleic acid construction is imported vegetable cell.When target was monocot plant cell, these other methods were particularly useful.Other transgenosis and method for transformation comprise, but be not limited to, by calcium-, polyoxyethylene glycol (PEG)-or the protoplast transformation taken in of the naked DNA of electroporation mediation (referring to Paszkowski etc. 1984, EMBOJ3:2717-2722, Potrykus etc. 1985, MGG 199:169-177; Fromm etc. 1985, the periodical 82:5824-5828 of institute of NAS; Shimamoto, 1989, natural 338:274-276) and the electroporation (D ' Halluin etc., 1992, vegetable cell 4:1495-1505) of plant tissue.The other method that vegetable cell transforms comprises microinjection, the DNA of silicon carbide mediation takes in (Kaeppler etc., 1990, vegetable cell report 9:415-418), with the micropellet bombardment method (referring to Klein etc. 1988, the periodical 85:4305-4309 of institute of NAS; Gordon-Kamm etc., 1990, vegetable cell 2:603-618).

For the summary of molecular biology of plants technology referring to, for example Weissbach and Weissbach, 1988, molecular biology of plants method, Academic Press, NY, joint VIII, pp.421-463; With Grierson and Corey, 1988, molecular biology of plants, 2d Ed., Blackie, London, Ch.7-9.

In the insect system, the clover crazing is examined moth polyhedrosis virus (AcNPV) night, and a kind of baculovirus is used as carrier and expresses donor gene in the fall army worm cell.The donor dna sequence can be cloned into the nonessential region (for example polyhedron gene) of virus and place under the control of AcNPV promotor (for example polyhedrin promotor).Then these recombinant viruses are used to infect the host cell that wherein inserts gene and expressed (referring to Smith etc. 1983, Journal of Virology 46:584; Smith U.S. Patent number 4,215,051).

In yeast, the multiple carrier that contains composing type or inducible promoter can with Saccharomyces cerevisiae (Saccharomyces cerevisiae) (Saccharomyces cerevisiae), grain wine fragmentation sugar yeast (Schizosaccharomyces pombe) (fission yeast), pichia pastoris phaff (Pichiapastoris) and multiform Hansenula anomala (Hansenula polymorpha) (debaryomyces hansenii) use together.For summary, referring to modern molecular biology method Vol.2,1988, Ed.Ausubel etc., Greene Publish.Assoc and Wiley Interscience, Ch.13; Grant etc. 1987, and zymic is expressed and secretion vector, in ferment method, and Eds Wu and Grossman, 1987, Acad Press, N.Y, Vol.153, pp.516-544; Glover, 1986, dna clone, Vol.II, IRL Press, Wash, D.C., Ch.3; And Bitter, 1987, the heterogenous gene in the yeast is expressed, Enzymology method, Eds.Berger and Kimmel, Acad.Press.N.Y., Vol.152, pp.673-684; With the molecular biology of sugar yeast, 1982, Eds.Strathern etc., Cold Spring Harbor Press, Vols.I and II.

In mammalian host cell, multiple mammalian expression vector can commerce be buied.In addition, can utilize a large amount of expression systems based on virus.Be used as in adenovirus under the situation of expression vector, the donor dna sequence can be connected to adenovirus and transcribe/translate on the adjusting mixture, as late promoter and trisected leader sequence.Then can mosaic gene be inserted in the adenoviral gene group by reorganization in external or the body.(for example will produce the recombinant virus that in infected host, to survive and can the expressing heterologous product in the insertion fragment of virus genomic nonessential region (for example distinguishing E1 or E3), referring to Logan and Shenk, 1984, the periodical 81:3655-3659 of institute of NAS).The replication orgin (Orip) of Epstein-Barr virus (EBV) and be used to produce shuttle back and forth episome cloning vector, for example EBO-pCD (Spickofsky etc. 1990, DNA Prot Eng Tech 2:14-18) as the EBNA-1 of trans-acting replicator.Virus vector based on retrovirus also may be utilized, and (Morgenstern etc. 1989, and nucleic acid section academic year comments, 12:47-65).On the other hand, can adopt bovine vaccine 7.5K promotor (referring to for example Mackett etc., 1982, the periodical 79:7415-7419 of institute of NAS; Mackett etc. 1984, Journal of Virology 49:857-864; Panicali etc. 1982, the periodical 79:4927 4931 of institute of NAS).

Can adopt the multiple choices system to be used for mammalian cell, comprise, but be not limited to herpes simplex virus thymidine kinase (Wigler etc., 1977, cell 11:223), hypoxanthine-guaninephosphoribosyl transferase (Szybalska and Szybalski, 1962, institute of NAS periodical 48:2026), and adenine phosphoribosyl transferase (Lowy etc. 1980, cell 22:817) gene can be respectively applied for tk, in hgprt or the aprt cell.And the metabolic antagonist resistance can be used as the basis of selecting Tetrahydrofolate dehydrogenase (dhfr), and it can make have the methotrexate resistance (Wigler etc., 1980, the periodical 77:3567 of institute of NAS; O ' Hare etc. 1981, the periodical 78:1527 of institute of NAS); Gpt, it can make has mould phenolic aldehyde resistance (Mulligan and Berg, 1981, the periodical 78:2072 of institute of NAS); Neomycin phosphotransferase (neo) can make to have aminoglycoside G-418 resistance (Colberre-Garapin etc. 1981, molecular biology magazine 150:1); And hygromix phosphotransferase (hyg), it can make has hygromycin resistance (Santerre etc. 1984, gene 30:147).

The present invention also provides the specific modification to host living beings that improves combination gene expression library performance.When the library was used to produce the purpose of secondary metabolites, the toxicity of compound can cause failing fully to represent in the library these to have the host living beings of productivity.Host living beings can be modified in one embodiment of the invention, to be subjected to the disadvantageous effect of generation of compound of interest few for the growth of host living beings and survival like this.The dosis tolerata that improves can reduce the loss that is producing the host living beings of possible medicine in screening stage and production phase.

One of host living beings preferred modify be in host living beings, introduce and/or excessive generation active medicine outside heat-extraction system.The energy drives that film is relevant comes outward in the drug resistance in most of biologies and plays main effect, and these biologies comprise bacterium, yeast and mammalian cell (Nikaido1994, science 264:382-388; Balzi etc. 1994, biological chemistry biophysics progress 1187:152-162; Gottesman etc. 1993, and bioid academic year is commented 62:385).Adorned host living beings with the outer heat-extraction system that strengthen to replenish can be secreted possible toxic chemical in a big way actively, has therefore reduced its accumulation in host living beings.Can avoid negative feedback mechanism, the end product that for example produces the pathways metabolism of compound suppresses.And because compound of interest does not have at the host living beings inner accumulation, so may become more effective to the separation of compound.

In bacterium, studied in a large number (efflux) system that effluxes, these outer heat-extraction systems can be discharged the incoherent molecule of large-scale structure, from for example polyketide microbiotic (colibacillary AcrAE group, Ma etc. 1993, bacteriology magazine 175:6299-6313), fluorescence quinoline and ethidium bromide (bmr of subtilis and the norA of streptococcus aureus, Neyfakh etc. 1993, biocide chemistry 37:128-129), Zorubicin (drr of pentamycin streptomyces verticillus) is to tertiary amine (qacE of aerogenesis Klebsiella pneumoniae and colibacillary mvrC).The non-limiting embodiments table of outer heat-extraction system sees Table III.Arbitrary this outer heat-extraction system can be used for the prokaryotic hosts biology.

In yeast, manyly make the outer heat-extraction system of the genes encoding with pleiotropy drug resistance, and can be used for the present invention.For example the bfrl+ gene is passed to grain wine fragmentation sugar yeast with the non-moral rhzomorph of mine-laying A resistance, and the CDR1 gene transmission ring hexanamide of white candiyeast and chlorampenicol resistant (Prasad etc. 1995, Curr Genet27:320-329).

For mammalian cell, can use to belong to ATP-(Juranka etc. 1989, FASEBJ, 3:2583-2592 in conjunction with the multi-medicine resistance protein of chlG class; Paulusma etc. 1996, science 271:1126-1128; Zaman etc. 1994, institute of NAS periodical, 91:8822-8826; Breuninger etc. 1995, cancer research 55:5342-5347, Koepsell EP 0699753).Many kinds of drug resistance genes of people mdr1 are functional expression (Kuchler etc. 1992, PNAS 89:2302-2306) in Saccharomyces cerevisiae.Any other outer heat-extraction system also can be used for eukaryotic cell.

Table III

By the outer heat-extraction system cationic dyestuff rhodamine of the concrete title of active medicine outflow system excretory compound table type of compounds-6G bmr

Ethidium bromide

Acriflavine acrAE alkaline antibiotic tetracycline bmr

Zorubicin drr, the hydrophilic microbiotic Vulkamycin. PA-93 of mdr acrAE

The hydrophobic microbiotic beta-lactam of macrolide class organic cation four Zhuan Ji Phosphonium bmr neutral taxol mdr

Paraxin bmr slightly acidic nalidixic acid emr

Mitiromycin mdr zwitter-ion fluorine quinoline bmr washing composition SDS acrAE

One or more outer heat-extraction systems can be introduced, induce or excessively produce in the host living beings.The gene that the system that coding can be effluxed forms imports in the host living beings and uses above-mentioned expression vector and method to make its expression.In some cases, it is favourable using inducible promoters to express outer heat-extraction system gene.5.1.4. make up natural approach expression library

The present invention relates to the structure and the purposes of combination gene expression library, wherein host living beings contains the genetic material from multiple donor biology of the natural biochemical route of coding or its part, and can produce the functioning gene product of donor biology.Thus, the biochemical route of donor biology or its part are functionally rebuild in the library of each host living beings.New compound active and these biochemical route can be easier to screen by drug discovery technology or method provided herein.

DNA or RNA can be used as initial genetic material and be used for preparing these libraries, and wherein these libraries can comprise the cDNA library, genome dna library and blended cDNA/ genome dna library.Dna fragmentation from the multiple donor biology of for example describing in 5.1.1 is imported in the host living beings storehouse, and each host living beings in the storehouse all contains the dna fragmentation from a donor biology like this.

If host living beings and donor biology have some hereditary feature, then may be favourable, for example similar GC content of DNA and common RNA montage mechanism, or physiological characteristic are as optimum growth temperature.Therefore may need to use the host living beings that is closely related with donor biological development genetics.For example, may more need the prokaryotic hosts biology to clone and express other protokaryon operon.

The donor biology that is unsuitable for conventional medicament discovery or drug development technology may be preferred.For example, most of marine bacterias are not characterized well, and are unsuitable for conventional Lu Sheng micro-biological process.The present invention can simplify the exploitation of production and purge process.

The donor dna fragment that shifts can comprise functional protein or its a part of coding region and relevant naturally regulatory region, for example promotor and the terminator of the complete biochemical route of encoding.The big prokaryotic gene group dna fragmentation that has the whole biochemical route possibilities of bigger coding by use can obtain optimum.If the natural functions and the structure of the dna fragmentation that shifts are preserved in host living beings, the gene of donor biology can correspondingly be expressed so.The external source regulatory region also is provided, and this regulatory region can link to each other with dna fragmentation guaranteeing the gene transcription that is transferred in the host living beings, thereby replaces or replenished from transcribing that natural promoter begins.

Interesting is to have found that many genes from marine bacteria use natural promoter to come expressive function albumen in intestinal bacteria.Therefore, the gene of marine microorganism even can when not needing to use the external source regulatory region, express.The embodiment table of the gene of the marine bacteria of its natural promoter of use is provided in the Table IV in intestinal bacteria.

Table IV

In intestinal bacteria, use the marine bacteria gene table gene of its natural promoter to belong to and plant and replace sporangium Barbeyron etc. 1994 with reference to document kappa carrageenan enzyme food siliquosa Pelvetia, the aerobic 139:105-109Na+/H+ of gene (cgkA) gram (-) oppositely transportation separates alginic acid vibrios Nakamura etc., 1994, give birth to (NhaA) thing chemistry and biophysics progress

1190:465-468 phosphodiesterase Fei Shi vibrios, amphibious Dunlap etc., 1993, bacterium (cpdP) is learned magazine 175 (15):

The 4615-4624 chitin replaces monospore mushroom, Tsujibo etc., 1993, bacterium

Bacterial strain 0-7 learns magazine 175 (1):

176-181 chlorination tributyl replaces the monospore mushroom, M-1, and Fukagawa etc., 1993, give birth to tin resistance gram (-) Rod thing chemistry and biophysics research

194 (2): the complementary tetraodotoxin of 733-740dagA-replaces sporangium Macleod etc., 1992, molecule

Gram (-) microbiology 6 (18):

The plain vibrio proteolyticus David of the molten born of the same parents of 2673-2681 vibrios etc., 1992, gene, (nprV) gram (-) 112:107-112 tetracyclin resistance Vibrio salmonicida Sorum etc., 1992, chemistry

Aerobic molecules 36 (3):

The synthetic Kao Shi of 611-615 melanocyte kills watt Salmonella Fuqua etc., 1991, gene, (melA) gram (-) 109:131-136DNA that grows nonparasitically upon another plant modifies multienzyme system raw silk sporangium Danaher etc., 1990

Happiness Wen Jiyin, 89:129-133

In a preferred embodiment, method of the present invention has been utilized prokaryotic organism, enters each independently functional relevant gene cluster in the genome as the gene organization of bacterium, is called the mode in the operon.In these bunches, the gene of the composition of coding biochemical route links to each other with the common regulatory region together.Functional relevant gene in the filamentous fungus (actinomycetes) is also by known clustering.Many bacteriums and actinomycetic gene cluster, and the separated and sign of a small amount of eucaryon fungal organism route of synthesis.For example, be used in bacteria Escherichia coli, to activate and produce synchronously (Perry etc., 1986, bacteriology magazine 168:607-612 at 12 kinds of protein that the living Erwinia de novo synthesis of grass produces carotenoid zeaxanthin and beta-cryptoxanthin; Hundle etc. 1991, photochemistry and photobiology 54:1:89-93).In addition, in independently bunch, also contain the prokaryotic organism amino acid biosynthetic pathway, the for example biosynthesizing of leucine and Isoleucine and glucose transfer system, therefore, prokaryotic organism are used as the donor biochron, and functional relevant gene may be separated in a clone in biosynthetic pathway.

Preferably has the tight genomic donor biology that contains the relatively small amount non-coding region.In many aspects, donor is biological for to have relatively little genomic bacterium, is 4400kbp length for intestinal bacteria for example, and is 2500-3500kbp for archeobacteria.Calculate the number (Clarke etc., 1976, cell 9:91-99) of the independent cloning that the library, requires to reach 99% the full sequence that may contain the donor gene group from following formula: $N=\frac{\ln (1-p)}{\ln (1-f)}$ Wherein

The number of essential recombinant clone in the N=library

The possibility that the p=sequence reappears

F=is genomic per-cent in single recombinant clone

For example intestinal bacteria have the DNA of about 4400kbp; Cosmid vector can be packed the DNA of about 40kbp.Calculate according to these, colibacillary full gene group is expected to reach lacking in cosmid library among 504 clones all to be represented.Because common DNA library can comprise 500,000 recombinant clones independently, so each such library can be represented the genome that is similar to the bacterial species of bacillus coli gene group up to 1,000 different having effectively.Therefore, comprise 1,000 or the gene expression library of the different genetic material of more kinds of bacteriums, can produce and evaluate considerable Chemical Diversity effectively by screening.

After the method for in common document, describing, Maniatis etc. 1989 for example, molecular cloning, the 2nd edition, Cold Spring harbor Press, New York; With Ausubel etc., the modern molecular biology method, Greene Publishing Associates and WileyInterscience, New York can then be used to make up the conventional molecular biology reaction in recombinant gene expression library.

