CN101084311A - Biosynthesis of phloroglucinol and preparation of 1,3-dihydroxybenzene therefrom - Google Patents

Biosynthesis of phloroglucinol and preparation of 1,3-dihydroxybenzene therefrom Download PDF

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CN101084311A
CN101084311A CN 200580034761 CN200580034761A CN101084311A CN 101084311 A CN101084311 A CN 101084311A CN 200580034761 CN200580034761 CN 200580034761 CN 200580034761 A CN200580034761 A CN 200580034761A CN 101084311 A CN101084311 A CN 101084311A
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phld
cell
phloroglucinol
reconstitution cell
enzyme
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约翰·W·佛罗斯特
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University of Michigan
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Abstract

The present invention provides methods, enzymes, and cells for the biosynthetic production of phloroglucinol from malonyl-CoA, which is ultimately obtained from simple starting materials such as glucose; also provided are methods for preparing derivatives of biosynthetic phloroglucinol, including, e.g., resorcinol.

Description

The biosynthesizing of Phloroglucinol and by its preparation 1, the 3-dihydroxy-benzene
Statement of government interest
The present invention partly finishes under the government of the N00014-02-1-0725 fund that research department of naval authorizes supports.Government can have certain right of the present invention.
The cross reference of related application
The application submitted the U.S. Provisional Application 60/617,959 of " biosynthesizing of Phloroglucinol and by its preparation 1,3-dihydroxy-benzene " and the continuation application of 60/618,024 part simultaneously on October 12nd, 2004.
Background technology
Phloroglucinol (1,3, the 5-trihydroxybenzene) and derivative thereof are widely used in the commerce.Phloroglucinol and derivative thereof are as TRIMETHYL PHLOROGLUCINOL, as medicament, as spasmolytic.Phloroglucinol is as the parent material or the intermediate of medicine, microbicide and other organic synthesis.Phloroglucinol is used as the tinting material of the microscopy sample (as wood sample) that contains xylogen, and is used for the production of dyestuff, comprises leather dyestuff, textile dye and hair dye.It is used to produce tackiness agent, is used as epoxy curing agent, and is used for the preparation of explosive, as thermostability and the high explosive of vibrations stability, 1,3,4-triamino-2,4,6-trinitrobenzene (TATB).Phloroglucinol is also as antioxidant, stablizer and corrosion inhibitor, and as the preservatives of surrogate, bone sample decalcifying agent and the flower of the Silver iodide of the coupling agent of sensitization copy paper, rainmaking.Phloroglucinol also can change into Resorcinol by catalytic hydrogenation.
Resorcinol (1, the 3-dihydroxy-benzene) is the useful especially derivative of Phloroglucinol, though Resorcinol does not prepare by above-mentioned path at present.The same with Phloroglucinol, Resorcinol is used for the preparation of dyestuff and tackiness agent, and as epoxy resin cure reagent; And it is as the parent material and the intermediate of medicine and other organic synthesis.Resorcinol and derivative thereof or separately or with other activeconstituents such as sulphur, further usually be used for makeup, and treat the local skin medicine that comprises acne, dandruff, eczema and psoriasis and so on disease, partly play sterilant and pruritus.Resorcinol is also as the linking agent of chloroprene rubber, the viscosity-increasing agent in the rubber combination, is used in the linking agent of organic polymer (as trimeric cyanamide and rubber) and in the preparation of cellulosic and other matrix material.Resorcinol is used for: the production of resin and resin glue, for example simultaneously as monomer and UV absorption agent; In the production of explosive, for example as styphnic acid (2,4,6-trinitrobenzene-1,3-diphenol) and the such high energy compound of heavy metal styphnate; And diazotizing dyes, softening agent, Sucrets and para-aminosalicylic synthetic in.
Modal in the Resorcinol resinoid is Resorcinol-aldehyde and Resorcinol-phenol-urea formaldehyde.The resorcinol resin of these types for example is used for: resin glue, composite matrix and the parent material of making regenerated fiber and nylon product.The example of matrix material comprises Resorcinol-formaldehyde carbon (or other is organic) particulate hydrogel, aerogel and xerogel, is used for for example metal and substrate material organometallic catalyzer.Resorcinol-formaldehyde resin and also be used for dentistry as root canal filling with its particle composite material.
Resorcinol-urea formaldehyde tackiness agent needing to be used in particular for comprising in the application of high bond strength, for example: wooden truss, joist, bucket and canoe; And aircraft.Resorcinol-acetaldehyde resin the tackiness agent of improvement is also as wound or surgical incision in local wound and the body, as the biological wound sealing agent composition of blood vessel otch.This is through being usually used in military open-air medication, for example, environmental exposure minimize, reduce hemorrhage and body fluid runs off and quickens the recovery from illness process.This improved resins tackiness agent comprises that for example, gel-Resorcinol-formaldehyde and gel-Resorcinol-glutaraldehyde composition, aldehyde wherein can separate with Resorcinol-gel component earlier to be deposited, and remix forms tackiness agent when needing then.
At present, Phloroglucinol and Resorcinol all are by using caustic material and hot conditions, and from the parent material of petroleum derivation, and the chemical organic synthesis method that produces a lot of environment-conscious refuses is carried out coml production.
So providing more effective cleaner method for the production of these valuable compounds therefroms will be the progress in present technique field.A kind of possible solution provides biosynthetic pathway and produces Phloroglucinol, and to biosynthetic Phloroglucinol selective hydration to obtain Resorcinol.Reported the biosynthesizing of the compound that relates to Phloroglucinol in plant, algae and the microorganism, for example: from the ethanoyl Phloroglucinol of Rhodopseudomonas (Pseudomonsa); Hyperforine (hyperforins), hyperfoliatins, hyperjovinols and hyperatomarins from hypericum (Hypericum); Pallidusol, dehydropallidusol, trans-resveratrol (pallidol), mallopallidol and homomallopallidol from mallotus (Mallotus); Garcinielliptone from garcinia plant (Garcinia); Toxifren compound (flavaspidicacids) from Dryopteris (Dryopteris); The macrocarpals and the sideroxylonals that belong to (Eucalyptus) from eucalyptus; From 1,3 of rose (Rosa), 5-trimethoxy-benzene; And the glycosides and the brown alga polyphenolic substance (phlorotannins) that contain Phloroglucinol.