Clone's step in recombinant natural approach gene expression library is shown among Fig. 3.Any cell from the donor biology all can be used as the nucleic acid source that is used to make up gene expression library.Can use genomic dna, it comprises chromosomal DNA and extrachromosomal genetic element, for example the plasmid of natural generation.On the other hand, can use the RNA of donor biology.Preferably extract purifying messenger RNA(mRNA) (mRNA) and be converted into complementary DNA (cDNA) by any method known in the art.It is synthetic that oligomerization (dT) primer and arbitrary sequence primer can be used for causing the first chain of cDNA.Optionally insert fragment by polymerase chain reaction (PCR) DNA amplification.

The method that can partly provide by 5.1.2 or by the known method extraction of prior art and purified genomic dna and RNA.For filamentous fungus and bacterium, these methods can comprise and comprise following method a), b) any or in several different methods c), a) the quick high salt lysis of SDS/ protoplastis and use the equilibrated phenol extraction immediately wherein, wherein the immature mycelium of protoplastis from be grown in liquid nutrient medium prepares; B) and guanidine thiocyanate in carry out the quick lysis of protoplastis, then super centrifugal in the Cscl gradient; Or c) the protoplastis separation of high molecular weight DNA from the agarose filler, preparing, the horizontal pulse electric field gel electrophoresis of going forward side by side.For bacterium, the another kind of molten born of the same parents' method by N,O-Diacetylmuramidase/stain remover is incubated with nonspecific protease, may be useful with a series of phenol/chloroform/primary isoamyl alcohol extraction then.

For optimum, preferred big any prokaryotic gene group dna fragmentation comes bigger possibility ground to contain whole operons or its essential part.Can use various Restriction Enzymes to cut genomic dna at the specific site enzyme.By partly being digested with common cutting restriction enzyme, genomic dna produces big dna fragmentation (greater than 20kbp) arbitrarily.The amount of required genomic dna changes according to the genomic complicacy of using.On the other hand, DNA can be sheared by physical property ground, for example by pore pin or supersound process.

Before inserting empty expression vector, can separate these DNA by standard method according to size and insert fragment, these methods including, but not limited to, agarose gel electrophoresis, dynamic density gradient centrifugation and column chromatography.The linear saccharose gradient of preferred 10-40%.By being connected in the expression vector with complementary sticky end, dna fragmentation can finish insertion.Be used for the carrier DNA of ligation and DNA and insert segmental amount and depend on their relative size, and can be determined by experiment by method well known in the prior art.Yet, be not present in expression vector if be used for the complementary restriction site of dna breakage, but the end of enzymatically modifying dna molecular for example produces flat terminal.In addition, can pass through nucleotide sequence, promptly joint or linker are connected to and produce required any site on the DNA end; These connected joints or linker can comprise the oligonucleotide of the specificity chemosynthesis of the restriction endonuclease recognition sequence of encoding.In another approach, can insert fragment by expression vector and the DNA that homopolymeric tailing comes modifying enzyme to cut.

After in carrier DNA being connected to DNA insertion fragment, expression constructs is imported host living beings, can adopt several different methods, comprise, but be not limited to conversion, transfection is infected, and engages, protoplastis merges, liposome-mediated transfer, electroporation, microinjection and micropellet bombardment method.In specific embodiment,, then allow these particles infect Bacillus coli cells phage or cosmid DNA are imported escherichia coli host by DNA being packaged in the phage particle external.Also can utilize the DNA transfer mechanism between the microorganism of other natural generation, for example bacterium engages.

Contain the host cell formation library of expression constructs in collection after, can and/or duplicate them by the known method amplification of prior art.The purpose of amplification provides the library that can be used repeatedly.By the picking library, make bacterial growth, and collect phage or bacterium and be used for storing and finish amplification.

In addition, the order that can stipulate is stored the library.The dull and stereotyped cultivation so that its formations is single can be chosen in the library of bulk when low density, independently plaque or bacterium colony, then with each plaque or colony lift in each hole of the porous mainboard of encoding, for example 96-hole flat board or 384-hole flat board.Allow each flora in the hole, grow under optimum conditions.The mainboard of coding can be used as the database that each clone is copied to respectively in one or more experiment flat boards and originate.Therefore, can each clone in the library be handled respectively and analyze.The dull and stereotyped bag in coded data storehouse also can be preserved to be used for purposes in the future.Available spininess replicator or be used for the hyperchannel device of fluid handling.Preferably the chamber auto navigator carries out whole or most transfer and operation (Bentley etc. 1992, genome 12:534-541) by experiment.

Can be by lyophilize, or in refrigerator cryopreservation (10 ℃～-100 ℃) or preserve library of the present invention down at liquid nitrogen (176 ℃～-196 ℃).

According to the host-vector system that adopts, can identify and be chosen in the host living beings that contains donor dna in the library by several different methods.In one approach, according to existing or not existing the function of marker gene to identify and select these host living beings, thymidine kinase activity for example, antibiotics resistance, kantlex for example, penbritin, bleomycin or thiostrepton resistance, chromogenesis, for example melanocyte, and methotrexate resistance.On the other hand, can adopt specified host living beings phenotype or the metabolic variation of the formation of inclusion body in the formation of the convergence point in the tissue culture or the baculovirus by metabolic test.In case the situation that has donor dna is selected, available clone carries out a series of enzymatic analyses or metabolic test with further sign.

Insert fragment in order to characterize the donor dna that contains in donor dna or its a part of clone library, the clone of available representative group carries out micropreparation and the restriction analysis of DNA.The result will provide the finger printing of donor dna size and can be considered to the scope and the big or small restriction map of the insertion sheet segment DNA of library expectation.Characterize the library 5.1.5. make up chimeric approach

The invention still further relates to the structure and the purposes of the chimeric approach expression library of combination, wherein host living beings contains the genetic material from any concatermerization of one or more donor biologies, and can produce the functioning gene product of donor biology.The a large amount of host living beings in library can contain any and unique gene combination from one or more donor biologies.The coexpression that is transferred gene can be subjected to the effect of the regulatory region that their natural separately regulatory regions or external source provide.In host living beings, from the new chimeric pathways metabolism and the new compound of several genes product interaction generation of different donor biologies.The new activity of these chimeric approach and compound can become by conventional medicine discover method or method provided herein and be more suitable in screening.

Be not subjected in the chimeric approach gene expression of combination library, how to produce any theoretical constraint of new way or compound, impel the vivo gene product to interact from the functional heterogeneic coexpression of one or more donor biologies, and interact with the moiety of host living beings.Interact by these, produce the biochemical reaction of new kind, some of them can act on simultaneously and form chimeric biochemical route.Heterologous gene products can run into and not be present in their substrates in the donor biology separately, cofactor and produce the molecule of signal.These substrates, cofactor and the molecule that produces signal can be provided by host living beings, provide by other heterologous gene products of coexpression in identical host living beings, or from substratum.

And, some heterologous gene products can by structural modification and during the biosynthesizing of host living beings by differently compartmentization and localization.Some heterologous gene products can be exposed to and be different from their host cell environment of donor separately.

Can predict that some heterologous gene products also can and change the host cell environment to host living beings generation effect.Can influence, or be subjected to the host cell factor of environmental of the function influence of heterologous gene products to include, but are not limited to salt concn, trace element, nutrition, oxygen, metabolite, energy derive, redox state and pH.Some heterologous gene products also can interact with the host gene product, thereby can cause the change of host's pathways metabolism.

Depend on the combination of heterologous gene, not being present in the natural new chimeric biochemical route and the compound of new kind can form in the host living beings library.In the chimeric approach expression library of combination, the genetic resources of donor biology is bred and is amplified, thereby the number of chemical structure that can not find in each biology is provided.So the library of preparation can use ordinary method or method provided by the invention to screen.Therefore, new approach and compound are become and are more suitable in drug screening.

Any donor biology that is described among the 5.1.1. can be used to the chimeric approach expression library of preparation combination.Can screen the donor biology according to they known biological characteristicses, or they can be the mixture of known and/or unidentified biology.

The chimeric approach expression library of combination of the present invention can assemble according to the principle of describing among the joint 5.1.3.In order to make from the dna fragmentation of multiple donor biology concatermerization at random, can reelect the library assembly method by comprising following step: produce less genomic DNA fragment, with the adjusting sequence, for example promotor is connected with terminator with the formation box gene, and the concatermer box gene.

Insertion sheet segment DNA can be the cDNA from mRNA, and/or genomic DNA fragment.The simultaneously DNA or the RNA of the different types of donor biology of purifying, or separate them respectively and then mix with specified proportion.Any mixing that can insert DNA in any stage before being inserted into clone or expression vector.

Can use methylated Nucleotide in cDNA is synthetic, for example 5-methyl-dCTP to be providing the antienzyme protection that short enzyme is cut, and permission is inserted segmental directed cloning at the cDNA that with respect to promotor and terminator fragment is sense orientation.

Can partly digest any fragment of the genomic dna that is produced as the 2-7kbp scope as the restriction enzyme of Sau3AI by using cleavage site with relative high frequency rate.Monitor and confirm part digestion by the aliquots containig of sample being carried out agarose gel electrophoresis.

The external source regulatory region can be provided, and for example composing type or inducible promoters and terminator cause the expression of gene that is transferred.When the host is incompatible with the donor expression system, must provide these to regulate series.PCR can be used to produce for the particular expression host to specific, and has defined the various promotors and the terminator fragment of restriction site at their end.Can adopt regulatory region is inserted any method that fragment is connected with DNA.Can adopt with the Nucleotide of Klenow fragment and part group and handle, promptly filling produces specificity only and is connected to promotor with compatible end and the insertion dna fragmentation on the terminator.

The invention provides a kind of relating to comprises the application method of inverted orientation at the box gene of the promotor of two copies of unique each side of restriction site, and any DNA that is inserted into this restriction site will transcribe on two chains by two promotors from both sides respectively.

The present invention also provides another kind to relate to comprise and is positioned at the application method that DNA inserts the box gene of the promotor of each side of fragment and terminator.If carry out the method for cDNA directed cloning, cDNA inserts segmental 5 ' end and 3 ' terminal restriction site that has 3 of promoter fragment ' end and the segmental 5 ' end of terminator that will have unique coupling respectively.

The genomic DNA fragment or the cDNA that have compatible restriction site at two ends are connected on the promotor, and in some cases, are connected on the terminator fragment, have the box gene that mean size is 1-10kbp thereby form.

By be similar to the concatermer that the method assembling of being adopted comprises a plurality of transcription units in peptide is synthetic.At an end subgroup in box gene storehouse is attached to solid phase, for example on the magnetic bead.Carrying out other end of wandering about as a refugee several times, successive " goes to protect " circulation to be connected with a series of transcriptional units residue storehouses.Each add circulation after, solid makes and separates in the never connected dna fragmentation of concatermer.After finishing concatermerization, by discharging concatermer with restriction enzyme insulation, intron nuclease for example, its enzyme are cut near the solid phase unique very rare site to reduce the possibility that enzyme is cut the DNA of concatermerization.Then the DNA of concatermerization can be inserted in the cloning vector with formation and be imported into expression constructs in the suitable host living beings; On the other hand, before importing host living beings, construction can be transformed into the intestinal bacteria recA minus strand bacterial strain that is used for increasing.

About promotor and terminator segmental synthetic, the preparation of box gene, the details that is connected that DNA inserts segmental assembling and inserts fragment and carrier is provided in the 5.4th and 5.5.

In case assembled the chimeric approach expression library of combination, then it can be stored with the same way as of describing among the joint 5.1.3 basically, and prescreen and screening are duplicated in amplification.5.1.6. bias combination expression library

In another specific embodiments of the present invention, the preselected dna fragmentation that can collect from one or more donor biologies together prepares the natural or chimeric approach expression library of bias combination.Substitute and only use biological gene DNA or the cDNA that all collects of donor, this method will reduce the percentage ratio that needs screened clone's number and increase the clone that will produce compound of interest.Preselected dna fragmentation comprises the gene of encoding part or all biological route of synthesis, and can be by will be from may be with to produce compound of interest a plurality of probes relevant or the known preparation of participating next preselected with the hybridization of initial DNA library.Initial DNA library, preferred cosmid library and not necessarily must be expression library can comprise the DNA from one or more donor biologies.For further prescreen,, the DNA in the positive colony can be transferred to the host who is used for preparing so and also express therein, for example intestinal bacteria or shallow Streptomyces glaucoviolaceus if initial library is an expression library.But prescreen is more than one initial library, and can collect from the DNA of all positive colonies, and is used to prepare bias combination gene expression library.

The initial library of can increasing, like this can be in multiple host living beings the DNA of prescreen donor biology.For example,, it can be imported and be used to the specific host living beings of expressing and screening, for example produce the S.rimosis of oxytetracycline, or produce the S.parlus of actinomycin in case produced the gene expression library in the streptomycete.If expression vector comprises the suitable sequence that is used for transgenosis in the plasmid of natural generation, these sequences can be used to by means of conjugal transfer the library be moved among the various compatible hosts.

The probe that is used for prescreen can be from any clone's biosynthetic pathway, polyketide biosynthesizing site for example, because these are biosynthesizing sites of preferably discerning, and it is conservative to have considerable sequence between in known bunch, actI (biosynthesizing-Malpartida of actinorubin etc. for example, 1987 natural 325:818-820), WhiE (biosynthesizing-the Blanco of spore pigment etc., 1993 gene 130:107-16) and eryAl (Donadio etc., 1991 science 252,675-679).Similarly principle can be used for other microbiotic or secondary metabolism biosynthesizing site.The peptide synthetase gene of cloning in for example low GC gram-positive microorganism, subtilis (Stachelhaus etc. for example, 1995 science 269:69-72) and high GC gram-positive microorganism, for example produce thiostrepton, virginiamycin, the actinomycetes kind of valinomycin and actinomycin can have and be similar to as probe to discern enough sequences in the new biosynthesizing site in two groups of bacteriums.Other clone's biosynthetic pathway, for example peptide synthetase and aminoglycoside synthetic enzyme also can be provided for the probe in the initial library of prescreen.

On the other hand, can screen initial DNA library by the probe that comes own coding to participate in the protein DNA of secondary metabolism.Can prepare these probes by the DNA that from all DNA, removes noncoding DNA and coding and elementary metabolism biological route of synthesis proteins associated matter.Remaining DNA is partial to thus to encode and participates in the proteinic coding region of secondary metabolism.The details of removing step is provided among the joint 5.3.5.5.2. screening combination expression library

Drug discovery of the present invention system further comprises the novel method of screening combination expression library.In the standard method of screening expression library, when for example the combination of antibodies and part also can be used with expression library of the present invention, the library can be adapted to expressed with identification the report mode of the host living beings of required approach and meta-bolites by tailing.

The claimed method of this paper can carry out minimum processing with the clone that allows to have in the library productivity effectively and the detection of high throughput carry out the management of a large amount of samples when separating.But the activity of the wide scope in prescreen library, a compounds of generation or the existence of associated dna sequence.In vivo with analyzed in vitro in, the library also can directly be used with target.Discerned or the isolated cells group can easily cultivate, carried out the quantity amplification, and carried out producing the further analysis of new compound.Coding causes producing the gene of the pathways metabolism of new activity or compound can be described by the genetic material that sign is imported among the separated clone.The information of related gene and approach and clone will be convenient to medicine optimization and production greatly

Adopt as this paper, term " library clone " or " library cell " refer to contain host cell or the biology at least one possibility coding donor pathways metabolism or the segmental combination gene expression library of its a part of donor dna.Term " positive colony " or " positive cell " refer to produce according to coverage the library clone or the cell of signal, term " productive cell " or " productive clone " refer to be different from the library remaining " cell that does not have productivity ", produce the host cell or the biology of activity interested or compound in the library.

Term " prescreen " refers to show the general biology or the biochemical analysis of the existence of interested activity or compound or gene.Term " screening " pointer is to specific disease or clinical disease and to adopt the specific of target be the biology or the biochemical analysis of purpose with the treatment.Term " target " is often referred to whole cell and macromole, enzyme for example, and test compound is exposed to wherein in screening.The application of prescreen and screening generally includes color and refers to detect or facies analysis automatically with the naked eyes of device, the fluoroscopic examination (FACS) by the fluorescent activation cell sorting or use the magnetic cell sorting system (MACS) that carries out when having coverage in the cell mass of library.

The inventive method provides generation another kind of but that do not repel mutually to be used to detect method with the detectable signal relevant with productive cell that separates these cells of interest purposes.Reporter can be the molecule that impels direct or indirect generation detectable signal.For example, reporter can be the light emission molecule, the cell surface molecule of other composition specific recognition of the scope that maybe can be in the news.Coverage comprises reporter and impels and support the mixture of reporter generation signal that coverage can comprise indicator cells alive or its part.The composition of coverage can be incorporated in the host living beings in library, or they can be wrapped quilt with single or library cell bank, the independently unit that is formed for screening altogether in the semisolid medium of permeability.

Detection for the compound of interest that is easy to describe hereinafter, can be with absorbent material, natural resin for example joins (Lam etc. in the culture of library cell as Diaion HP 20 or Amberlite XAD-8 resin, 1995, industrial microorganism magazine 15:453456).Because many secondary metabolites are hydrophobic molecule, the release of these metabolites or secretion may cause producing precipitation in outside.The inclusion of these resins in the substratum causes taking place sequestration in resin, this resin can be removed from substratum to be used for wash-out and screening.

In one embodiment of the invention, host living beings is operated and comprises chemistry and reply construction, and this chemistry is replied construction and comprised coding operability ground and the gene of the compound of the required type that will screen or chemistry that the metabolite generation is replied being replied the relevant reporter molecule of promotor in expression library.When having required activity or compound, the chemistry in the positive colony is replied promotor transcribing by the reporter gene of inducing the startup operability to link.Positive cell is discerned by the detectable signal that the expression owing to reporter gene produces.

In another embodiment, can use in each cell, owing to have required activity or compound, the physiological probe that produces signal as physiological being changed generation reply.This probe can be the precursor of reporter molecule, and this precursor is by directly or indirectly being converted into reporter molecule by activity or compound in the biochemical route of seeking.With productive cells contacting the time, physiological probe or report precursor produce and impel identification and/or the isolating detectable signal that the productivity cell is arranged.Contact can be finished by directly probe or precursor being joined in the cell of library.On the other hand, contact can be applied and during screening probe or diffusion of precursor be finished in the cell of library by bag.

In another embodiment of the invention, indicator cells can be used to report the generation of required activity or compound, thereby can discern and/or separate the productive cell in the library.Can with all live or the fixed indicator cells, or some part of its cell is mixed with each library cell or library cell bank or is wrapped quilt altogether.Select indicator cells according to the biological property that they are replied the existence generation of required activity or compound.Indicator cells can be the target cell of required compound.On the other hand, indicator cells can be used to produce detectable signal with reporter.

Characterize host living beings comprehensively, after the possible donor of sign comprehensively biology, selecting the prescreen and the screening in each library.Wherein the positive analysis of host living beings is underproof, and wherein the biological positive analysis of donor is considered to acceptable library prescreen or screening.The substrate that is preferably the target of the enzyme relevant with required biosynthesis ability can be used as and show and exist DNA to transcribe and translate active another incoherent target (for example amylase, beta-galactosidase enzymes) in specific cloning.

In another embodiment of the invention, antibiotics resistance can be used as the indicator that generation maybe may produce secondary metabolites interested.When library clone was placed in one group of microbiotic, antibiotics resistance can show and have self-protective mechanism, efflux pump for example, and its is usually as to being found near the secondary metabolites biosynthetic pathway from the toxic protection of body.These are cloned in when detecting may not show the generation secondary metabolites, but has the possibility that contains the adjacent biosynthetic pathway that can further be operated or detect as required of increase.

The present invention also provides and is substituted in culturing bacterium in the culture dish and in the method for the effective high throughput of the bag quilt of limited space culturing cell.The viable cell of flat type is the intensive form of laboratory and entity, and the cell of bag quilt is easily grown in the liquid medium within, and its advantage is that the various cells that separate are brought together, and is easy to detect interested secondary metabolites therefrom.Another advantage of bag quilt is can independently carry out prescreen or screening in the unit like this with composition and/or other indicator cells of library cell envelope coverage.The bag of cell be applied can be easily by using material, as agarose, the heat of alginic acid or carrageenin or ionic gel turn into and are used for carrying out.

FACS be a kind of method of knowing according to particulate fluorescent characteristic separating particles (1-130 μ m size) (Kamarch, 1987, Enzymology method, 151:150-165).The principle of work of FACS is the laser excitation of the fluorescence part in each particle.Positive fluorescence causes a little charge is passed to particle.This variation makes can carry out electromagnetic separation with the positive and negative particle from mixture.Separated particle can directly be run up in each hole in 96 holes or the 384 hole flat boards.

MACS is a kind of method of knowing (Dynal, 1995) according to the binding ability separating particles agent of they and magnetic microsphere body (0.5-100 μ m diameter).Can carry out multiple useful change to the magnetic microsphere body, comprise that covalency adds specific recognition cell-surface antigens or haptenic antibody.On the other hand, for the magnetization of coated cell, coverage can be mixed in the host cell that produces magnetic report protein such as ferritin.In this case, produce the effect of the coated cell magnetize microsphere of positive signal.The microsphere of selecting can be exposed in the magnetic field and carry out physical treatment.For example, can the microsphere of selecting be carried out sequester by the outside that magnet is placed on reaction vessel.5.2.1. report construction

According to the present invention, the host living beings in the library can be comprised by transformation and contain the operability ground chemistry relevant with reporter gene and reply the chemistry of promotor and reply the report construction.Host living beings and/or construction can comprise other coding and participate in regulating the proteinic gene of assisting a ruler in governing a country of replying transcribing of promotor or signal generation from chemistry.

It is any double chain DNA sequence that chemistry is replied promotor, and it can be in conjunction with RNA polymerase, and only starts or regulate transcribing of reporter gene that operability links when the compound of active or some kind that has some kind.Preferably, when not having induced activity or compound, chemistry replys that promotor does not have or only have low-level composing type background transcriptional activity in host living beings.Also can use by reducing or reducing transcriptional activity the passive chemistry of replying of existence generation of activity or compound is replied promotor.

Being used for promotor of the present invention can include, but are not limited to, and is used for pathways metabolism, biodegradation pathway, the promotor of cytopigment and stress reaction (Orser etc., 1995, external toxicology 8:71-85), for example heat shock protein.For example, can adopt between pseudomonas TOL plasmid the positive regulon XylS albumen (Ramos etc. that a series of benzoates and halogen or alkylaryl compounds are induced and regulated that pass through of the Pm promotor of position lytic pathway and it, 1988, FEBS Letters226:241-246; De Lorenzo etc., 1993 gene 130:41-46; Ramos etc., 1986, the periodical 83:8467 8471 of institute of NAS; Mermod etc., 1986, bacteriology magazine 167:447-454).The example that other nonrestrictive chemistry is replied promotor is the promotor (Metcalf etc., 1993, bacteriology magazine 175:3430 3442) relevant with phosphatic utilization, to the promotor (Rothmel, 1990) of suitable-suitable muconic acid sensitivity; Promotor (Cohen etc., 1993, bacteriology magazine, 175:7856-7862 to microbiotic and Whitfield's ointment sensitivity; Cohen etc., 1993, the bacteriology magazine, 175:1484-1492), from the promotor of arsenate with from the cadmium operon (Corbisier etc., 1993, FEBS Letters 110:231-238) of streptococcus aureus; SfiA (Quillardet etc., 1982, the periodical 79:5971-5975 of institute of NAS), and zwf (Orser etc., 1995, above).

The reporter gene coding can directly or indirectly produce the reporter molecule of detectable signal.This comprises reporter molecule and any luminous reporter that produces color or produce magnetic, the biological example shiner, can use chemoluminescence or fluorescence protein, it comprises, but be not limited to green fluorescent protein (the GFP) (Chalfie etc. of Victoriaaequona, 1994, science 263:802-805), have the improved GFP (Heim etc. that strengthen fluorescence, 1995, natural 373:663-4), the luciferase of Vibrio harveyi (LuxAB gene product) (Karp, 1989, biological chemistry biophysics progress 1007:84 90; Stewart etc., 1992, the General Biology magazine, 138:1289-1300) with from the fluorescence worm, the luciferase of photinus pyralis (De wet etc., 1987, molecular cytobiology 7:725-737).Can use any generation fluorescence or colorific enzyme, it is including, but not limited to beta-galactosidase enzymes (LacZ, Nolan etc., 1988, the periodical 85:2603-2607 of institute of NAS); And alkaline phosphatase.Can use any cell-surface antigens, for example, escherichia coli thioredoxin-flagellin fusion rotein, be colibacillary Trx (trxA gene) (Lu etc. with the fusion protein form expression that forms with the flagellin (fliC gene) on coli flagellum surface, 1995, biology/technology 13:366-372).

The example that chemistry provided herein is replied the report construction is pERD-20-GFP, it comprises Pm promotor and the XylS gene (Ramos etc. of pseudomonas, 1988, FEBS Letter226:241-2476), they produce the benzoate of some type and reply, thereby cause that reporter GFP's transcribing and translating (expression) (see figure 6).

Different promoter sequences can link to each other by the PCR generation and with the coding region of GFP or flagellin-Trx reporter.The DNA purification process that can use standard comprises the genome and the plasmid DNA of interested promotor from relevant kind purifying, and resuspending is in TE.Can corresponding to 5 of the promoter region of other sequence with restriction site ' and 3 ' border synthetic primer with the facilitation subclone.At the template and the primer that are defined as for selecting is under the acceptable terms, carries out amplified reaction in thermo cycler.By the agarose gel electrophoresis reaction product isolated, and the promotor series of using TA clone's box (Invitrogen, La Jolla) to carry out the subclone amplification can be cloned into the GFP of cloning vector of common purpose herein or 5 ' end of flagellin-Trx again.5.2.2. physiological probe and report precursor

The physiological probe that this paper adopted is fluorescence or colorific reagent, and they are in contact or when entering, and produces the signal that the variation of the physiological of library cell or indicator cells and/or metabolizing parameters is replied.

Probe can be the enzyme substrates that links to each other with fluorescent reagent.For example, the fluorescence alkyl oxide can be incubated with these cells.If cell produces many aromatic hydrocarbon, but this hydro carbons induced microparticle body dealkylase, and the enzyme of this enzyme own is cut the fluorescence alkyl oxide, produces fluorescence-causing substance.

Fluorescent probe can be selected to detect the variation of following physiology and metabolizing parameters, for example, but be not limited to those that describe in (bioid academic year are commented 219:169-81) such as Shechter etc. (1982, FEBS Letters 139:121-124) and Bronstein.

Active specific examples (type of compounds) membrane potential of reason bacterial strain/substrate utilization reduces pressure, damage BacLight bacterial strain

It is (lipophilic that (Virahol) (semi-permeable nucleic acid staining) internal pH physiological changes BCECF-AM

The acetoxyl methyl esters of fluorophenol) cytopigment mediations passes through prescreen and the screening of inducing tonka bean camphor (fluorescence alkyl oxide) 5.2.3. library of many aromatic hydrocarbon (naphthalene) to the MC dealkylase of increase of 7-oxyethyl group-heptadecyl oxygenizement

Combination gene expression library of the present invention can come prescreen or screening by several different methods, these methods comprise, but be not limited to, visual inspection, automated imaging is analyzed, (Tyaei etc. 1996, Nature Biotechnol, 14:303-308) fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS) with the molecular marker DNA probe hybridization.Screening can be carried out in the large volume culture in library that do not increase or increase.

In specific embodiment of the present invention, during prescreen or screening, each library cell or cell bank are coated on inertia with droplet form, in the stable and porous semisolid matrix.Semisolid matrix is to gas, and liquid and macromole are permeable, and allows the cell growth and the division of bag quilt.The embodiment of suitable matrix can include, but are not limited to agarose, alginic acid and carrageenin.Can droplet form cultivate and detect the library cell of bag quilt, but and the maintenance visual inspection, can from droplet, reclaim cell like this and be used for further operating.Matrix can be chosen wantonly and be exposed in for example antibiotic material, and this material can select to comprise the library cell of alternative mark.Also droplet can be placed nutrition to support the growth of library cell.Following embodiment is used for illustrating and not limiting the present invention in any way.

Can wrap in many ways and be applied, according to the detection method that during afterwards prescreen or screening, adopts, preparation is thick drips (0.5-2.5mm's drips) or droplet (10-250 μ m's drips).In forming the process of dripping, can control the size and the composition that drip.Preferably, each slightly drips or droplet comprises 1 to 5 library cell.

For example, can drip by the preparation of following use sodiun alginate is thick: the applying pressure mixing tank is dissolved in sodiun alginate in the 100ml sterilized water with 1% concentration with 2000rpm.With the intestinal bacteria or the zymic library cell of 1 volume, for example grain wine fragmentation sugar yeast and sugar yeast class; Or the spore of strepto-mushroom, subtilis and filamentous fungus, for example aspergillus and Neurospora class join in the solution of sodium alginate, like this in every bag by 1-5 cell.Allow mixture leave standstill venting at least 30 minutes, then extrude by causing forming independently any device of drop.Wherein a kind of device is the syringe with 25 metering syringe needles.Form drop by solution of sodium alginate being added drop-wise in the beaker that stirs gently that the 135mM calcium chloride solution is housed.Allow droplet solidify 10 minutes, then transfer in the sterilization flask of having removed calcium chloride solution and having replaced with the suitable growth substratum.The library cell of bag quilt can be grown under standard conditions.

Can prepare droplet by any method or the equipment that produces droplet.For example, but be not limited to two-phase annular atomizer, the static droplet generator shakes orifice flow system and emulsifying effect.Prior art is known other and is prepared semi-solid method of dripping; Referring to routine Weaver, U.S. Patent number 4,399,219.

Following embodiment is to use emulsifying technology to prepare the example (Monshipouri etc. 1995, and little bag is by magazine 12:255-262) of droplet.Use pressure mixer, 0.6g sodium polyphosphate and 2% sodiun alginate are dissolved in the 100ml sterilized water, allow solution of sodium alginate exit 60 minutes as 2000rpm.By with 300ml oil, for example Histamine, monohydrochloride acid or sweet oil mix at least 30 minutes preparation oil phases with the soybean lecithin of 1.0g purifying.Prepared the slurry that in 10ml 50% ethylene glycol, comprises 1.9g calcium sulfate at least 15 minutes by supersound process.The library cell that will produce an every 1-5 cell that to starch immediately before importing oil phase with 1 volume is admixed in the alginic acid solution.Emulsion process started by the alginic acid mixture slowly being transferred in the oil phase and with the 580rpm mixing in 10 minutes.Then add the 500ml sterilized water and allow mix and continue 5 minutes.By the centrifugal droplet that from oil, separates.The washing droplet also is suspended in it in suitable growth substratum again, promptly can be used for the cultivation under the standard conditions if desired.Can measure the size of droplet by phase microscope.For sorting purposes,, can use the filter membrane of required size restriction to carry out the size selection to dripping if drop in outside the necessary required magnitude range of sorting by FACS or MACS.

According to the present invention, the composition of coverage or the target of drug screening also can be coated on and have in the dripping of library cell.Before forming foregoing thick or droplet, can or comprise bioassay with whole report cell, enzyme, or the cell of reporter molecule part and the library cytomixis that is suspended in the substratum.The compound of interest that the library cell produces can be built up in dripping and indicator cells or the reporter of diffusion to reach common bag quilt, and produces signal.The indicator cells that wraps quilt altogether can be the maneuvering target of required compound, for example anti-infective at pathogenic agent, or antineoplastic agent at tumour cell.Any variation in the metabolic condition of indicator cells, for example dead, or growth-inhibiting, formed signal and also can in dripping, detect by several different methods well known in the prior art.This method can include, but are not limited to use the physiological probe, for example vital staining, or the mensuration of the optical characteristics of dripping.