Yet these plants of report and the production of the Phloroglucinol in the microorganism only are more complicated degraded products, therefore more a spot of and/or more valuable parent material.Referring to, for example: L.Schoefer etal., Appl.Environ.Microbiol.70 (10): 6131-37 (2004); D.Baas ﹠amp; J.R é tey, Eur.J.Biochem.265:896-901 (1999).In addition, the microorganism biological synthetic production of diacetyl Phloroglucinol has been proposed as a kind of raising and has been discharged in the agricultural environment method of anti-mycotic activity that the recombinant bacteria of phytopathogen is resisted in as biocontrol agent.United States Patent (USP) 6,051,383 and M.G.Bangera ﹠amp referring to Thomashow etc.; L.S.Thomashow, J Bact.181 (10): 3155-63 (1999).But the anabolic biosynthesizing production of Phloroglucinol approach does not for example but provide from the production approach that cheap parent material such as glucose begin.
Recently, elaborated an alternative route (Fig. 2) of Phloroglucinol (1a), this approach comprises that the microorganism catalysis from glucose to nitrilotriacetic lactone (3a) is synthetic; Yet, find that nitrilotriacetic lactone (3a) is converted into Phloroglucinol (1a) needs a plurality of chemical steps.Referring to W.Zha et al., J.Am.Chem.Soc.126 (14): 4534-35 (2004); With C.A.Hansen ﹠amp; J.W.Frost, J.Am.Chem.Soc.124 (21): 5926-27 (2002).So, this approach is at most that part biological is synthetic, the approach of part chemosynthesis.
Therefore, so far, biosynthetic pathway does not come the report of commercial production Phloroglucinol (1,3, the 5-trihydroxybenzene) itself completely.The enzyme or the encoding gene that do not have catalysis to form Phloroglucinol itself are identified out.
Summary of the invention
The invention provides a kind ofly from malonyl CoA, and finally is method, enzyme and the cell for preparing Phloroglucinol from the biosynthesizing that the simple parent material such as glucose begins.Specifically, the invention provides first complete biosynthetic metabolic pathway of synthesizing of Phloroglucinol synthetic that is used for, this approach does not need four whole phlABCD operon enzymes, and can only carry out business-like Phloroglucinol production with phlD enzyme or other Phloroglucinol synthetic enzyme.The preparation method who comprises as the derivative of the biosynthetic Phloroglucinol of Resorcinol also is provided.The application in the preparation Phloroglucinol of described enzyme system, reconstitution cell and method; And prepared Phloroglucinol.Described enzyme system, reconstitution cell and method are preparing such as the application in the Phloroglucinol derivative of Resorcinol; Prepared Phloroglucinol and such as the derivative of Resorcinol.The application in preparation compound or composition of described enzyme system, reconstitution cell, method, Phloroglucinol or derivative, described compound or composition be for example: explosive or propellant compounds and composition; Described enzyme system, reconstitution cell, method, Phloroglucinol or derivative the preparation on-explosive, the compound of non-propelling agent and the application in the composition, the compound of described on-explosive, non-propelling agent and composition for example: medicine, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, matrix material or laminated or bonded material.Prepared explosive or propellant compounds and composition; Prepared on-explosive, the compound of non-propelling agent and composition.The present invention further provides:
A kind of isolating or the reorganization PhlD +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one; PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one; Genetic design improves the wherein PhlD of the expression of PhlD +Reconstitution cell, wherein enzyme system and reconstitution cell can be converted into Phloroglucinol with malonyl CoA; Be PhlA -, PhlB -And PhlC -Enzyme system and cell; The enzyme system and the cell that further comprise the enzyme of at least a synthetic malonyl CoA;
A kind of method for preparing anabolic Phloroglucinol comprises the enzyme system or reconstitution cell and malonyl CoA or other carbon source that provide such, and wherein said other carbon source can be converted into malonyl CoA by described enzyme system or reconstitution cell; Can under the condition of method for synthesizing phloroglucinol malonyl CoA be contacted with described enzyme system or reconstitution cell, perhaps can make described carbon source be converted into malonyl CoA and can condition by its method for synthesizing phloroglucinol under, other carbon source is contacted with described enzyme system or reconstitution cell; In the method, described carbon source is simple carbon source, for example: carbohydrate, aliphatic polyhydroxy compound or its combination; In the method, cell cultures is in the substratum that contains described carbon source, or described cultivation implements with the extractive fermentation method, and/or described cultivation adopts the many temperature curves such as the two temperature curve to carry out; And by the Phloroglucinol of described method preparation;
A kind of enzyme that malonyl CoA can be converted into the method for synthesizing phloroglucinol of the isolating of Phloroglucinol or reorganization; Comprise aminoacid sequence SEQ ID NO:2 or the aminoacid sequence of at least 70% homology is arranged, as enzyme as described in the conservative replacement variant of aminoacid sequence SEQ ID NO:2 with SEQ ID NO:2;
A kind of Nucleotide of isolating or reorganization of the open reading frame that comprises at least one coding PhlD enzyme, and be phlA -, phlB -Or phlC -In at least one; The Nucleotide of open reading frame that further comprises the enzyme of the synthetic malonyl CoA of at least one coding; Nucleotide, the ORF of the PhlD that wherein encodes comprises the base sequence of SEQ ID NO:1, have the base sequence of at least 80% homology, RNA base sequence correspondingly with SEQ ID NO:1 or with its redundant codon sequence;
A kind of phlD that has transformed with Nucleotide +Reconstitution cell, described cell can be expressed the Phloroglucinol synthetic enzyme by described Nucleotide; Be phlA -, phlB -Or phlC -In at least one cell; Be phlA -, phlB -And phlC -Cell; Further be phlE -Or at least one cell among the phlF;
A kind of method for preparing Resorcinol, comprise provide with described isolating or the reorganization enzyme system or the biosynthetic anabolic Phloroglucinol of reconstitution cell, and hydrogen and rhodium catalyst, Phloroglucinol is contacted with rhodium catalyst with hydrogen; And by the Resorcinol of anabolic Phloroglucinol preparation;
Described anabolic Phloroglucinol, or Resorcinol prepared therefrom, the application in producing medicine, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, matrix material or laminated or bonded material; Contain described anabolic Phloroglucinol or Resorcinol prepared therefrom, or the medicine, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, matrix material or the composition laminated or the bonded material that obtain by the chemically modified of described anabolic Phloroglucinol or Resorcinol prepared therefrom;
Can comprise with coding PhlD Nucleotide transforming this cell that described Nucleotide can be expressed by described cell, or is included in phlD a kind of the preparation from the method for the reconstitution cell of malonyl CoA biosynthesizing Phloroglucinol +Deactivation gene in the cell, this step comprise and are provided as phlA +, phlB +Or phlC +In at least one phlD +Cell, and deactivation at least one in phlA, phlB or the phlC gene wherein; Be the reconstitution cell of microorganism, described microorganism is for example bacterium, and its example comprises colon bacillus (Escherichia coli) and Pseudomonas fluorescens (Pseudomonas fluorescens); Enzyme system or reconstitution cell comprise the enzyme of synthetic malonyl CoA in the described method; Described method comprises provides phlABCD +Cell, and deactivation at least one phlA, phlB or phlC gene wherein, and/or insert at least one phlD therein +Nucleotide, described phlD +Nucleotide is phlA -, phlB -Or phlC -In at least one; PhlD in the described method +Reconstitution cell is phlA -, phlB -And phlC -Described method comprises provides phlABCD +Cell, and wherein whole phlA, phlB and the phlC genes of deactivation; Described method comprises provides phlABCD -Cell also inserts the phlD gene therein; PhlD in the described method +Nucleotide is positioned in the genomic dna of cell; PhlD in the described method +Nucleotide is positioned among the genome DNA outward of cell.
A kind of method for preparing propelling agent or blasting agent cpd, comprise providing and come the anabolic Phloroglucinol of biosynthesizing by described enzyme system or reconstitution cell isolating or reorganization, and this anabolic Phloroglucinol of chemically modified, or chemically modified Resorcinol prepared therefrom;
Described anabolic Phloroglucinol, or the application of Resorcinol prepared therefrom in the production of explosive or propelling agent; And comprise described anabolic Phloroglucinol or Resorcinol prepared therefrom, or the explosive or the propellant compositions that obtain by the chemically modified of described anabolic Phloroglucinol or Resorcinol prepared therefrom.
Description of drawings
Fig. 1 provides diagram 1, illustrates the approach of bibliographical information: (a) do the biosynthesizing of the ethanoyl Phloroglucinol of intermediate without Phloroglucinol, see M.G.Bangera ﹠amp; L.S.Thomashow, J Bact.181 (10): 3155-63 (1999); (c) S.Eckermann et al. is seen in the biosynthesizing of nitrilotriacetic lactone, Nature396:387 (1998), J.M.Jez et al., Chem.Bio.7:919 (2000); W.Zha et al., J.Am.Chem.Soc.126:4534 (2004).Also shown in this work hypothesis and identify with Phloroglucinol as the biosynthesizing path (b) of the ethanoyl Phloroglucinol of intermediate and (b ').
Fig. 2 provides diagram 2, illustrates: the commercial chemical route of synthesis (a, b, c) that Phloroglucinol is common; The rapid approach of previously presented multistep (d, e, f, g) from the glucose method for synthesizing phloroglucinol; The common commercial chemical route of synthesis of Resorcinol synthetic first-selection (i, j); The common commercial chemical route of synthesis of Resorcinol synthetic second (k, l).Also illustrated: the complete biosynthetic pathway of reporting in (1) this work (pointing out) that is used for Phloroglucinol production with the circle asterisk with the circle arrow; (2) the chemical hydrogenation effect (h) from the Phloroglucinol to the Resorcinol.Shown specific reaction or reactions steps are: (a) Na 2Cr 2O 7, H 2SO 4(b) Fe, HCl; (c) H 2SO 4, 108 ℃; (d) referring to W.Zha et al., J.Am.Chem.Soc.126:4534 (2004); (e) Dowex 50 H +, MeOH; (f) Na, methyl alcohol, 185 ℃; (g) 12 N HCI; (h) H i) 2, load on Al 2O 3On Rh, ii) 0.5 M H 2SO 4, reflux; (i) SO 3, H 2SO 4(j) NaOH, 350 ℃; (k) HZSM-12 zeolite, propylene; (l) i) O 2, ii) H 2O 2, iii) H +
Fig. 3 shows according to response path of inferring of the present invention, by this paths malonyl CoA by the Phloroglucinol synthetic enzyme by biosynthesizing be converted into Phloroglucinol, perhaps by enzyme activated 3,5-diketone pimelate (diketopimelate) (3,5-diketone pimelate (diketoheptanedioate)), perhaps by enzyme activated 3,5-diketone hexanoate (diketohexanoate) (3,5-diketone hexanoate (3,5-diketocaproate)).
Fig. 4 has illustrated the various exemplary approach that utilizes different carbon sources in anabolic Phloroglucinol building-up process.Dotted arrow is represented selectable utilization of carbon source approach; Be may absent variable intermediate in some approach in the square brackets.
It should be noted that accompanying drawing given herein is used for the general feature in illustration instrument of the present invention, material and the method, purpose is to describe these embodiments at this.These figure may accurately not reflect the feature of any embodiment that provides, and the embodiment in the unnecessary qualification or the restriction scope of the invention.
Embodiment
The invention provides the method, material and the organism that produce Phloroglucinol and derivative thereof.The present invention is based on the working foundation in the past; in this work, be surprisingly found out that monogenic expression just can produce the Phloroglucinol with suitable content; rather than by the polygene in microorganism ethanoyl Phloroglucinol path, as the expression of phlABCDEF.Publication; for example U.S. Patent No. 6,051, and 383; draw such conclusion: if Phloroglucinol produce with such gene, so probably preferably or only approach of so doing will be in this path, to add a kind of enzyme again so that the ethanoyl Phloroglucinol takes off acetyl at least.
Yet now unexpected the discovery is named as " Phloroglucinol synthetic enzyme " and the expression of the single enzyme that gets from phlD genetic expression can directly cause the formation of Phloroglucinol at this, that is to say, without the intermediate of acetylizad or diacetylation.Thereby this endonuclease capable leaves other any other phl operon gene and is expressed separately, reaches Phloroglucinol synthetic conspicuous level.In addition, this Phloroglucinol synthetic approach is by 3, and 5-diketone caproic acid thioesters intermediate carries out, rather than by 3,5, the sad thioesters of 7-triketone.The expression of further finding the Phloroglucinol synthetic enzyme itself that separates with other any phl operon gene be proved to be able to now the Phloroglucinol produced than any other biosynthesizing of proposition before this and half a lifetime the thing route of synthesis all many significantly; Therefore, Phloroglucinol is can be by producing than any other biosynthesizing that has proposed and all simpler, the more effective and more economical mode of thing route of synthesis in half a lifetime.
Only be used for the theme of disclosure of the present invention is carried out general tissue at these these titles used (as " background technology " and " in the invention ") and subtitle, rather than be used for limiting open or its any aspect of the present invention.Specifically, the technical elements that disclosed subject content can be included in the scope of the present invention in " background technology " is not quoted and do not constitute formerly technology.Disclosed theme is not the thoroughly detailed of four corner of the present invention or its any embodiment or fully open in " summary of the invention ".