In the time of in dripping the composition that is exposed to coverage, can diffuse through semisolid medium generation signal by the library cell of bag quilt and the metabolite and the compound of report composition generation, for example, the physiological probe can be joined among a collection of of sorting method that then suits.If the library cell is allowed to separate in dripping, the offspring of initial positive cell is held togather in small colonies, thereby produces a stronger signal.Preferred semisolid medium is an optical compatible with the signal that is produced by reporter, only transparent to the wavelength of certain limit for example, and signal can be detected effectively like this.

Thick dripping can allow to drip by the screening point by eyes or use, and any equipment with the positive thing ability of separation, uses the reporter of adding lustre to carry out sorting.Can use FACS or MACS to come the sorting droplet.The FACS operation can be carried out on any suitable machine (for example Becton-Dickinson TACStar Plus) by qualified operator.Particle suspension density (cell or drip) is adjusted to 1 * 10 ⁶Grain/ml.In all cases, positive thing can directly be sorted in the porous flat plate in 1 clone/hole.Use MPC-M magnetic test-tube stand to carry out MACS (Dynal, 5 Delaware Drive, Lake Success, New York 11042) according to the explanation of manufacturers.

Being found to be the male coated cell in prescreen or screening can reclaim by dripping to be placed on to cultivate maybe will drip to be dissolved in the Trisodium Citrate on suitable agar or the liquid growth medium.After cultivating for some time, positive cell can grow out from drip.In order to drip the convenience of handling and storing, the cultivation of back can be carried out in porous flat plate.

Be reduced to can be then than the positive thing of the prescreen of microcommunity exist under the situation of ethylene glycol freezing and store or in porous flat plate, grow.These can be used to use the spininess replicator genome to be transferred in various types of analysis flat boards (division culture medium for example, selective medium, the antimicrobial or analysis lawn transformed).In these microtitration flat boards, also can carry out specific analysis, and the dull and stereotyped reader by standard or any other method of being adopted in the high throughput triage techniques are at present read.

For the clearness of discussing, following sub-chapters and sections have described in detail and have related to prokaryotic organism and eukaryote, the of the present invention different example of donor and host living beings.Following example is not for limiting to the present invention for example.5.3. from the biological method for preparing high-quality nucleic acid of donor

Is very important as the high-quality DNA of initial substance or the availability of RNA in the structure in DNA library, and this DNA has represented the genetic information of donor biology.This section provides from the culture of donor biology or has extracted from environmental sample, selects and prepare the method for high-quality nucleic acid.The preparation method who is used for the dna probe that is removed in prescreen DNA library in order to concentrate the DNA relevant with secondary metabolites has also been described.5.3.1. guanidinium isothiocyanate separate nucleic acid

Can be by the mechanical mill device, or in addition by mortar and pestle existence grind very carefully glass or float stone with hand with lyophilize or not cryodesiccated material fragmentation.The cryodesiccated material that grinds with every 1-2 gram immediately after the grinding mixes corresponding to the amount of the molten born of the same parents' damping fluid of 10ml.Molten born of the same parents' damping fluid is the 5M guanidinium isothiocyanate, 50mM Hepes pH 7.6,10mM EDTA and 5% beta-mercaptoethanol (or 250mM DTT).Mix being incorporated in 50 ℃ down insulation before centrifugal, solution is poured in 4% sarkosine after 5 minutes with 8000g, mix, and 50 ℃ of insulations more than 5 minutes.If supernatant is visible as muddiness, can use rotating speed is 27, and 90 minutes centrifugation step of 000g do not need carbohydrate with sedimentation.In addition, can adopt 15, the rotation of 000g is to remove the lysate that does not need pollutent.After centrifugal, pour into supernatant among the 1.42M CsCl (0.15gCsCl/ml) and previously prepared 5.7M CsCl/TE (10mMTris-HCl/1mM EDTA) the solution higher slice in the ultracentrifugation pipe.20 ℃, can 160,000g carried out ultracentrifugation 18 hours.Behind the ultracentrifugation, the limpid layer as colloid on 1.42M/5.7M CsCl interface is DNA, and whole cell RNAs is present in the bottom of pipe with limpid pellet form simultaneously.DNA from the ultracentrifugation step can be dialysed to the TE damping fluid, change 0.1M NaCl into and dialyse, with 2.5 volume ethanol precipitation, drying is laid equal stress on new soln in the TE of appropriate volume.If the DNA layer be white, it can be shifted out and in CsCl/ biphenyl imide gradient centrifugal again 8 hours to remove residual carbohydrate.Can be by with 85% washed with isopropyl alcohol 2-5 time removal dyestuff, and as top, dialyse and handle.

RNA can be dissolved in again again in the damping fluid that suspends (5M, guanidinium isothiocyanate, 50mM Hepes, pH 7.6,10mM EDTA), with 50mM Hepes pH 7.6,10mM EDTA solution dilution is to the 1.33M guanidinium isothiocyanate.Whole if desired RNA then add the RNA sample that 2 volume ethanol or 1 volume isopropanol precipitating dilute.With sedimentary RNA ethanol rinsing, drying also is suspended in the water or in the methane amide again, and-70 ℃ of storages are until use.5.3.2. contain the separation of polyadenylic acid RNA

Because most eukaryote mRNA molecules contain poly-(adenosine) acid of a slice at 3 ' end, length is 250 bases nearly, and it can carry out purifying by the affinity chromatography of using oligomerization-dT cellulose matrix.Oligomerization-dT the matrix that can use a large amount of commerce to buy, including, but not limited to simple gravity post, paramagnetic particle, spin and propelling post.Isolating mRNA can be dissolved in water or the methane amide or-70 ℃ of following exsiccant forms and store.5.3.3. enrichment from the non-ribosomal sequence of total RNA

The enrichment of non-ribosomal sequence may be an important step the useful RNA quantity of the biological acquisition of the donor that maybe can not cultivate from difficulty.On the neutral saccharose gradient fractional separation of RNA can be used for purifying from other RNA kind, be purified into remarkable ribosome-RNA(rRNA) (R.McGookin1984, molecular biology method Vol.2 nucleic acid Humana Press, pp.109-112).After centrifugal, abandon comprising the sample of maximum ribosome-RNA(rRNA), dialysis and the remaining part of precipitation.

Utilization has or does not have other method of arbitrary primer of widow-dT primer of any tailing and PCR can be used to increase a spot of RNA in the raw material.5.3.4. use the segmental filling-in of Klenow

Use e. coli dna polymerase, or other lacks the Klenow fragment of the archaeal dna polymerase of 3 ' → 5 ' exonuclease activity, it is the standard method that is commonly used to produce the flush end dna molecular after digestion that Nucleotide is added to 5 ' cohesive end, when being used when not having perfect kernel thuja acid group, this activity can be used to produce own incompatible with them but compatible being connected in the end mutually.

This technology has been used to prepare gene library and construction (Hyug etc., 1984, the nucleic acids research 12:1863-1874 that height is tired; Zaborovsky etc. 1986, gene 42:119; Foster, 1991, Ph D dissertation, the University of California, Santa Barbara, Loftus etc. 1992, biotechnology 12:172-175).

Available Klenow damping fluid (50mM Tris-HCl pH 7.5,10mM MgCl ₂, 50mg/ml BSA, 1mM dNTP), enzyme, and carried out filling-in in 3-4 hour 37 ℃ of insulations.After the reaction, can pass through the several different methods purify DNA, including, but not limited to, affinity chromatography, ethanol sedimentation and column spinner precipitation.5.3.5. be used for the preparation method of the dna probe that is removed of prescreen

Can be from having the not young of complicated life cycle, the biological culture thing isolation of RNA of middle logarithmic phase less than what break up.This RNA storehouse with participate in not differential growth and elementary metabolic gene complementation.RNA is carried out biotin labeling and excessively shears arbitrarily with the quilt from the genomic dna of homology or closely-related allos kind external, and hybridize by the fragment of the big minispread of gene, cause having removed at interface and elementary metabolism RNA complementary genome sequence with this mixture of phenol extraction, this process can be repeated once.The single stranded DNA fragment that obtains is made up of with (+) and (-) chain that comprises other gene relevant with secondary metabolism (+) chain of elementary metabolic gene.Make this DNA mixture sex change, and hybridize 5-10 half CotS again under highly rigorous condition, so only Xiang Guan sequence can be hybridized again to form double-stranded DNA.Can be by combining with hydroxyapatite or by removing remaining single stranded DNA with the mung bean nuclease enzymic digestion.Can then adopt any initiation mark to represent the isolating double-stranded DNA of non-elementary metabolism related gene, and come the prescreen library used as probe.5.3.6. purification of nucleic acid from soil or other mixed environment sample

With pedotheque quick freezing and being stored under-70 ℃ in liquid nitrogen until use.In addition, pedotheque is frozen and is stored under-20 ℃.Immediately sample is being melted on ice before the use, or before operation, be frozen drying.

Depend on the physical properties and the source of material, extract whole nucleic acid by several different methods with minor modifications.Directly lyophilize and processing are dried to semiarid sample; Very Shi sample is carried out quick freezing and lyophilize; Before operation, the oily sample is diluted with phosphate buffered saline buffer.Can adopt following any method: Ogram etc. 1987, micro-biological process magazine 7:57-66; Steffan etc. 1988, practical environmental microbiology, 54:137-161; Werner etc. 1992, bacteriology magazine 174 (15): 5072-5078; Zhou etc. 1996, practical environmental microbiology 62 (2): 316-322.

In brief, the molten born of the same parents' damping fluid of guanidinium isothiocyanate (see joint 5.3.1) by dropping to heat is directly with the molten born of the same parents of 5g sample, and carries out the antimony chloride purifying.In addition, in the 50ml centrifuge tube with sample and 13.5ml DNA extraction damping fluid (100mM Tris-HCl pH 8.0,100mMEDTA, the 100mM sodium phosphate, 1.5mM NaCl, 1%CTAB (bromination hexadecyl first ammonium) and 100 μ l 20mg/ml Proteinase Ks mix, and under 37 ℃, vibrate 30 minutes with the velocity level of 225rpm.After the vibration, add 15ml 20%SDS, 65 ℃ were carried out a tail-tail vibration down in every 15-20 minute with sample insulation 2 hours.Under 20 ℃, by collecting supernatant in centrifugal 10 minutes with 6000 * g.Extract damping fluid and 0.5ml 20%SDS by adding 4.5ml, vortex vibration 1 minute is then 65 ℃ of insulations 10 minutes and centrifugal again pellet is extracted 3 times again down.To extract twice with chloroform-primary isoamyl alcohol (48: 1) from the supernatant that 3 leaching process compile.By adding the Virahol of 0.6 volume, then be incubated 1 hour, and at room temperature came precipitate nucleic acids in centrifugal 20 minutes with 16,000 * g.The nucleic acid pellet that then will slightly carry is suspended in 10nM Tris-HCl pH 8.0 again, among the 2mM EDTA.By the EDAE chromatography DNA is further purified if desired.Come from the pellet of slightly carrying, to obtain whole RNA (Ausubel etc. 1990, Greene Publishing Associates and WileyInterscieuce, New York by RNA being carried out selective precipitation with 4M lithium acetate or acid phenol extraction; Hoben etc. 1988, practical environmental microbiology, 54:703-71).5.3.7.DNA reparation

By at first making any cut terminal flush endization repair the DNA (New England Biolabs) that band is incised or is degraded with the T4 archaeal dna polymerase.Under 37 ℃, with DNA at flush end damping fluid (50mM Tris-HCl pH 7.8,10mM MgCl ₂, 40 μ MdNTPs, 5U/10 μ g T4 archaeal dna polymerase) and middle the processing 1-2 hour.3M sodium acetate by adding 1/10 volume and 100% ethanol of 2.5 volumes carry out ethanol sedimentation to DNA.

Precipitation and suspend in water again after, under 16 ℃, with the DNA sample at intestinal bacteria ligase enzyme damping fluid (50mM Tris-HCl pH 7.8,10mM MgCl ₂, 10mMDTT, 26 μ M NAD+ and 25 μ M BSA, 10U intestinal bacteria) in handled 1-2 hour with e. coli dna ligase.After the processing, with DNA sample 20mM Tris-HCl pH 8.0,5 times of 0.3M sodium acetate solution dilutions, and use phenol and chloroform extraction respectively once.Add 2.5 volume ethanol deposit D NA to aqueous phase.With sample rinsing twice, be suspended in sterilized water or 10mM Tris-HCl with 70% ethanol again, pH 8.0, among the 1mM EDTA, and-70 ℃ of storage extremely uses certainly down.5.4. be used for the method in prokaryotic expression library

This section provides uses the side of prokaryotic hosts and biological natural approach expression library of preparation of donor and chimeric approach expression library to go.The high-quality DNA of the purifying that the method for describing by joint 5.3.1-5.3.4 obtains can be used in the following method.5.4.1. bacterial species, bacterial strain and culture condition

Good especially expressive host biology is restricted-minus, endonuclease enzyme defect and recombination deficient mutant.For intestinal bacteria, preferred bacterial strain be XLl-MR (genotype: McrA-, McrCB-, McrF, Mrr, hsdr, cndal-, recA-).For streptomycete, preferred bacterial strain is shallow Streptomyces glaucoviolaceus TK64.For subtilis, preferred bacterial strain is subtilis PB 168 trpC2; Subtilis PB5002 sacA, degUhy; Subtilis PB 168 delta trpC2, pksdelta 75.8; Subtilis ATCC 39320 and 39374.

The donor biology is a bacterial species.Some are used to select their to produce the ability of the unique compounds that can detect by present analysis.Other then since they be present in may interested environmental sample in and selected.In some instances, marine bacteria obtains from Harbor Branch OceanographicInstitute and Scripps Institute of Oceanography.They usually from high sea offshore place greater than 200 miles collections.The test of metabolic test and gram and colonial morphology proceeded to guarantee that sample has the degree of classification difference.

When thing is stocked in the preparation library, allow intestinal bacteria 37 ℃ of growth and expression under 30 ℃ down.Allow the kind of marine actinomycete genus and streptomyces only grow down at 30 ℃.5.4.2. the preparation of donor gene group DNA

Cultivation is from the 10ml culture of the bacterium of each kind.Also be suspended in 10mM Tris, 5mm EDTA (TE) again by the centrifugation bacterium.But can perhaps the bacterial precipitation thing can be dissolved in the SDS/ Proteinase K by the method purify DNA of describing among the joint 5.1.2,, and use isopropanol precipitating with phenol-chloroform extraction.The purify DNA that obtains suspended in TE spend the night.

The aliquots containig of the DNA of each purifying is carried out agarose gel electrophoresis to confirm integrity and to determine DNA concentration.

In order to prepare any big dna fragmentation that is used for natural approach expression library, by under 37 ℃, insulation is 1 hour in 1X enzyme buffer liquid and every micrograms of DNA 0.01-0.5 unit enzyme, the DNA that 20 μ g are various types of with frequent nickase (frequent-cutting enzyme) for example Sau3A partly digest.Can be determined by experiment used enzyme amount to produce required magnitude range.The DNA that collection is digested, phenol: chloroform extraction, and use ethanol sedimentation.This mixture of 100ug is used for each library of a large amount of natural gene group of needs dna fragmentation.This mixture optionally comes size classification to separate by saccharose gradient.Can separate the less dna fragmentation of selecting to be used for chimeric approach expression library by size classification simultaneously.

Agarose gel electrophoresis confirms to digest and by size fractional separation by the aliquots containig of sample is carried out.5.4.3. the segmental preparation of prokaryotic promoter

In one embodiment, the synthetic oligonucleotide is used to make up the fragment of the beta galactose glycosides sugar promotor (lac) that contains two copies, an end that is positioned at unique BamHI site, each copy that is located in the lac that directly transcribes is towards the BamHI site at center (Fig. 4 A).The synthetic oligonucleotide by synthesizer by phosphorylation.Before room temperature connects 30 minutes with the T4 dna ligase, made the various oligonucleotide renaturation of 400ng in 30 minutes to 25 ℃ by boiling also slow cooling in 5 minutes.To connect mixture and carry out agarose gel electrophoresis, excision 2-7kbp fragment is also used Gene Clean purifying.By the Smal site that in 1 * ligase enzyme/PEG damping fluid, will connect pairing and correct localized box insertion pBSK plasmid vector in 16 hours with the insulation of T4DNA ligase enzyme at 15 ℃.To connect mixture imports in the XLl-MR cell.Analyzing each clone by the restriction enzyme analysis method also optionally checks order to confirm location and accuracy.