Not constituting those reference at this reference of quoting is technology or admitting of any dependency arranged with patentability of the present invention disclosed herein formerly.The generality general introduction that in background technology, only is used to provide the author of these reference to advocate to any discussion of citing document content, and do not constitute admitting to the exactness of these reference contents.All reference that the embodiment of this specification sheets is partly quoted by reference, its whole contents is incorporated in this.
Description and specific embodiment when the explanation embodiments of the present invention are just to illustrative purposes rather than in order to limit the scope of the invention.And the quoting of a plurality of embodiments with definite feature is not other embodiment of having supplementary features in order to get rid of, and perhaps other has merged these and determines other embodiment of the various combination of feature.
Refer to embodiment that some benefit in the present invention in some cases be provided as word " preferably " and " preferably " of using herein.Yet other embodiment also can be preferred under same or other situation.In addition, one or more quoting and do not mean that other embodiment is useless preferred embodiment neither be used for getting rid of other and come from embodiment in the scope of the invention.
" comprising " and variant as the word of using herein, is nonrestrictive, and just quoting of Listed Items can not got rid of other similar item useful in material of the present invention, composition, apparatus and method like this.
Refer to used Nucleotide operation at this used word " reorganization ".Therefore, phrase is meant to small part it is to operate the entity that produces by Nucleotide as " reorganization " Nucleotide, " reorganization " polypeptide and " reorganization " cell.Comprise " intracellular " at this used term " intravital ", wherein said cell is meant viable cell.
Sequence homology
In preferred embodiment, has the aminoacid sequence of homology of exercising the polypeptide at least 50% of said function with original and this mutant according to the polypeptide of sudden change of the present invention.For example, Phloroglucinol synthetic enzyme according to the present invention has the aminoacid sequence with SEQ ID NO:2 at least 50% homology; In the preferred implementation, this sequence has the homology with SEQ ID NO:2 at least 60%; In the preferred implementation, this sequence has the homology with SEQ ID NO:2 at least 70%; In the preferred implementation, this sequence has the homology with SEQID NO:2 at least 80%; In the preferred implementation, this sequence has the homology with SEQ ID NO:2 at least 90%.
In one embodiment, as mentioned above, be the polynucleotide that any coding and original polypeptide of exercising said function have the polypeptide of homology according to the recombination of polynucleotide of coding desired polypeptides of the present invention.In one embodiment, according to the recombination of polynucleotide of coding desired polypeptides of the present invention, have with coding and exercise and original Nucleotide of the polypeptide of mutant identical function aminoacid sequence greater than 80% homology.In preferred embodiment, these polynucleotide have the homology with original Nucleotide at least 85%; In preferred embodiment, these polynucleotide have the homology with original Nucleotide at least 90%; In preferred embodiment, these polynucleotide have the homology with original Nucleotide at least 95%.
Sequence homology is meant between the amino-acid residue of two sequences, or the consistent degree between the Nucleotide of two sequences.This can determine by the visual comparison of two sequences, perhaps utilize the algorithm collating sequence of information biology compare or definite institute comparative sequences between the per-cent of homology.Useful automatic algorithm can obtain in GAP, BESTFIT, FASTA and TFASTA computer software module in the Wisconsin genetics software package (can from heredity computer software group, the Madison of Wisconsin, USA).Automatic permutation algorithm in those modules comprises Needleman ﹠amp; Wunsch, Pea rson ﹠amp; Lipman and Smith ﹠amp; Waterman series arrangement algorithm.The useful algorithm that other series arrangement and homology are determined is automatically in following software, comprising: FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL V; Referring to, for example, N.P.Brown et al., Bioinformatics:Applications Note, 1998,14:380-81; The http://www.ncbi.nlm.nih.gov/Tools/index.html of American National biotechnology information center (the U.S.National Center forBiotechnology Information); With United States Patent (USP) 6,790,639, wherein provide to be used for the definite software parameter setting of homology herein.
, can determine as the sequence homology as shown in Nucleotide and the nucleotide analog by nucleobase polymer by the check of the hybridization between the complement of first sequence and second sequence.Any known hybridization check can be used for this purpose, and the example of these checks comprises those at United States Patent (USP) 6,767, and that describes in 744 and 6,783,758 is limited to those hybridization checks under " highly rigorous " hybridization conditions.
The conservative replacement
In addition, Bao Shou aminoacid replacement can appear at according in the polypeptide of the present invention.Term " conservative aminoacid replacement " refers to the arbitrary amino acid of given amino-acid residue is replaced, to such an extent as to wherein this replacement residue reduces to the chemically so similar substance of polypeptide function (as enzymic activity) that can not cause of this given residue.Conserved amino acid is substituted by well known in the art, and its example is described in, and for example, United States Patent (USP) 6,790 is in 639,6,774,107,6,194,167 or 5,3 50,576.In preferred embodiment, conserved amino acid replaces in any one that will appear in following six groups:
1. little aliphatics, substantial non-polar residue: Ala, Gly, Pro, Ser and Thr;
2. big aliphatics, non-polar residue: Ile, Leu and Val; Met;
3. polarity, electronegative residue and aminocompound thereof: Asp and Glu;
4. polar aminocompound, electronegative residue: Asn and Gln; His;
5. polarity, positively charged residue: Arg and Lys; His; And
6. big aromatic residues: Trp and Tyr; Phe.
In preferred embodiment, conservative aminoacid replacement will be any one of following original residue (the conservative replacement) centering: Ala (Ser); Arg (Lys); Asn (Gln; His); Asp (Glu); Gln (Asn); Glu (Asp); Gly (Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe) and Val (Ile; Leu).
As polypeptide can contain the conserved amino acid replacement, polynucleotide herein also can contain conservative codon and replace.Codon is substituted in to produce when expressing and is considered to guard under the situation of aforesaid conserved amino acid under replacing.The codon that causes not having the degeneration of aminoacid replacement replaces, and also is used for according to polynucleotide of the present invention.Therefore, for example, the polynucleotide of the selected polypeptide that is used for embodiments of the present invention of encoding can replace by the codon of degenerating and suddenly change, and this codon usage frequency to be showed near the expression host cell that is used for transforming perhaps is used for improving its expression.