With pBSK-(lac/lac) _nClone's (wherein n is the integer of 2-10) cultivates with the amount of 0.3l, and uses plasmid to prepare box (Qiagen) plasmid purification.The pBSK-(lac-lac) of and purifying selected in 1 * damping fluid with Saml complete digestion 40 μ g _nThe DNA of digestion is carried out agarose gel electrophoresis, downcut lac/lac promotor dimer, and, in 1 * damping fluid, use the BamHI complete digestion with Gene Clean purifying.Referring to Fig. 4 B and 4C.Promotor monomer phenol with digestion: chloroform extraction.Ethanol sedimentation handles dephosphorylation with CIAP in 1 * CIAP damping fluid.Extract as previously mentioned and precipitate the promotor that the quilt of dephosphorylation digests, and be suspended among the TE again with the concentration of 20ng/ μ l before using down or further storing-20 ℃.

In another embodiment, the promoter fragment of preparation is mixed with the joint that does not comprise promoter sequence of similar preparation, flushing and be used for and being connected of donor gene group DNA.Transcribing at antisense is that this feasible generation only has the box of a promotor under the situation of the problem considered of needs.5.4.4. be used to make up the preparation of the box gene of chimeric approach expression library

In an example, genomic dna (mean size is 3.5kbp) BamHI-BamHI fragment is mixed with excessive dephosphorylation promoter fragment, then connect.The mol ratio of promotor and genomic DNA fragment is 20: 1.Unit (the lac/ genomic DNA fragment/lac) will have the mean size that is about 4kbp that produces.Spendable other prokaryotic promoter comprises other escherichia coli promoter (Harley etc., 1987, nucleic acids research 15:2343-2361) and the streptomycete promotor (Strohl, 1992, nucleic acids research V20:961-974) that is used for the expressive host of streptomycete kind.When the host has not definite or recombinates ability significantly, need to use a series of different promotors, any clone who comprises some boxes like this will contain some different promotors.5.4.5. the preparation of solid support

The super Streptavidin particle that is connected and fixed is available from Pierce (catalog number (Cat.No.) 53113).3MEmphaze Biosupport Medium ABl " blank bead " is available from Pierce (catalog number (Cat.No.) 53112).Similar solid support from other seller can replace them to be used for this purpose.

Oligonucleotide is available from Life Technologies (Gibco-BRL).Oligonucleotide " bead-connection-5 " is 5 ' vitamin H-GCC GAC CAT TTA AAT CGG TTA AT3 '." bead-connection-3 " is 5 ' phosphoric acid-TAA CCG ATT TAA ATG GTC GGC3 '.When annealing, these oligonucleotide comprise a SwaI restriction endonuclease sites (being shown in the underscoring part).The bead of renaturation-connection oligonucleotide also stays next AT overhang at 3 ' end.This overhang representing in oligonucleotide bead-connection-5 with black matrix.

Vitamin H-GCC GAC CAT TTA AAT CGG TTA AT

CGG?CTG?GTA?AAT?TTA?GCC?AAT

Various particles connection oligonucleotide with equimolar amount in the eppendorf pipe mix.With add in the pipe 5M NaCl to ultimate density be 300mM.To react insulation 1.5 hours at 60 ℃.Use unannealed oligonucleotide in contrast, confirm annealing by agarose gel electrophoresis.

In order to prepare blank bead, the bead that 100mg is done is suspended in the 1ml phosphate buffer normal saline (PBS) again.Adding bovine serum albumin (BSA) to ultimate density is 1mg/ml.Under the room temperature bead was rotated 4 hours.The centrifugation bead washed 3 time to seal unreacted azalactones site with it with 1M Tris-HCl pH8.0 under the room temperature in 2 hours.Of short duration centrifugation bead is also used the PBS thorough washing.Under 4 ℃, blank bead is stored among the PBS until use.

For the oligonucleotide that allows bead connect combines with the streptavidin bead, in 1 * binding buffer liquid (PBS, 500mM NaCl), with the oligonucleotide and the super fixed Streptavidin bead mixing that is connected of 20 μ l of 10 μ g pre-annealings.Opposite with the suspension of maintenance bead, under the room temperature bead is incubated 3 hours.The precipitation bead is also used 1ml binding buffer liquid washing 3 times.Then with bead with 1 * connection damping fluid (50mM Tris-HCl pH 7.8,10mM MgCl ₂, 10mM dithiothreitol (DTT), 1mM ATP, 25 μ g/ml BSA) and washing and balance.Store bead until use for 4 ℃.5.4.6. make up the assembling of chimeric approach expression library

Box gene is connected on the magnetic bead: in 1 * kinase buffer liquid, use T4 polynucleotide kinase phosphorylation box gene.Ethanol sedimentation phosphorylation fragment also is suspended among the TE again.It 1/10 is connected on the mixture of unphosphorylated synthetic linker of two weak points.Remaining 9/10 is used for later step.Each joint will have in two rare nickases, Not1 or Srf1.In addition, the joint that contains Not1 is used biotin labeling when synthetic oligonucleotide.The transcriptional units of Not1 and Srf1 joint and phosphorylation respectively with 100: 100: 1 mixed, and is connected 16 hour with the T4 dna ligase at 15 ℃ in 1 * ligase enzyme/PEG damping fluid.Allow this mixture combine, and the method that provides with manufacturers is removed bead-in conjunction with transcriptional units from connect mixture with avidin bonded MPG magnetic bead.

In the mixture of the DNA that connects, about 1/2 will have biotin labeled Not1 joint and have biotin labeled Srf1 joint at another end at an end.The Not1 end will combine with particle by avidin-vitamin H connection.The fragment that has the Not1 joint at two ends does not participate in further Connection Step.The fragment that has the Srf1 joint at two ends is not retained in the magnetic separating step.

Be used for the preparation of the dna library that is connected with bead bonded DNA: as above the transcriptional units with remaining 9/10 phosphorylation only is connected with the Srf1 joint, then uses the Srfl complete digestion, dephosphorylation, and purifying is also used ethanol sedimentation.

The deprotection of bead bonded DNA: in 1 * Srf1 damping fluid, use Srf1 enzyme complete digestion and bead bonded transcriptional units.This reaction of heat inactivation and logical magnetic separate removes bead.

Concatermerization: then bead is joined the transcriptional units that contains dephosphorylation Srf1-Srf1 digestion the connection mixture 1 * connect in the damping fluid.Before heat inactivation ligase enzyme and magnetic separate bead, start and be connected and carried out 60 minutes at 25 ℃ by adding the T4 dna ligase.Connect between the bead bonded DNA of main phosphorylation and the unphosphorylated transcriptional units and take place.By the transcriptional units on the T4 polynucleotide kinase phosphorylation particle, heat inactivation, magnetic separates, and gets back in the connection mixture by adding more T4 dna ligase.

By before cutting polymer with Not1 digestion from the bead enzyme, should circulate and repeat 10 times.The DNA that the ethanol sedimentation enzyme is cut is suspended among the TE again, and before in being inserted into SuperCos 1 or other carrier, according to expressive host, observes character and magnitude range to estimate it on sepharose.As save among the 5.4.5 and describe, concatermer is used to produce the protokaryon library in relevant expressive host.5.4.7. make up the assembling of natural approach expression library

It is reservation size to be the segmental clay SuperCos 1 of insertion of 30～42kpb that the expression vector that is used for the intestinal bacteria library is hoped.Carry out being inserted into dna fragmentation among the SuperCos 1 and using the Gigapack extract to pack according to the explanation (Stratagene) of manufacturers.

In brief, with the SuperCos 1 phage-infect XL1-MR host cell that comprises the DNA library.It according to as get off to carry out: 37 ℃ are supplemented with 1% maltose, 10mMMgSO at 5ml ₄The LB substratum in 300rpm the XL1-MR cell cultures is spent the night.With 1: 10 culture of spending the night of dilution, and under 37 ℃, with 300rpm at LB/10mM MgSO ₄The middle cultivation 3 hours.With 800 * g centrifugation culture, and be suspended in again among the 5ml LB.Under the room temperature, 600 μ l suspension are incubated 30 minutes with the library that 500cfu is packaged in the phage particle, then under 37 ℃, with the LB of 8 volumes with 300rpm insulation 60 minutes.

For the expression library that increases, infected host cell is layered on contains 50ml LB, in the 150mm culture dish of 50 μ g/ml penbritins.These are dull and stereotyped by dry 48 hours in advance under the room temperature.After the coating, allow dull and stereotyped incubated overnight under 37 ℃.Scrape the plate of making even, contain 3ml 15% ethylene glycol, the 85%LB bacterium colony that suspends again with every flat board.This bacterial suspension is stored in-70 ℃ in order to further using.

In order to prepare the library that is used to screen each clone, infected host cell is layered on contains 50ml LB, in the 150mm culture dish of 50mg/ml penbritin.Under the room temperature with dry in advance 48 hours of flat board.After the coating, allow dull and stereotyped incubated overnight under 37 ℃.Transfer in the porous flat plate with bacterium colony and ground, a hole that the toothpick picking of sterilizing produces.75/ μ, 1 LB is contained in each hole of 384 hole working plates, 50 μ g/ml penbritins, 7% ethylene glycol.The row (80 holes altogether) of outside is not inoculated, but loads onto substratum similarly to be provided at the evaporation barrier between insulation afterwards and pool period.Under 37 ℃, the main ware nonoscillatory of these inoculations was placed 16 hours.The master's 384 hole plates that spend the night are used as the source plate and duplicate and draw among one or more work 384 hole plates or the Omni-Trays.Then should lead 384 hole plates wrap respectively by and down freezing at-80 ℃.Duplicate with 384 syringe needle copying instruments.Before each the use and afterwards, 384 syringe needle copying instruments were sequentially immersed in the sodium hypochlorite solution 20 seconds, in the water 30 seconds, then before burning, immersed in the ethanol 5 seconds.The method of library assembling depends on the selection of carrier and expressive host.5.4.8. the prescreen of expression library

Three types prescreen is arranged, in the born of the same parents, differential and selection.

In brief, first type, prescreen need import the library by genetic modification and comprise among the host of report construction of chemical responsiveness in the born of the same parents.Reporter is GFP (green fluorescent protein) or beta-galactosidase enzymes, selects by fluorescence-activated cell sorting (FACS) or slightly drip sorting to carry out.

Second type, the differential prescreen need be with the library among the physiological tracer of fluorescence or the chromogen insulation host, then by FACS or slightly drip sorting.

The third type is selected prescreen, needs to use selective reagent, and for example antibiosis usually is incubated the library among the host, follows by FACS or slightly drips sorting and discern survival or proliferating cells.

For all methods, before detecting each culture, in the culture in a large amount of amplifications library, carry out cell sorting.

Can be by FACS or slightly drip sorting and come the prescreen library.The storehouse of cultivating the host cell that contains the DNA library in one or more modes that improve high or low density microenvironment.

In first method, identify the cell in amplification library by individual cells.Before precipitation, 30 ℃, with 300rpm intestinal bacteria library aliquots containig was cultivated 4 hours in the substratum of 20 volumes, be suspended in the sterilization ddH of 1 volume again ₂O is incubated (if desired) with fluorescent probe, and is placed on ice to transfer on the FACS equipment.

In the second approach, before transferring to FACS or slightly dripping size separation equipment, 5.2.3 is described as joint, the aliquots containig in packing and cultivation amplification library when having substrate or selective agent.

For the culture that detects with fluorescent tracing thing or substrate, the method according to manufacturers before FACS will be suspended in ddH again ₂Culture dyeing among the O, following carrying out usually: be incubated 15 minutes in the dark under the room temperature, then in 1.5ml microfuge pipe, precipitated 5 minutes and be suspended in again the cold ddH of 1 volume ₂Among the O.

After the sorting, will cultivate in the 0.5L nutritional medium from the 1-1000 clone bank or thick the dripping of the selection of expression library.Produce about 20mg-1g extract/rise thick organic extraction of culture by the bacterium cultivated and substratum being handled to be used for the chemical analysis rotary evaporation with the 0.5L ethyl acetate extraction.Purifying clone's of the same race DNA also is transferred in the host cell again to confirm the location of the sequence relevant with clay.The chemical example that produces by the expression of library clone can be by using a series of posts (positively charged ion, negatively charged ion, anti-phase) HPLC and measure by the quantitative chemical analysis of use NMR subsequently.5.4.9. duplicate metabolic determination to ocean gram (-) bacterium/intestinal bacteria library by flat board

Before preparation DNA library, detect the order of various wild-types ocean kind earlier with the metabolic determination that prevents repetition and help determining in the library of finishing, carrying out.

In order to prepare the library that is used to screen each clone, with infected host cell, for example intestinal bacteria XLl-MR is layered on and contains 50ml LB, on the 150mm culture dish of 50mg/ml penbritin.Under the room temperature in advance with dry 48 hours of flat board.After spreading out, under 37 ℃, allow dull and stereotyped incubated overnight.Transfer in the 384 hole flat boards with bacterium colony and ground, a hole that the toothpick picking of sterilizing produces.75 μ l LB are contained in each hole, 50 μ g/ml penbritins, 7% ethylene glycol.The row (80 holes altogether) of outside is not inoculated but is loaded onto substratum similarly with the insulation that is provided at the back and the evaporation barrier of cooling period.Under 37 ℃, the main ware nonoscillatory of this inoculation was placed 16 hours.The main 384-hole plate that spends the night is used as the source ware and copies among one or more work porous plates or the Omni-Trays.Then wrap respectively by main 384 hole plates ,-80 ℃ of storages.Duplicate with 384 syringe needle copying instruments.Before each the use and afterwards, sequentially 384 syringe needle copying instruments were immersed in the sodium hypochlorite solution 20 seconds, in the water 30 seconds, then before burning, immersed in the ethanol 5 seconds.

Work porous flat plate or Omni-Trays are used as the source plate and the DNA library are copied to a series of differentials and/or select (for example siderophore detects substratum or antibiotic substratum) on the substratum.The compilation result also compares with the collection of illustrative plates of the wild-type marine bacteria that is used for the constructed dna library.Penetrated mensuration 5.4.10. generation carries out in ocean gram (-) bacterium/intestinal bacteria library by thick bag

Cloned by taking by weighing sodiun alginate and being dissolved in to wrap in the 100ml sterilized water with 1% concentration with 2000rpm by pressure mixer.Add the library suspended substance of 1 volume so that every bag by 1-5 clone.Allow mixture leave standstill at least 30 minutes with venting.Then be pressed through and allow it to form any equipment of single.One of them example is the syringe with No. 25 syringe needles.These are splashed in the beaker that stirs gently that 135mM calcium chloride is housed.Allow a hardening also then be transferred in the flask of sterilization in 10 minutes, remove calcium chloride, replace with LB/Amp substratum and substrate (for example x-glycosamine).Then will contain the flask shaken overnight of dripping down at 30 ℃, detect by the positive colony that exists blue look bacterium colony to represent the next morning.