In addition, contain the polymkeric substance of nucleic acid base sequence, for example nucleic acid can preferably include those codings according to one or more enzyme of the present invention.And these polymkeric substance can comprise, for example, have shared the nucleic acid of at least 80% sequence homology with the nucleic acid of the given enzyme of coding.For instance, Phloroglucinol synthetic enzyme encoding sequence according to the present invention can have the base sequence with SEQ ID NO:1 sequence at least 80% homology; In preferred embodiment, this sequence has the homology with SEQ ID NO:1 sequence at least 85%; In preferred embodiment, this sequence has the homology with SEQ ID NO:1 sequence at least 90%; In preferred embodiment, this sequence has the homology with SEQ ID NO:1 sequence at least 95%.
The production of Phloroglucinol and derivative thereof
As shown in Figure 3; the Phloroglucinol synthetic enzyme is identified and characterizes at present; and this enzyme catalysis Phloroglucinol synthetic mechanism is unexpectedly found to carry out according to following series of steps; perhaps by another other mechanism; wherein provide to be transformed and form illustrated thioesters (SR) first malonyl CoA of the group of bonding; malonyl is provided, rather than ethanoyl.
● the ethanoyl activation-the first step comprises the activation of ethanoyl.This decarboxylation by malonyl CoA to be transferring to enzyme with the ethanoyl that produces, thereby forms enzyme activated ethanoyl thioesters (part that " R " among Fig. 3 represents enzyme or link to each other with enzyme); In another embodiment, the first step comprises the activation of whole malonyl, to form enzyme activated malonyl thioesters;
● the extension-next stage of chain comprises that two successive malonyl CoA decarboxylations form enzyme activated 3-ketone butyric acid thioesters so that ethanoyl is further shifted, and form enzyme activated 3,5-diketone caproic acid thioesters then; In another embodiment, successive shifts and forms the enzyme activated: 3-ketoisocaproic thioesters and 3,5-diketone pimelic acid thioesters;
● cyclisation-final step comprises 3, and the cyclisation of 5-diketone caproic acid thioesters intermediate forms Phloroglucinol; In another embodiment, taken place 3, the decarboxylation of 5-diketone pimelic acid becomes Phloroglucinol with cyclisation.
Three all steps are all by the catalysis of Phloroglucinol synthetic enzyme.
Enzyme system according to the present invention comprises at least one Phloroglucinol synthetic enzyme.In preferred embodiment, this Phloroglucinol synthetic enzyme obtains from pseudomonas; In preferred embodiment, from the member that Rhodopseudomonas (Pseudomonas) is planted, obtain: in preferred embodiment, from Pseudomonas fluorescens (P.fluorescens), obtain.In preferred embodiment, from Pseudomonas fluorescens Pf-5, obtain.The aminoacid sequence of the Pseudomonas fluorescens Pf-5 Phloroglucinol synthetic enzyme of identifying in this work is shown in SEQ IDNO:2, and its original encoding sequence is shown in SEQ ID NO:1.
In preferred embodiment, further contain at least one enzyme according to enzyme system of the present invention, this endonuclease capable or separately or with the formation of the common catalysis malonyl CoA of other enzyme.Malonyl CoA can prepare in biosynthesizing, for example, enzyme with synthetic malonyl CoA prepares from acetyl-CoA, the enzyme of synthetic malonyl CoA for example comes from the malonyl CoA synthetic enzyme (MatB) (referring to GenBank Accession AAC83455[gi:3982573]) of rhizobium leguminosarum (Rhizobium leguminosarum), and it is converted into malonyl CoA with malonyl; Come from rhizobium leguminosarum malonyl CoA decarboxylase (MatA) (referring to GenBank Accession AAC83456[gi:3982574]), it is converted into malonyl CoA with malonic semialdehyde; The perhaps transcarbocylase activity of acetyl-CoA carboxylase (EC 6.4.1.2), this enzyme carboxylation acetyl-CoA forms malonyl CoA.These parent materials of propanedioic acid, malonic semialdehyde or acetyl-CoA can be, and preferably biosynthetic; For example, but the acetyl-CoA biosynthesizing derive from such as in the various sources of glucose, photosynthetic 3-phosphoglycerate etc. any one.
Can in vivo also can be outside organism according to enzyme system of the present invention.When the enzyme of synthetic malonyl CoA is not provided, with in malonyl CoA adding and the substratum that cell and/or enzyme contact.In one embodiment, the Nucleotide of coding Phloroglucinol synthetic enzyme can be transformed in the cell of the organism that can synthesize malonyl CoA, and Phloroglucinol can generate therein in this case.The example of the synthetic malonyl CoA of organism comprises plant, algae, animal and human.Vitro system comprises, for example enzyme suspension or (absorption or covalently bound) immobilized enzyme bio-reactor in batches.System comprises in the body, for example, immobilized cell biological reactor, continuously ferments and fermentation in batches." fermentation " of herein using refers under any effective condition the cultured cells growth, and not necessarily under the condition of for example anaerobic condition or anaerobic metabolism, such condition only is another embodiment here.In any one embodiment, the source of malonyl CoA can offer the Phloroglucinol synthetic enzyme, and no matter this source is (for example external source) malonyl CoA or biology in situ synthetic (for example endogenous) malonyl CoA that adds.
Can express at least a Phloroglucinol synthetic enzyme according to reconstitution cell of the present invention, with or express or do not express the enzyme of at least a synthetic malonyl CoA, but neither whole phlABCD operons neither all three phlA, phlB and phlC genes.In preferred embodiment, reconstitution cell is a kind of cell of Phloroglucinol synthetic enzyme of express recombinant therein.In preferred embodiment, the reconstitution cell that can express the Phloroglucinol synthetic enzyme and can express or can not express the enzyme of synthetic malonyl CoA is a lepocyte.The example of lepocyte comprises vegetable cell, yeast/fungal cell, bacterial cell, Archimycetes cell and some protobionts.In one embodiment, reconstitution cell can be does not have pipe plant (as liver moss), protobiont (as algae), yeast, fungi, bacterium or Archimycetes cell.In one embodiment, reconstitution cell can be the microorganism of reorganization.In one embodiment, reconstitution cell can be yeast, fungi, bacterium or Archimycetes cell, more preferably yeast, fungi or bacterial cell.In preferred embodiment, this reconstitution cell can be bacterial cell.In preferred embodiment, this reconstitution cell can be protein mycetocyte (proteobacterial cell).Preferably, this reconstitution cell shortage is expressed and is come from phlABC, the ability of the functional enzyme of phlE and phlF gene.In preferred embodiment, described cell can be phlABC -, phlE -And phlF -Cell.The host cell of reorganization can comprise at least a Nucleotide according to Phloroglucinol synthetic enzyme of the present invention.In preferred embodiment, described Nucleotide can exist with the form of carrier, as plasmid or transposon.
Be used to form useful phlD +In the embodiment of reconstitution cell, by any gene knockout technology (that is to say any technology that causes cell can not make and remove or suddenly change), with phlD by the gene of the functional expression product that knocks out preceding genes encoding +And phlA +, phlB +And/or phlC +Cell can be made into phlA -, phlB -And/or phlC -Cell.Preferably, phlA all in the cell, phlB and phlC gene are knocked out.The cell that obtains can keep its PhlD +Phenotype.PhlE and/or the phlF gene knockout that exists in the described cell can or not do not knocked out yet.One preferred embodiment in, phlABCD +Cell can be made into phlABC -In one embodiment, phlD -And phlA -, phlB -And/or phlC -Cell in can insert the Nucleotide of effable PhlD coding, perhaps be inserted in its genomic dna or as the part of karyomit(e) other unit, as plasmid, perhaps the both has, thereby makes phlD +Cell.One preferred embodiment in, phlABCD -Cell can be made into phlD +
In some embodiments, PhlD +Original or the reorganization cell, as phlD +Cell can further be replenished with additional phlD gene, transforms by the Nucleotide with the open reading frame that contains one or more effable coding Phloroglucinol synthetic enzyme.PhlD +Cell can be PhlA -, PhlB -, and/or PhlC -Cell is as phlA -, phlB -And/or phlC -Cell; Perhaps can be PhlA +, PhlB +And/or PhlC +Cell is as phlA +, phlB +And/or phlC +Cell (phlABCD for example +Cell).The reconstitution cell that can express additional phlD gene of gained can show enhanced Phloroglucinol synthesis capability.
Similar to reconstitution cell, enzyme system isolating or reorganization according to the present invention comprises at least a Phloroglucinol synthetic enzyme, also can comprise or not comprise the enzyme or a cover enzyme of at least a synthetic malonyl CoA, but do not comprise whole three PhlA, PhlB and PhlC enzyme, and preferably do not comprise in PhlA, PhlB and the PhlC enzyme any one.Therefore, comprise at least a Phloroglucinol synthetic enzyme and can comprise or not comprise the enzyme of at least a synthetic malonyl CoA, but comprise be less than whole three PhlA, PhlB and PhlC enzyme according to reconstitution cell of the present invention and enzyme system, be meant PhlA -, PhlB -And PhlC -In at least one, PhlABC preferably -PhlD +Entity.
The method of producing Phloroglucinol comprises the Phloroglucinol synthetic enzyme is contacted with malonyl CoA.The method of producing Resorcinol is included in carries out hydrogenation on the biosynthetic Phloroglucinol, for example use hydrogen and rhodium catalyst.The Phloroglucinol of producing with the method according to this invention and Resorcinol can be used to or as or prepare, such as medicine, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, propelling agent, explosive, matrix material and composition laminated or the bonded material.
Full cell fermentation pattern
The full cell fermentation of reconstitution cell herein can carry out in any training mode, preferably adds in formula or continous way (or semi continuous, just the inoculate formula) pattern at batch-type, stream and carries out.In some embodiments herein, by cultivating the processed Phloroglucinol that extracts of the exhausted substratum that contains Phloroglucinol that reconstitution cell according to the present invention produces.Yet, in some cases, Phloroglucinol itself, when in growth medium, having reached threshold concentration, can be to institute's cultured cells toxigenicity in being called as the process of end-product inhibition.The cell viability that can reduce the cells produce of Phloroglucinol when this inhibition exists and can cause reducing.Therefore, in some embodiments, preferably stage that cell is produced Phloroglucinol actively from still useful in addition growth medium to Phloroglucinol with also can extract to Phloroglucinol derivative (if present).Such embodiment is called " extractive fermentation " at this.
In this collection, get fermentation and can carry out with useful any pattern known in the art.For example; some embodiments have used dispersive extractive fermentation pattern; wherein can extract in the substratum that the extraction absorption agent of Phloroglucinol or absorbent liquid or particulate be introduced into the reconstitution cell growth mutually, extract particulate or liquid regions there and contacted and extract Phloroglucinol.As any extraction process, can be nonspecific, preferential or specific with this absorption agent or absorber material to the extraction of this product, and the preferably preferential or specific extraction to Phloroglucinol.
After " carrying " Phloroglucinol, " carrying " extraction phase of is shifted out from substratum, for example by centrifugal, filter, the magnetic force of magnetic or magnetizable particulate collected and/or by phase disengagement method, rise on the body of substratum as extraction phase or drop under it.In some embodiments, adverse current or crossing current abstraction technique can be used for extracting Phloroglucinol from substratum, comprise as extraction liquids or particulate phase as adverse current or crossing current with respect to medium flow.
In some embodiments, the membrane type extractive fermentation is that substratum is undertaken by the spe membrane such as ion-exchange membrane.In some embodiments, pillar extractive fermentation formula is that substratum is undertaken by column extractor, and column extractor is hollow fiber membrane extraction device post or fibrous or pearl resin column for example.Cultured cells can be passed through pillar in substratum, and perhaps part or all of cell was removed before substratum is by pillar, for example by filtering.In any extractive fermentation method, extractive fermentation can carry out once, repeatedly or during the fermentation carry out continuously.In adverse current, crossing current, membrane type and pillar extractive fermentation pattern, the substratum that some of them or whole (preferably most ofs' or whole) Phloroglucinol has been removed is got back in the fermenting container again then.
In preferred embodiment, pillar extractive fermentation technology is used for removing Phloroglucinol at biosynthetic process from substratum.Useful medium for this purpose comprises anionic exchange medium, as anionresin pearl, anion-exchange fibre and anionresin tubular fibre.Same, anion-exchange membrane and anionic exchange medium particulate are respectively applied in membrane type and the dispersive extractive fermentation pattern.When using particulate anionresin matrix, though fixed bed pattern also can selectively be used the preferred fluidized-bed extractive fermentation pattern that adopts.
Useful anionic exchange medium can comprise any upholder, and is organic or inorganic, and it comprises or the covalent attachment anion exchange groups.In some embodiments, can use organic upholder, as vinylbenzene-Vinylstyrene, polystyrene, polyethylene, acrylic acid or the like, P-F, silicoorganic compound or cellulosic polymer backbone, wherein said skeleton comprises or is connected with anion exchange groups.