Observe dripping to be placed in the big clean dish and with eyes with individual layer.Take out positive bacterium colony and be positioned in the 96 hole master's wares that comprise LB/Amp and 50mM Trisodium Citrate pH 7.4 and drip, and allow its grow overnight at 37 ℃ with dissolving.These master who spends the night 96 hole plates are used as the source plate and copy among one or more work porous plates or the Omni-Trays.Wrap respectively by main 96-hole plate, and freezing down at-80 ℃.Positive colony can be sent to be used for specific product and to detect or send to and carry out another and take turns prescreen or screening.Can screen by duplicating further of carrying out with spininess head copying instrument.Before each the use and afterwards, sequentially spininess head copying instrument was immersed in the sodium hypochlorite solution 20 seconds, in the water 30 seconds, then before burning, in ethanol, immersed 5 seconds.5.4.11. by the droplet bag by to the metabolic determination that ocean gram (-) bacterium/carries out in the intestinal bacteria library

Can produce droplet by following method.With 2000rpm applying pressure mixing tank 0.6g sodium polyphosphate and 2% sodiun alginate are dissolved in the 100ml sterilized water.Allow this mixture exit 60 minutes.Then with 1.9g calcium sulfate supersound process at least 15 minutes in 10ml 50% ethylene glycol.Import at least 30 minutes the oil phase of soybean lecithin pre-mixing (sweet oil) added the 1.0g purifying will starch immediately before and will produce 1-5 cell/droplet the library suspension of 1 volume be admixed in the alginic acid solution.This emulsion process is by transferring to the alginic acid mixture lentamente oil phase and starting in 10 minutes with the 580rpm mixing.Then add the 500ml sterilized water and allow mix and continue 5 minutes.Can droplet be shifted out from oil and wash by centrifugal, be suspended among the LB/Amp again.For sorting,, can use filter membrane to carry out the size selection to dripping with required size restriction if drop in outside the essential required magnitude range of sorting by FACS.Can be at 30 ℃, in containing the LB/Amp substratum of fluorogenic substrate, the clone swayed and cultivated 2 hours.

After the insulation, by centrifugal, the washing and with 1 * 10 ⁶Drip/density of ml is suspended in again and prepares the sample that is used for by the FACS sorting in the sterilized water.Can measure the size of dripping by phase microscope.On Becton-Dickinson FACStar Plus, carry out the FACS operation by qualified operator, and positive colony directly is sorted in the porous flat plate that contains LB/Amp, positive colony is divided into 1 clone/hole.Allow dull and stereotyped the growth down from bead, grow (1-2 days) until the clone at 37 ℃.These flat boards that spend the night are used as the source flat board and copy among one or more work porous flat plates or the Omni Trays.Wrap respectively by main porous flat plate and freezing at-80 ℃.Can then positive colony be sent to the detection that is used for specific product or send to and carry out another prescreen of taking turns or screening.Can finish further screening by duplicating with 96 or 384 syringe needle copying instruments.Before each the use and afterwards, sequentially copying instrument was immersed in the sodium hypochlorite solution 20 seconds, in the water 30 seconds, then before burning, immersed in the ethanol 5 seconds.5.4.12. duplicate metabolic determination to actinomycetes/carry out in shallow Streptomyces glaucoviolaceus library by flat board

Before preparation DNA library, measure various educable wild-type actinomycetes kinds earlier and repeat, and have and utilize the order of determining the metabolic determination that in the library of finishing, carries out to prevent taxonomy.In order to prepare the library that is used to screen each clone, with transformed host cells, shallow Streptomyces glaucoviolaceus TK66, coating contains in the 150mm plate of F10A.Under the room temperature with dry in advance 48 hours of plate.After the coating, under 30 ℃, allow the plate incubated overnight.Start selection by coating thiostrepton.Transfer in the 96 hole plates with bacterium colony and ground, a hole that the toothpick picking produces.Every hole comprises the F10A substratum.Under 30 ℃.The main ware of these inoculations was placed 1-4 days.The main 96 hole plates that spend the night are used as the source plate and copy among one or more porous plates or the Omni-Trays.Then wrap respectively by main 96 hole plates and freezing at-80 ℃.Duplicate with spininess head copying instrument.Before each the use and afterwards, sequentially spininess head copying instrument was immersed in the sodium hypochlorite solution 20 seconds, in the water 30 seconds, then before burning, immersed in the ethanol 5 seconds.

Work porous plate or Omni-Trays are used as the source plate DNA library are copied in a series of differentiation and/or the selection substratum (for example microbiotic plate or antibiotic substratum).Compile the result and compare with the collection of illustrative plates of the wild-type bacterium that is used for the constructed dna library.5.4.13. metabolic determination is carried out in actinomycetes/shallow Streptomyces glaucoviolaceus library by the thick bag that drips

Cloned by wrapping as the method for describing among the joint 5.4.10 that is used for the intestinal bacteria library.Allow a hardening also then be transferred in the sterilization flask in 40 minutes, remove calcium chloride and use the F10A substratum and substrate (for example x-gal) replacement.Under 30 ℃, will contain the flask that drips and vibrate 1-5 days, and measure by the positive colony that exists blue look bacterium colony to represent.

Observe dripping to be placed in the big clean dish and with eyes with an individual layer.Extract positive bacterium colony and be placed in the 96 hole master's wares that comprise F10A 50mM Trisodium Citrate pH 7.4 and drip, grew 2 days down at 30 ℃ subsequently with dissolving.These master who spends the night 96 hole plates are used as the source plate and copy among one or more work porous plates or the Omni-Trays.Bag is by main 96 hole plates and freezing down at-80 ℃ respectively.Be used for specific product and detect or send to and be used for another and take turns prescreen or screening along with positive colony being sent to.Can by as top joint 5.4.9 in describe duplicate further and screen.5.4.14. the prescreen in the library that is carried out by common bag with indicator cells

By the suitable dilution of plating and carry out enumeration and come the titration library clone.With the library cytomixis of capacity in 1% alginic acid to produce about 1 cell/thick dripping.In addition, the indicator cells that adds capacity is to produce about 50 target cells/drip.As save 5.4.10 and describe thick of ground preparation, and under the condition that is suitable for library and indicator cells, cultivate.

Usually,, in R5 or F10A, cultivate shallow Streptomyces glaucoviolaceus library and slightly drip,, in LB or B3, cultivate intestinal bacteria and slightly drip at 30-37 ℃ at 30 ℃.Can regulate substratum and temperature to adapt to the physiological requirements of indicator cells.For of the effect of visual inspection library cell, make following coverage:, add toluylene red or Congo red in order to detect necrocytosis to indicator cells; Exist in order to detect cell, add the substrate relevant (for example for E.faecalis X-glucopyranoside) with indicator cells; Activation produces the beta galactose glycosides report activity of replying to promotor in order to detect, and adds 80mg/ml X-gal in substratum.As joint 5.4.10 in describe to separate positive thick dripping after, by adding, usually eliminate indicator cells but indicator cells is not had optionally antibiosis to the library cell.Then store the library cell and/or further measure on demand.5.5. be used for the method in eukaryotic expression library

This section has been described the method for the combination gene expression library that can be generally used for preparing eucaryon donor biology.The related step of preparation in the chimeric approach gene expression of combination library is shown among Fig. 5 A-5F in the eukaryote.

Good especially expression eucaryon host biology is stable, and is non-thread, and sufficiently characterized the purpose that is used for genetic expression with activation by genetic manipulation.To yeast and fungi, preferred kind is for being grown in 30 ℃ of following grain wine fragmentation sugar yeasts (C.Guthrie and G.R.Funk, yeast genetics and molecular biology guide, Enzymology method, Vol.194, Academic Press).Arabidopis thaliana (A.thaliana) and tobacco (N.tabacum) cell be preferred host (C.P.Lichtenstein and J.Draper, genetically engineered plant, dna clone Vol.II, pp.67119).5.5.1. density gradient centrifugation is removed the satellite genomic dna

The eukaryotic gene group has a large amount of mainly by the rrna coding region or there is not the repetition DNA that the sequence of manifest function is formed usually.Therefore, from the biological preparation of eucaryon donor genomic dna the time, be expected to remove from the library with these noncoding DNA sequences.Before the clone, there is dna binding dye, the standard C sCl genomic dna purification process of Hoechst 33258 can be used for separating various types of genomic dnas.5.5.2. the segmental preparation of promoter in eukaryote and terminator

By using the PCR that changes the sequence specific primers that comes from the open sequence of known promotor and terminator can prepare promotor and terminator gene fragment.The selection of promotor and terminator sequence can be decided by the host living beings that adopts.If for example adopt grain wine fragmentation sugar yeast as expressive host, can adopt natural promoter, for example nmt 1 or ura 4 and non-natural promotor, for example from those of virus, as CMV, SV40 (Forsburg, 1993 nucleic acids research 8:4321-4325).Or from the people, for example chorionic gona dotropin or somatostatin (R.Toyama, H.Okayma 1990, FEBS Letters 268 (1) pp.217-221).Also can adopt the promotor (Faryar etc. 1992, Curr Genet21:345-349) that is similar to the genetic modification of in can inducing the tsiklomitsin system, finding.

Can on the PCR instrument that commerce can be buied, use the PCR reaction conditions of standard and the archaeal dna polymerase of high reliability and productivity, for example, carry out the PCR reaction but be confined to Pfu polysaccharase (Stratagene) or Vent polysaccharase (New England Biolabs).Because be not have primer sets all to use identical reaction conditions, so available method well known in the prior art is determined condition accurately with experiment.The PCR Oligonucleolide primers can commercial be buied or synthesize by the method for knowing in the prior art.

Promotor and terminator fragment that PCR produces may comprise restriction site at 5 ' end.This paper adopts Bgl II, and Xho I and BamHI are to illustrate principle of the present invention.As long as this site does not occur, can use any restriction site in promotor or promoter gene sequence.

Insert the compatible cloning site of fragment in order to produce with cDNA or genomic dna, cut 5 ' end that the promoter gene fragment will be created in them with BglII and Xho I enzyme and have Bgl II site, have the promoter gene fragment in Xho I site at their 3 ' end.Only the terminator general with Xho I cutting only has Xho I site at their 5 ' end.5 ' and 3 ' direction based on transcriptional orientation along the expectation of promotor or terminator gene fragment.Referring to Fig. 5 B.

Utilize the part filling-in of e. coli dna polymerase 1 big subunit (Klenow fragment) and deoxynucleotide subgroup (being dCTP and dTTP here) can be used to produce to be undertaken by their Xho I end the promotor and the terminator fragment that self are connected.Owing to lacking base complement, the Bgl II end of promoter fragment is unaffected, and the terminal 5 ' end that does not expose of the segmental BamHI of terminator is utilized by the Klehow fragment.

Use Phosphoric acid esterase, for example calf intestine alkaline phosphatase is handled and will be prevented that Bal II self from connecting, and the similar end that is provided for being connected in promotor and terminator fragment.By in first chain is synthetic, mix 5 '-methyl dCTP protection cDNA fragment avoids the digestion (Short, J.M1988, nucleic acids research 16:7583-7600) of NotI.

In another embodiment of the invention, when DNA inserted fragment from mRNA, directed cloning can be used to improve clone's efficient.Can be to have the direction of justice cDNA to be inserted fragment is unidirectional to be connected to promotor and terminator fragment.These can be different by producing on promotor and terminator fragment, the end that can not connect and realizing.Bgl II, Xho I, Xma I and BamHI are used to illustrate the present invention.Can use and produce compatible end and can be right by the protected any enzyme that methylates.

Replaced Xho I site in the segmental 5 ' terminal Xma I of terminator site, and the preparation of promoter fragment does not change.Adopt Xma I to be because by inserting Klenow fragment and dCTP, it is compatible with Not I.This has produced the terminator fragment with two base dCTP-dCTP 5 ' projections, and the cDNA gene fragment that it and suitably prepd Not I digest is compatible.Referring to Fig. 5 A.5.5.3.DNA insert segmental preparation

The encoding gene fragment that is used for the eucaryon library will be from two main DNA resources, and promptly genomic dna (gDNA) or enzymatic are from the complementary DNA of messenger RNA(mRNA) (mRNA).The method for preparing gDNA or cDNA is closely similar, but inequality.

Use obtainable standard method in the document, or especially the explanation of manufacturers prepares complementary DNA from messenger RNA(mRNA) and/or total RNA.Can finish separating of total RNA and genomic dna simultaneously by the guanidinium isothiocyanate method that joint 5.3.1 describes, and mRNA can separate by the order affinity chromatography on widow-dT Mierocrystalline cellulose.

Synthetic can the use at 5 ' end of first chain cDNA comprises cloning site, for example the widow in Not I site-dT dna primer.The synthesizing of first chain that in comprising, also can be used for random primer at the oligonucleotide of the stochastic sequence in Not I site near 5 ' end.Use this alternative primer to avoid 3 ' deviation for big mRNA.Methylated deoxynucleotide, for example 5-methyl-dCTP can with polysaccharase, for example Pfu uses together so that the protection that avoids restrictive diges-tion (Short etc., Supra to be provided; G.L.Costa, 1994, Strategies 7:8).Exist only in the initial primer do not methylate the site in addition enzyme cut, therefore guaranteed 3 ' end of the definition of cDNA.Methylated cDNA also can prepare by handling with methylation, thereby but because all obtainable sites all will be methylated and enzyme cut produce resistance and will lose the directivity of cloning.

By catenation sequence specificity connector, for example the BamHI connector with 5 ' phosphoric acid of Xiu Shiing prepares 5 ' end of the definition of cDNA.When its complementary oligonucleotide was annealed, connector only comprised the dGTP-dATP 5 ' overhang and the smooth 5 ' phosphate terminal of two bases.The connector of modifying can be connected to as on the cDNA in the standard method with Pfu or the processing of T4 archaeal dna polymerase.At the BamHl connector that has connected modification with after with Not I digested cdna, banded cDNA can be used Klenow fragment and dGTP handle, produce the cDNA gene insertion fragment that is used to be connected to suitable promotor for preparing and the segmental orientation that is defined of terminator.Segmental direction is such, and promptly 5 of cDNA ' end is towards 3 ' end of promotor, and 3 of cDNA ' end is towards 5 ' end of terminator.Referring to Fig. 5 C.

By full gene DNA being used the restriction enzyme of cutting usually, for example Sau 3AI partly digests the acquisition genomic DNA fragment.This enzyme is widely used in this purpose, and carries out fractional separation after the part digestion, though saccharose gradient is a very method of standard.The sheet phase library that can use three kinds of different digestion that change from initial enzyme concn is so that the susceptibility difference of the enzyme in the genome.

After size fractionation separation and purifying, fragment can be handled to protect any intrinsic BamHl site with the BamHI methylase, then with Klenow fragment and dATP and dGTP processing.This is created in the BamHI site by the methylated gene fragment in inherence, and only has the dATPdGTP overhang.Referring to Fig. 5 D.These fragments can not self connect, and only can be connected to the promotor and the terminator gene fragment of suitable preparation.5.5.4. insertion sheet segment DNA is connected with promotor and terminator

Under 16 ℃, with the cDNA of suitable preparation, promotor is connected with the terminator fragment spends the night.In ligation, can adopt 10 promotors (P): 1 cDNA: the ratio of 10 terminators (T).Optimum proportion can be determined by experiment by method well known in the prior art.The directed cloning method only provides a kind of connection product, promptly correct directed promotor-have justice to insert fragment-terminator box gene.

Can be under 16 ℃, with the genomic dna that different ratios is prepared, promotor is connected with the terminator gene fragment.Owing to do not have a kind of coordinator self to connect, can be determined by experiment optimum proportion.Half of connection product that assesses formation is directly available, and 1/4 product of formation can not connect circulation, and 1/4 product only can be connected once before growing chain stops.

Combination (P=promotor, frag=5 ' → 3 ' genomic DNA fragment, T=terminator garf=3 ' → 5 ' genomic DNA fragment) below:

1.P-frag-T 5.P-gaff-T

2.T-frag-P 6.T-garf-P

3.P-frag-P 7.P-gaff-P

4.T-frag-T 8.T-garf-T

The construction that 1,6 and 2,5 combination representatives are required, but because to insert segmental direction be at random, being expected 50% construction is correct direction (1 and 6) for any given gene.

Can form terminator/terminator box gene, but can not participate in any follow-up clone's step, because owing to lacking the 5 ' end that exposes at their the 3 ' terminal BamHI that is not cut by flush endization ends.