Useful anion exchange groups can be any cation group, and preferred non-metal cations group is as organic ammonium, sulfonium and phosphine groups.The preferred cation group comprises organically: tertiary amine (for example DEAE-cellulose), quaternary ammonium, pyridinium salt, uncle's sulfonium and season phosphine groups.In one embodiment, the anion exchange groups in the described anionresin matrix is quaternary ammonium or pyridinium salt group.The resinoid example of quaternary ammonium comprise AG-1 X8 resin (from Bio-Rad Laboratories Inc., Hercules, CA, USA) and the DOWEX1 resin (from The DOW Chemical Co., Midland, MI, USA); The resinoid example of pyridinium salt comprises polyethylene-alkyl-pyridinium salt resin, available haloalkane processing polyethylene-pyridine resin such as REILLEX HP are (from Reilly Industries, Inc., Indianapolis, IN, USA) obtain, perhaps directly obtain, as gathering (4-vinyl N-methyl iodate pyridine) (from Polymer SourceInc., Montreal by commercially available source, QC, CA).
One preferred embodiment in, before use, handle anionic exchange medium to prepare the phosphate complex with cation group of this matrix.When anionic exchange medium is reused, (does not interfere and therefrom remove Phloroglucinol), perhaps with the Continuous Contact of given fermentation in, preferably replace with enough frequencies, do not cause that end product suppresses the level that takes place so that the concentration of the Phloroglucinol in the substratum can not rise to anionic exchange medium new or that upgrade; Preferably be not higher than about 2g/L, or be not higher than the Phloroglucinol of about 1.5g/L.
By extractive fermentation or fermentation back extracting process, anionresin matrix optimization water, acidifying water, acidifying alcohol (for example tart ethanol) or its combination of having carried Phloroglucinol are carried out carrying out washing treatment and are removed Phloroglucinol.Preferred technology is that washing is washed with acidifying alcohol then earlier.(preferably) after most of or whole Phloroglucinol is removed from anionresin matrix, can prepare this matrix utilizes in the extraction of Phloroglucinol again, for example before utilizing again, form cation group-phosphoric acid salt mixture by carry out balance with phosphate solution.The Phloroglucinol that exists in these washes can further separate and/or purifying with any technology known in the art, for example, is separated solvent evaporates etc.
Full cell fermentation condition
The full cell culture that is used for producing according to the method for Phloroglucinol of the present invention will utilize condition that allows the cell growth and the condition that allows the anabolic Phloroglucinol of cells produce of growth.In some cases, the Phloroglucinol synthetic enzyme can be expressed in running through whole cell culture period, for example constitutes and expresses; Yet under many circumstances, desirable is only near just beginning to express the Phloroglucinol synthetic enzyme exponential phase of growth during (EGP) terminal point.Expect when the later stage expresses, can when about EGP of 70% to 100% be over and done with, be activated or derepress at the Phloroglucinol synthetic enzyme encoding sequence of regulating under the promotor control, preferably be activated or derepress when over and done with to about 80% EGP when about 70% to about 90%, more preferably about 70%.The example that is used for the promotor of this purpose comprises tac, T5 and T7 promotor; P preferably wherein T7Can induce with lactose or gratuitous induction agent such as IPTG (sec.-propyl-β-D-thio-galactose pyran-glucoside).
Herein some preferred embodiment in, the microorganism cells of reorganization can be used as the whole-cell biological catalyzer as the recombinant bacteria host cell.The preferred bacterium that is used for this purpose comprises the protein bacterium; The example of preferred mycetozoan comprises the γ mycetozoan, as enterobacteria and pseudomonas; Escherichia, as colon bacillus (E.coli), and Rhodopseudomonas, as Pseudomonas fluorescens, be wherein preferred.Preferred microorganism is those shortages or treated to reduce or to eliminate those and can degrade according to the microorganism of the protease activity of the enzyme of Phloroglucinol synthetic enzyme of the present invention and/or synthetic malonyl CoA.In bacterium, Lon and OmpT are two proteolytic enzyme that preferably lack or be lowered or eliminate, as passing through sudden change.E.coli bacterial strain BL21 and W3110 are the phlABCD that preferably is used for inserting the phlD gene -The example of cell; The bacterial strain Pf-5 of Pseudomonas fluorescens is preferred phlABCD +The example of cell, this cell is the phlA, the phlB that are used to have or do not have the insertion of additional phlD gene and/or the deactivation of phlC,, perhaps be used to have the deactivation of the phlABCD that inserts additional phlD gene,, perhaps be used to have replenishing of additional phlD gene.E.coli bacterial strain BL21 is obtainable to be: and BL21 STAR (DE3) ONE SHOT (Invitrogen Corp., Carlsbad, CA.USA); Perhaps ULTRA BL21 (DE3) (Edge BioSystems, Gaithersburg, MD, USA).E.coli bacterial strain W3110 is obtainable be ATCC No.27325 (American Type Culture Collection, Manassas, VA, USA); The bacterial strain Pf-5 of Pseudomonas fluorescens is obtainable to be ATCC No.BAA-477.
Under the situation of E.coli, preferred leavening temperature is from about 20 to about 37 ℃, preferred about 25 to about 37 ℃, and more preferably from about 30 ℃ to about 37 ℃.Have been found that in anabolic Phloroglucinol synthetic situation, during the EGP or at least at the higher temperature during the fore portion of inducing of EGP, with during the maintenance cultivation stage of small part (for example, after running through the inducing of all or part, or all or part keeps stage phase) the combination of lesser temps be the key character of Phloroglucinol production decision preferably.Therefore, one preferred embodiment in, during the EGP, the E.coli cell of reorganization is grown under about 35～37 ℃, preferably at about 36～37 ℃, more preferably under about 36 ℃ at EGP or before inducing; In the maintenance phase or the period after inducing, the E.coli cell of reorganization is grown under about 30～34 ℃, preferably at about 30 to about 33 ℃, more preferably at about 33 ℃ or under about 30 ℃.In some embodiments, can well betide the maintenance phase, for example, finish the back from cultivating beginning in about 15 hours at EGP to cryogenic conversion.Therefore, under cell (as E.coli) the cultivation situation when EGR ends to begin about 15 hours from cultivation, can occur in from the conversion of comparatively high temps for forming the two temperature fermenting characteristic to lesser temps, for example, about 11 or about 12 hours the time (for example, approximately with 70% or 80%EGR induce a while), or about 15 hours the time, or even when beginning about 30 hours from cultivation.Perhaps, the comparatively high temps that for example is used for EGR in such two temperature embodiment is useful temperature to whole culture cycle.Under the situation of Pseudomonas fluorescens, preferred temperature is from about 20 to about 30 ℃, and preferred comparatively high temps is from about 27 to about 30 ℃, and preferred lesser temps is from about 24 to about 27 ℃.