Because the terminal 5 ' phosphoric acid (first round) that lacks of Bgl II, promotor/promotor construction will be in the back only with in being connected of the BamHI end of other exposure be cloned.Because lack 5 ' phosphoric acid, being connected in the new box gene of the Bgl II end of back and exposure is rare.The BamHI end that exposes only can be formed on the chain and can not produce in the new box gene of introducing at inherent.Therefore be expected by with on another chain near this promotor of BamHI site cyclisation/the promoter gene box is with terminating chain, these cyclic actions are expendable.If this promotor/promoter fragment becomes an obvious problem of joint efficiency, the processing of fixed growth middle-of-chain kinases will allow promotor/promoter fragment to connect product elongation growth chain by forming Bgl II/Bgl II before adding new box gene so.Kinases is handled and is promoted that Bgl II/Bgl II is connected with Bgl II/BamHI in the solid phase, the growing chain that it participates in cyclisation.5.5.5. the series of box gene is connected to form concatermer

Insert being connected of box gene that fragment forms with carrying out being positioned at the gene DNA of its side or cDNA by promotor/terminator combination with the similar approach that the front is described procaryotic DNA.Here main this method that is not both uses endonuclease BamHI to produce 3 ' restriction site of the exposure that is used for cloning later.The BamHI site of using BamHI methylase or 5-methyl-dCTP to guarantee to insert among the DNA is protected.Referring to Fig. 5 E.

After the 5-10 endless chain connects, with concatermer growing chain deprotection, and handled with Klenow fragment and dATP and dGTP to prepare and be used for and being connected of expression vector with BamHI.This makes that all ends of growing chain can not be interconnection, thereby has eliminated the loss of any cyclic action and concatermer chain.

Can carrier DNA be connected on the concatermer chain 5: 1 mol ratios.Also can adopt other ratio.They can be carried out under 16 ℃ 8-12 hour, perhaps under 22 ℃, carry out 4 hours.After the connection, but washing granule and it is suspended in intron nuclease restriction damping fluid again.According to producing digesting of specification sheets description.Can use any intron nuclease.Preferred enzyme CeuI, because it produces the non-palindrome 3 ' overhang, this overhang can be used for preventing that self from connecting.Referring to Fig. 5 F.5.5.6. cyclisation and comprise the conversion of the carrier of concatermer construction

By impelling the concatermer-carrier molecule that discharges from solid phase to carry out the intramolecularly connection 100 times of CeuI digestion mixture dilutions with 1 * ligase enzyme damping fluid.Can add the T4 ligase enzyme, and can reaction be carried out 4-6 hour, or 16 ℃ are spent the night at 22 ℃.Referring to Fig. 5 F.Can concentrate the construction that produces by micro-filtration or lyophilize, and import in grain wine fragmentation sugar yeast bacterial strain or intestinal bacteria or the shallow Streptomyces glaucoviolaceus bacterial strain by standard method.Any method be can use, electroporation and improved calcium phosphate method for transformation included, but are not limited to.5.5.7. the preparation of the preparation carrier of in yeast, expressing be connected

This section is described and can be generally used for preparing the method for yeast as the combination gene expression library of host living beings of using.

In order to prepare the library at grain wine fragmentation sugar yeast, one is possible, but of course not unique carrier be intestinal bacteria/grain wine fragmentation sugar yeast shuttle vectors pDblet (Brun etc. 1995, gene, 164:173-177).This carrier has the advantage that has a plurality of cloning sites and f1 phage replication starting point, and it is expressed with suitable high copy number, and highly stable in intestinal bacteria and grain wine fragmentation sugar yeast.

For the present invention, the multiple clone site (MCS) that can modify pDblet is to be fit to the BstXI site of known array.Referring to Fig. 6 B.This is owing to be used for discharging from solid phase 3 ' Nucleotide overhang (3 ' GATT of the intron nuclease generation defined nucleotide sequence of concatermer chain ...).After enzyme was cut, the BstXI site with transformation of sequence C CACCTAACTGG produced suitable CTAA-3 ' overhang.

In order to modify pDblet, can at first cut to remove the BstXI site that exists that does not have correct sequence with SacI and NotI.Can will once mix with pre-synthetic oligonucleotide by the pDblet plasmid that rotates chromatography or other method purifying, this oligonucleotide also comprises the compatible overhang with NotI-of new NcoI site and SacI-except that the correct sequence that contains the BstXI site.Referring to Fig. 6 C.After connecting and transforming, by detect the clone's of micropreparation exactness with NcoI digestion.Correct clone is identified in existence by BstXI and NcoI site.Use BstXI, then the pDblet generation of handling this modification with the XhoI site comprises the carrier of 5 ' XhoI site and 3 ' CTAA BstXI overhang.Referring to Fig. 5 E.This enzyme can be cut carrier handles so that it can not self be connected with dTTP with dCTP with the Klenow fragment.This carrier can be used to accept the concatermer chain.5.5.8. expression of plants library

This section is described and can be generally used for using vegetable cell to prepare the method for combination gene expression library as donor and/or host living beings.

In order to prepare donor dna from plant, general step below adopting: (1) in cold diethyl ether the pre-treatment plant tissue to promote the plant fragmentation; (2) by with sand, glass grain or aluminum oxide grind together will organize mechanical homogenate; (3) filter to remove cell debris with sieve; (4) extract DNA with the method for describing among the 5.1.2.Describe as 5.5.3, modify the DNA of the purifying that is produced.By preparing CaMV 35S or rouge alkali synthetase fragment and rouge alkali synthetase terminator fragment as the PCR that describes among the joint 5.5.3.Promotor is linked to each other with dna fragmentation with the terminator fragment, and as joint 5.5.5 and 5.5.6 in describe it is connected with the DNA of plants carrier.

Preferred DNA of plants carrier is Bin19 or its varient, it makes in T-DNA edge and the Agrobacterium tumefaciems transactivation function in virus district of the common Ti-plasmids that exists with donor transfer of genetic material (Bevan in the nuclear gene group of plant host cell, 1984, supra).Can use to comprise a plurality of cloning sites, for example can commerce buy the Bin19 carrier of the modification of pBI 121 or pBI 221 (Clontech, Palo Alto).Kalamycin resistance and/or betagalactosidase activity are used as mark and monitor conversion and prescreen.

Compile Acadcmic Press as Potrykus etc. 1988 at " molecular biology of plants method " Weissbach and Weissbach, that describes in p.376-378 prepares plant protoplast from the tobacco plant leaf.By Power etc. 1988, compile Academic Press at " molecular biology of plants " Weissbach and Weissbach, the conversion of the use polyoxyethylene glycol of describing in p.388-391 imports expression constructs in the protoplasm somatocyte.By antibiotics resistance, for example anti-kantlex is selected the protoplastis of conversion, and can be as the coated prescreen of describing among the joint 5.4.10 that is used for.

6. embodiment: the structure of combination gene expression library and screening

Following trifle is described the mixture that uses Lu Sheng microorganism or marine microorganism and is prepared and prescreen combination gene expression library as donor is biological.Shallow Streptomyces glaucoviolaceus is used in the library, and intestinal bacteria and grain wine fragmentation sugar yeast are as host living beings.The result shows that some library cells show the metabolic activity of donor biology, shows interested donor pathways metabolism to be effective in host living beings.In addition, show that a library clone comprises that known enzyme has the marine bacteria protein DNA of sequence homology in coding and the pathways metabolism.6.1. material and method

The reagent that is used for present method normally can commercial be buied.For example:

Gene C lean, and the Genome box (Bio 101, Vista, CA); Restriction enzyme, PCR reagent and damping fluid (Promega, Madison, WI; New England Biolabs; Stratagene, La Jolla, CA): TA clone box (Invitrogen, La Jolla, CA); Bacteria culture medium (Difco, Inc); Mira TIP (Hawaiian ocean import firm); The pBSK plasmid, the XLl-MR cell, SuperCos 1 clay, the Gigapack packaging extract (Stratagene, La Jolla, CA); QiagenQIAprep plasmid purification box (Qiagen, Inc, Chaworth, CA); Avidin is in conjunction with magnetic sintered glass (MPG) pearl (CPG, Inc., New Jersey); Plate, 96 and 384 hole flat boards, Omni-Trays (Nunc), 96 and 384 syringe needle copying instruments and template (V﹠amp; P Scientific, San Diego, CA); Penbritin (IBI, Inc, CA); Green fluorescent protein and GFP cDNA (Clontech, Inc); Oligonucleotide (Genset, La Jolla, CA); Other places do not have indicated bacterial species and dna sequence dna (American type culture collection, Rockville, MD); 7-oxyethyl group-heptadecyl tonka bean camphor, and BCECF-AM (molecular probe, Oregon); 3-tolyl acid salt, the 3-toluene(mono)chloride, meta-toluic acid salt, tsiklomitsin, paraxin, acetyl aminophenol, arsenic, antimony, suitable-along muconate, and other chemical reagent (Sigma) except as otherwise noted; And Dynabeads, and MPC-M (Dynal, Inc., Lake Success, NY).6.1.1. the preparation of substratum

Usually be used in the purified water (ddH in substratum and the solution ₂O) by softening, reverse osmosis and deionization are come purifying.(La Jolla CA) obtains also to filter before use Pacific Ocean seawater (seawater) from Scripps Institute ofOceanography.By adding salt (45.2mm NaF, 48.8mm SrCl ₂, 0.324mM H ₃BO ₃, 0.563mMKBr, 6.25mM KCl, 4.99mM CaCl ₂, 0.7mM Na ₂SO ₄, 16.4mMMgCl ₂, 268mM NaCl, 45.8mM Na ₂SiO ₃, 1.10mM EDTA, 1.58mM NaHCO ₃) and ocean trace element (0.01%Mira Trip) from ddH ₂O prepares synthetic sea water (SSW).

With 1% trypsinase peptolysis, 0.5% yeast extract, 1%NaCl is from ddH ₂O prepares the LB substratum.Use 0.25% peptone, 0.15% yeast extract, 0.6% (vol/vol) ethylene glycol prepares W2-B1 from 75% seawater or SSW.

From containing 2.5% solubility potato starch, 0.2% glucose, 0.5% yeast extract, 0.5% peptone, (Nutrition Products Co.Louisville, KY), 0.3% lime carbonate, pH are adjusted to 7 ddH to 0.5%Distiller solution ₂O prepares F10A.6.2. duplicate and the thick bag that drips is made up natural approach expression library by prescreen actinomycetes/shallow Streptomyces glaucoviolaceus by flat board

Be accredited as 34 kinds of actinomycetes kinds of kind of #501-534, be used as the donor biology.Respectively with microorganism culturing at the F10A substratum, and as joint extraction and the purified genomic dna described among the 5.3.1.

Obtain about 100 μ g genomic dnas/kind, and as joint describe among the 5.4.2 they are mixed the part restrictive diges-tion that is used for Sau 3A.Genomic DNA fragment is carried out size fractionation by the close heart of saccharose gradient separate, collect and contain the segmental fraction of 20-40kb, and partly mend flat with the Klenow fragment with compatible (Korch 1987, nucleic acids research 15:3199 3220 with the carrier of following similar preparation; Loftus etc. 1992, biotechnology 12:172-175).The fragment of in many batches 0.5-3.0 μ g being collected is connected to 0.5-3.0 μ g with (Hopwood 1985, supra) among the pIJ 922 of BamHI or XhoI preparation and the pIJ 903.Connected expression constructs is transformed among the shallow Streptomyces glaucoviolaceus bacterial strain of the host living beings TK64, and this bacterial strain becomes by remove cell walls with N,O-Diacetylmuramidase that suitable (Hopwood 1985, supra).Produce about 11,000 independent clones, amplification also is stored in 20% ethylene glycol with the mycelium form, and is stored in-70 ℃ down in 50% ethylene glycol with the spore suspension form.

In order to prepare the library that is used to screen each clone, the TK64 host cell that transforms is spread out in the 150mm plate that F10A agar is housed.After spreading out, 30 ℃ allow plate be incubated 21 hours.With 5 μ g/ml, the thiostrepton of 1ml/ flat board covers plate and selects.After 48-72 hour, transfer in the 96 hole plates with sterilization toothpick picking colony and ground, a hole.The F10A substratum is contained in each hole.At 30 ℃, allow the main ware of inoculation place 1-4 days.The main 96-hole plate that will spend the night copies among one or more work 96 hole flat boards or the Omni-Trays as the source plate.Then wrap respectively by main 96 hole plates and freezing at-80 ℃.Being used in the 96-syringe needle copying instrument of sterilizing with burning before each use duplicates.

The 96 hole plates of will working copy to a series of differentiation as the source plate with the library and/or select substratum and indicate in the flat board.The selectivity microbiotic comprises erythromycin, Vulkamycin. PA-93 and Xin Meisu.Division culture medium comprises F10A and the R5 substratum that contains substrate X-glycopyranoside and X-glyconic acid.Indicate plate to comprise and be grown on the F10A, then with faecalis (E.faecalis), the library clone that the indication lawn of subtilis (B.subtilis) or SOS Chromotest (having X-gal) covers.The compilation result also compares with the collection of illustrative plates of streptomycete host TK64.

Also wrap by the prescreen library clone by thick dripping.For each prescreen, the method bag of describing among usefulness as the joint 5.4.13 is by the clone of 50,000 amplifications in library.6.3. made up chimeric approach expression library by prescreen actinomycetes/intestinal bacteria by the thick bag that drips

To be used in the chimeric approach gene expression of shallow Streptomyces glaucoviolaceus preparation combination library as saving 6.2 gene DNAs of describing that obtain from 34 unwrapping wire bacterial classifications (being accredited as kind of a 501-534).Collection comprises the fragment of 2-7kbp of the genomic dna of Sau 3A digestion.

As save the aliquots containig of describing among the 5.5.3 and be connected on the different promotors to form box gene respectively with genomic DNA fragment.8 round-robin that are used for the box gene of each round-robin different sink by use connect and deprotection formation concatermer, and the concatermer of Chan Shenging has 8 box genes that comprise 8 different promotors that link to each other with genomic DNA fragment separately like this.

Be connected with 0.5 μ g SuperCos, 1 carrier with 10 μ g concatermer cyclisation and in the BamHI site forming expression constructs, according to manufacturer's explanation (Stratagene) at this construction of external packing to be used for the infection of e. coli host cell.Obtain about 1,000,000 single clone, amplification is also collected the library that forms amplification.The library is stored in-70 ℃.As save cell that the bag described among the 5.4.10 is amplified and as joint 5.4.14 in describe carry out prescreen.6.4. made up chimeric approach expression library by prescreen fungi/grain wine fragmentation sugar yeast by the thick bag that drips

Use is two chimeric approach expression libraries of combination of the biological preparation of fungi donor below the ATCC acquisition: wood mould (Trichoderma reesei), fusarium oxysporum, Lou Ge Faerteshi mould, few spore head mold, Neurospora crassa, the cloth Laplace must be mould, Aspergillus fumigatus, flavus, Emericellaheterothallica, chactomium globosum (C.gracile), some mould, Penicllium chrysogenum.

With medium rotating speed with various respectively at 500ml potato agar glucose (PDA; Difco) malt extracted solution agar (MEA; Difco) cultivated 48-72 hour in, 1 * 10 ⁴-1 * 10 ⁶The spore inoculating thing of spore/ml is put in 1 liter of 500ml potato extracting solution or malt extracted solution meat soup in the culture flask, and at 22 ℃, growth is 48-72 hour under 225 revolutions per.

Collect culture by filtering under the vacuum with Miracloth (Calbiochern).With the mycelium solid collected with 2 liters of ddH ₂O washing, and before lyophilize dry air 10 minutes.As save among 5.3.1 and the 5.3.2 and describe, extract and purifying fungal gene group DNA and mRNA from mycelium.Store down with the mycelium lyophilize of part collection and at-70 ℃.

As save the described preparation fungal gene of 5.4.2 group dna fragmentation.According to standard method fungi mRNA is converted into cDNA (Sambrook etc. 1989, Watson CJ and JacksonJF (1985) dna clone: hands-on approach 79-88, IRL Press).From various, collect equiponderant dna fragmentation to produce genomic dna storehouse and cDNA storehouse.

Each storehouse that will comprise about 5-10 μ g DNA is used for assembling the chimeric approach expression library of reorganization independently.As save below the preparation of describing among the 5.5.2 compatible promotor and the terminator of grain wine fragmentation sugar yeast: CMV speed and send out/early stage, SV40 is early stage, RSV, HSV thymidine kinase, CaMV, nmtI, adh1 and uva4 promotor.As save describe among the 5.5.4 promotor and terminator fragment are mixed with cDNA and genomic dna storehouse.As to save what describe among the 5.5.5 be each box gene concatermerization of 5kb with mean size.With the final concatermer cyclisation of each self-contained 8 box gene and insert among the pDblet of carrier modification (Brun etc., 1995, gene, Vol.164 pp.173-177).Lithium acetate method by means of Gietz and Woody is transformed into (FD Gietz and RA Woody, zymic molecular genetics: hands-on approach, the 8th chapter, pp 121-134) in the grain wine fragmentation sugar yeast cell with expression constructs.When selecting the existing of Ura4 mark, obtain and the clone of 110,000 the grain wine fragmentation sugar yeasts that increase.Collect the clone and form the library that is amplified of preparing with prescreen.Carry out following prescreen: enzyme substrates test, antimicrobial acivity, antibiotics resistance.6.5. duplicate prescreen ocean gram () bacterium/intestinal bacteria library by flat board

The marine bacteria that obtains near the seawater of collecting the Bahamas island is provided by Harbor BranchOceanographic Institute.Preparation DNA library with determine repeatability and have be used in carry out the order of prescreen in the library of determining to finish before, detect the painted marine bacteria kind of Gram-negative of every kind of agriotype

Carry out lower surface analysis in the ocean of parent's kind Gram-negative bacteria/Escherichia coli library, the result is as follows: positive kind Chromazurol S (CAS) 27 streptococcus pyogenes, 0 enterococcus (E.faecalis) the 3 proteus mirabilises 1 sarcina aurantiaca 10 staphylococcus aureuses 6 starch digestion liquid 17 in analyzing 37 kinds

In these are analyzed, below step be selected at intestinal bacteria; CAS; Streptococcus aureus; Sarcina aurantiaca; Carry out in the cell of the combination gene expression library in the starch digestion liquid.

In brief, each of 40 kinds of parent's kinds is inoculated in the 5ml B3 substratum and at 30 ℃, with 300 rev/mins rotating speeds overnight incubation in Falcon 2059 pipes.The culture that spends the night of precipitation also extracts full gene group DNA with standard method.By developing quantitate gene group DNA in sepharose, each of 40 kinds of 5 μ g is put into the storehouse that is total up to 200 μ g.As save the natural footpath expression library that connects of the assembly of describing among the 5.1.4 in intestinal bacteria.Explanation (Stratagene) according to SuperCos 1 manufacturer partly digests DNA, is connected among the SuperCos 1 and is packaged in lambda particles phage, to import intestinal bacteria.Produce 5 * 10 ⁶Single clone increases 7 * 10 by standard method ⁸/ ml cfu.Under-70 ℃, the storage liquid that increases is stored in 15% ethylene glycol in order to the back use.

In order to prepare the library that is used to screen each clone, the library cell that increases is layered on contains 50mlLB, in the 150mm plate of 100mg/ml penbritin and 50mg/ml kantlex.Under the room temperature in the dark with dry in advance 24 hours of plate, with 7 * 10 ⁸/ ml cfu stores and is diluted to 500cfu/ml with LB.1ml is coated with in each 150mm plate, after the coating, under 37 ℃, allows the plate incubated overnight.Transfer in the 384 hole plates with bacterium colony and ground, a hole that the toothpick picking of sterilizing produces.Select 6400 bacterium colonies and pile heap.75 μ l LB are contained in each hole, 50 μ g/ml penbritins, 7% ethylene glycol.Do not inoculate the row (80 holes altogether) of outside, but substratum is housed similarly to provide an evaporation barrier during insulation and the lyophilize in the back.37 ℃, the main ware nonoscillatory of these inoculations was placed 16 hours.The master's 384 hole plates that spend the night are used as the source flat board and copy among one or more work porous plates or the Omn1-Trays.Then wrap respectively by main 384-hole plate and freezing down at-80 ℃.Duplicate with spininess head copying instrument, before each the use and afterwards, before burning, sequentially 384 syringe needle copying instruments were immersed in the sodium hypochlorite solution 20 seconds, in the water 30 seconds, then in ethanol 5 seconds.

Work porous plate or Omni-Trays are used as the source plate the DNA library copied to a series of differentiation and/or to select (for example siderophore detects substratum (CAS) or antimicrobial lawn) in the substratum.The compilation result and with the collection of illustrative plates of the wild-type marine bacteria that is used for the constructed dna library relatively.

Separate 6 to the active male clone of starch digestion.Detect the ability that these clones suppress streptococcus aureus or sarcina aurantiaca, find that a clone suppresses the growth of sarcina aurantiaca.This clone is further analyzed, comprise dna sequence analysis, and find that it comprises those homologous protein DNA sequences in coding and the polyketide route of synthesis.Figure 10 shows the consistence from the actinorhodine dehydrase gene of the aminoacid sequence of the prediction of the dna sequence dna of clone CXC-AMN 20 and streptomyces coelicolor.

By with organic solvent extraction to further analyzing from this clone's activeconstituents and carrying out purifying according to antimicrobial mensuration.

Further measure to determine parent's kind of the same race by the dna sequence dna that composite PCR contains this clone.Sequence selection and synthetic pcr primer thing according to the clone.The ribosome-RNA(rRNA) primer sequence of high conservative is used as positive control in PCR.The fragment of the about 2kb of this positive control deposits yields.The amplicon that produces from this clone or its parent's kind of the same race is less than 600bp.Originally, composite PCR reaction is carried out in the standard method of the genomic dna in one group of four storehouse by using parent's kind.Genomic dna from storehouse 1-3 produces amplicon when increasing.Referring to Figure 11.Genomic dna with each parent's kind repeats composite PCR reaction.Figure 12 shows that in PCR reaction from storehouse a kind #6, the genomic dna of storehouse 2 kind #18 and storehouse 3 kind #31 is positive.This shows that certified dna sequence dna may be from any kind of of these three kinds of marine bacterias.

Therefore, the result shows that the combination gene expression library comprises and has from marine bacteria the clone of the genetic material of the pathways metabolism interested of encoding.And, show that also these clones in the library can be identified and separate by prescreen.6.6. by thick prescreen ocean gram (-) bacterium/intestinal bacteria library of dripping

By the weighing sodiun alginate (Protanol LF 20/60, Pronova Biopolymer, Drammer, Norway) and the applying pressure mixing tank with 2000 rev/mins of concentration it is dissolved in and wraps in the 100ml sterilized water by 30,000 clones with 1%.Add 1ml and contain the library suspension of 30,000 cells so that imbed 1-5 clone in every.Allow mixture leave standstill venting in 30 minutes.Then allow mixture from No. 25 pins, extrude.In the beaker that these fluid drop are stirred gently to the 0.5L that 135mM calcium chloride is housed.Allow a hardening also transfer to subsequently in the flask of sterilization in 10 minutes, and remove calcium chloride, replace with LB/Amp substratum and 80ug/ml substrate X-glucosaminide.Other substrate is the X-acetate, X-glycopyranoside, X-gal and the specific conventional substrate relevant with the polyketide approach.Under 30 ℃, will contain the flask shaken overnight of dripping and detect positive colony the next morning by the appearance representative of blue look bacterium colony.Also as will cloning of describing in the joint 5.414 with its bag quilt of indicator cells.Indicator cells comprises streptococcus aureus, sarcina aurantiaca.

Be placed on a big clean dish and observe dripping with eyes with individual layer.Reclaim the positive bacterium colony of X-glucosaminide, be suspended in 15% ethylene glycol again and-70 ℃ of storages.Reclaim other positive bacterium colony, and be placed in the 96 hole master's wares that contain LB/Amp and 50mM Trisodium Citrate pH 7.4, and under 37 ℃, allow its grow overnight with dissolved matrix.These master who spends the night 96 hole plates are copied among one or more work porous plates or the Omni-Trays as the source plate.Then bag is also-80 ℃ freezing by main 96 hole plates respectively, positive colony is sent to product is carried out particular detection or send to carrying out another and taking turns prescreen or screening.Further screening is finished by using spininess head copying instrument to duplicate.

Disclose exemplary embodiment of the present invention thus, it only is exemplary those skilled in the art will recognize that disclosed, can carry out various other selections within the scope of the present invention, improves and revises.Therefore, the specific embodiments that the present invention is not given an example by this paper limits, but only is subjected to the restriction of following claims.

Claims (41)

1. combination gene expression library, comprise the expression constructs storehouse, each expression constructs comprises from the cDNA of multiple donor biology or genomic DNA fragment, and wherein cDNA or genomic DNA fragment operability ground links with one or more regulatory regions of the genetic expression of being encoded by cDNA in the suitable host living beings or genomic DNA fragment that cause.

2. one kind is made up chimeric approach gene expression library, comprise the expression constructs storehouse, each expression constructs comprises cDNA or the genomic DNA fragment from the concatermerization at random of one or more donor biologies, and wherein concatermer cDNA or genomic DNA fragment operability ground links with one or more regulatory regions of the genetic expression of being encoded by concatermer cDNA in the suitable host living beings or genomic DNA fragment that cause.

3. the combination gene expression library of a bias, comprise the expression constructs storehouse, each expression constructs comprises cDNA or genomic DNA fragment, they some since special properties by preselected from multiple donor biology, wherein cDNA or genomic dna operability ground causes that with one or more regulatory regions by the genetic expression of cDNA in the suitable host living beings or genomic DNA fragment coding link.

4. the gene expression library of claim 1,2 or 3, wherein expression constructs comprises plasmid vector, phage, virus vector, cosmid vector or artificial chromosome.

5. the gene expression library of claim 4, the wherein shuttle vectors of carrier in different host cell kinds or bacterial strain, duplicating

6. the gene expression library of claim 1,2 or 3, wherein cDNA or genomic DNA fragment be from bacterium, fungi, algae, lichens, plant, protozoon, metazoan, coelenterates, insect, mollusk, sponge, worm, Amphibians, Reptilia, tunicate, bird or Mammals.

7. the gene expression library of claim 1,2 or 3, wherein the donor biology comprises the mixture of Lu Sheng microorganism or marine microorganism, or the mixture of Lu Sheng microorganism and marine microorganism.

8. the gene expression library of claim 1,2 or 3, wherein cDNA or genomic DNA fragment are from environmental sample.

9. the gene expression library of claim 7, wherein cDNA or genomic DNA fragment comprise operon or its part of one or more donor biologies.

10. the gene expression library of claim 9, wherein operon or the complete or part pathways metabolism of its part coding.

11. the gene expression library of claim 1,2 or 3, wherein each expression constructs is included in the host cell.

12. the gene expression library of claim 11, wherein host cell is by importing, and induces or the outer heat-extraction system of excessive generations activity is modified.

13. the gene expression library of claim 11, wherein host cell is by by importing, and induces or the interested known pathways metabolism of excessive generation or its part are modified.

14. the gene expression library of claim 11, wherein host cell is bacterium, fungi, vegetable cell, insect cell or zooblast.

15. the gene expression library of claim 14, wherein host cell is intestinal bacteria, subtilis, shallow Streptomyces glaucoviolaceus, streptomyces coelicolor, Saccharomyces cerevisiae, grain wine fragmentation sugar yeast, Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, tobacco, COS cell, 293 cells, the VERO cell, NIH/3T3 cell or Chinese hamster ovary celI.

16. the gene expression library of claim 1, wherein host cell further comprises by (tailored) of montage and is suitable for identifying the report system (regimen) of expressing the clone of required pathways metabolism or compound in the library.

17. the gene expression library of claim 16, wherein the report system comprises the DNA of the reporter gene that coding operability and the regulatory region that can be induced by the required pathways metabolism of host cell expression or compound or regulate link.

18. the gene expression library of claim 11, wherein host cell is arranged in the matrix of report system of the clone's who contains the required pathways metabolism of expression that is suitable for identifying the library or compound montage.

19. method for preparing the combination gene expression library, comprise dna vector is connected with the cDNA or the genomic DNA fragment that obtain from multiple donor biology, produce the expression constructs library, wherein the gene operability that comprises in cDNA or the genomic DNA fragment links with their the natural or allos regulatory region that causes that gene is expressed in suitable host cell.

20. method for preparing chimeric approach gene expression library, comprising will be from the biological cDNA that obtains of one or more donors or genomic DNA fragment concatermerization randomly, and the dna fragmentation of concatermerization is connected with dna vector to produce the expression constructs library, and the gene operability ground that wherein is included in cDNA or the genomic DNA fragment links with their natural or allos regulatory regions that cause that gene expresses in suitable host cell.

21. method for preparing bias combination gene expression library, comprise dna vector is connected with the cDNA or the genomic DNA fragment that obtain from one or more donors biologies, some of them are because the selected generation expression constructs of special properties library, and the gene operability ground that wherein is included in cDNA or the genomic DNA fragment links with their the natural or allos regulatory region that causes that gene is expressed in suitable host cell.

22. the method for claim 19,20 or 21, wherein dna vector is plasmid vector, phage, virus vector, cosmid vector or artificial chromosome.

23. the method for claim 22, the wherein shuttle vectors of carrier in different host cell kinds or bacterial strain, duplicating.

24. the method for claim 19,20 or 21, wherein cDNA or genomic DNA fragment be from bacterium, fungi, algae, lichens, plant, protozoon, metazoan, coelenterates, insect, mollusk, sponge, worm, Amphibians, Reptilia, tunicate, birds or Mammals.

25. the method for claim 19,20 or 21, wherein the donor biology comprises Lu Sheng microorganism or marine microorganism mixture, or the mixture of Lu Sheng microorganism and marine microorganism.

26. the method for claim 19,20 or 21, wherein cDNA or genomic DNA fragment are from environmental sample.

27. the method for claim 25, wherein cDNA or genomic DNA fragment comprise operon or its part of donator microorganism at least.

28. the method for claim 27, the wherein complete or part pathways metabolism of operon coding.

29. the method for claim 19,20 or 21 further comprises the expression constructs library is imported host cell.

30. the method for claim 29, wherein host cell is bacterium, fungi, vegetable cell, insect cell or zooblast.

31. the method for claim 30, wherein host cell is intestinal bacteria, subtilis, shallow Streptomyces glaucoviolaceus, streptomyces coelicolor, Saccharomyces cerevisiae, grain wine fragmentation sugar yeast, Spodopterafrugiperda, Aspergillus nidulans, Arabidopsis thaliana, tobacco, COS cell, 293 cells, the VERO cell, NIH/3T3 cell or Chinese hamster ovary celI.

32. the method for claim 29, wherein host cell further comprises the clone's who is suitable for identifying required pathways metabolism of expression in the library or compound of montage report system.

33. the method for claim 32, wherein the report system comprises that coding operability ground and required pathways metabolism that can be by host cell expression or compound are induced or the DNA of the reporter gene that the regulatory region regulated links.

34. the method for claim 29, wherein host cell is modified by importing, induce or excessively producing active discharge system.

35. the method for claim 29, wherein host cell is by by importing, and induces or the interested known pathways metabolism of excessive generation or its part are modified.

36. the method for the compound of interest in the identified gene expression library comprises:

(a) gene expression library of cultivation claim 11; With

(b) screening produces the cloned genes expression library of compound.

37. a method of screening the gene expression library of compound of interest comprises:

(a) gene expression library of cultivation claim 16; With

(b) detect the signal that the report system produces;

Thereby identify the clone who produces compound.

38. a method of screening the gene expression library of compound of interest comprises:

(a) gene expression library of cultivation claim 18; With

(b) detect the signal that the report system produces;

Thereby identify the clone who produces compound.

39. a method for preparing compound of interest comprises:

(a) cultivate the clone that claim 36 is identified; And

(b) from the clone's that identifies culture, reclaim compound.

40. a method for preparing compound of interest comprises:

(a) cultivate the clone that claim 37 is identified; And

(b) from the clone's that identifies culture, reclaim compound.

41. a method for preparing compound of interest comprises:

(a) cultivate the clone that claim 38 is identified; And

(b) from the clone's that identifies culture, reclaim compound.

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