Carbon source
The embodiment that is used for producing cell, enzyme system and the method for Phloroglucinol according to the present invention will utilize carbon source.Wherein the carbon source except that malonyl CoA contacts with cell or enzyme system, can utilize any other carbon source that provides according to enzyme system of the present invention or reconstitution cell, enzyme system or the reconstitution cell according to the embodiment of putting into practice can become the carbon source metabolism material that can be used as anabolism described herein ground synthetic Phloroglucinol like this.Except that the Phloroglucinol synthetic enzyme, which kind of carbon source the character of other zymetology and the composite reactive that is present in the selectable malonyl CoA in the reaction mixture will determine to utilize.Fig. 4 has illustrated from the many representative approach of the anabolism ground method for synthesizing phloroglucinol that different carbon sources begin.
Therefore, concerning most of carbon sources, cell or enzyme system or will at first decompose carbon source or fix it provide simple organic molecule, and by these simple organic molecules, cell can form acetyl-CoA.Perhaps, carbon source can be converted into acetyl-CoA.For example, for the acetate carbon source, acetate can be converted into acetyl-CoA, perhaps can be converted into acetyl phosphate earlier.Acetyl-CoA itself also can be used as carbon source.In case obtain or be provided, acetyl-CoA can be used for synthetic malonyl CoA by the effect of acetyl-CoA carboxylase.When the degraded of carbon source produces metabolic propanedioic acid or metabolic malonic semialdehyde, or when propanedioic acid or malonic semialdehyde are present in the carbon source, malonyl CoA synthetic enzyme or malonyl CoA decarboxylase that they can be present in respectively in the enzyme storehouse of cell or enzyme system are converted into malonyl CoA, perhaps they can be used for the synthetic of acetyl-CoA by metabolism, and subsequent transformation is a malonyl CoA.In case obtain or be provided, malonyl CoA just is used as the substrate of Phloroglucinol synthetic enzyme, forms Phloroglucinol.
When carbon source contained biomolecules class carbon compound, this carbon compound is primary metabolite type compound preferably.The example of primary metabolite type compound preferably includes any one in the following compounds: preferred C1～C18: lipid acid, wax, list-, two-, the Three-glycerol ester; Polyol; Aliphatic hydroxyl acid; Phosphatide; Phosphonic acids; Monose (for example triose, tetrose, pentose, hexose, heptose etc.); Amino acid; And Nucleotide; And the hydrolysis homologous and allogenic oligomer (for example comprising dipolymer) and the polymer that derive from these compounds; Biologically active form (for example acetyl-CoA) with these compounds.Biomolecules type compound can be any source, or biogenetic derivation or synthetic.Other preferred compound comprises any little or uncomplicated organic compound, the aliphatics of just common C1～C18, alicyclic and aromatics etc., preferred per 18 carbon atoms have four or carbon-to-carbon branch point still less, for example: C1～C18 aliphatic hydrocarbon and their single acid and polyacid, single pure and mild many alcohol, monoamine and polyamines, single carbonyl and many carbonyls; And the hydrolysis homologous and oligomeric thing and the polymer that therefrom form.
Comprise so little/uncomplicated organic compound, and/or primary metabolite type compound, and do not have the carbon source of the secondary metabolites of essence concentration (concentration that preferably is less than the carbon of 10wt%) or big haplotype or complicated organic compound to be called " simply " carbon source at this yet.As using herein, secondary metabolites comprises, for example: alkaloid; Tonka bean camphor; Polyketide; Terpenoid; Isoprenoid; Sterol; Steroid; And prostaglandin(PG); Catecholamine; Porphyrin; Xanthone; Flavonoid; Phenylpropyl alcohol alcohols (phenylpropanoids) and phenol compound (comprising for example benzenoid and polyphenol) etc.Big or complicated organic compound is aliphatic, alicyclic and aromatic compound etc., and they have greater than the monomeric compound size of C18 and/or the complicacy monomeric compound greater than 4 carbon-to-carbon branch points in per 18 carbon atoms.
In preferred embodiment, carbon source is simple carbon source.Preferred simple carbon source comprises 0% to about 5%, and more preferably 0% to about 2%, or 0% to about 1%, or 0% to about 0.5%, or the secondary metabolites of preferred about 0% weight and bigger or complicated organism; Perhaps preferably there are not or do not have at least in fact secondary metabolites and bigger/complicated organism.In some embodiments, simple carbon source comprises primary metabolite type compound.Comprise at this preferred example of primary metabolite type compound of using: carbohydrate, preferred monose and/or disaccharides; And polyol.Glucose, wood sugar and pectinose are the examples of the preferred monose in this used carbon source; Glycerine is a preferred example of polyol.In this a embodiment, glucose, wood sugar and/or pectinose can be used as carbon source, are preferably used as exponential phase of growth of running through cell cultures and the carbon source during the maintenance phase.In one embodiment, use the combination of monose (preferably glucose, wood sugar and/or pectinose) and glycerine, for example, with the weight ratio of 1: 1 or 2: 1; Preferably, such combination only is used in the maintenance phase, and wherein monose (not having glycerine) was used in during exponential phase of growth.
Embodiment
Phloroglucinol (1a, diagram 1, just Fig. 1) is found to be the substituting group in the multiple natural product.Yet the Phloroglucinol 1a conduct independently biosynthesizing of molecule was not was not also recorded and narrated.As the active part Study of this biosynthesizing, analyzed the ethanoyl Phloroglucinol (6 and 7, diagram 1) that in Pseudomonas fluorescens Pf-5, forms.B.Novak-Thompson et al.，Can.J.Microbiol.40：1064(1994)。Detected the Phloroglucinol biosynthesizing.The heterogenous expression of Pseudomonas fluorescens phlD has afterwards caused Phloroglucinol 1a gathering in the colon bacillus culture.Except the biosynthetic inference about the ethanoyl Phloroglucinol, the benign synthetic basis of new environment of Phloroglucinol and Resorcinol has been set up in these discoveries.
Activated 3,5-diketone hexanoate (diketonhexanoate) 2 (is an activated 3,5-diketone hexanoate (3,5-diketocaproate)) be likely the simplest polyketide natural product on two structures, the precursor of Phloroglucinol 1a and nitrilotriacetic lactone 3a (diagram 1), for example, from the conversion of the 2-pyrone synthetic enzyme catalysis malonyl CoA of African chrysanthemum (Gerberahybrida) to the nitrilotriacetic lactone.S.Eckermann et al.，Nature 396：387(1998)；J.Jez et al.，Chem.Bio.7：919(2000)。Avtive spot tyrosine is changed to the formation that phenylalanine has caused the nitrilotriacetic lactone, unique biosynthetic products as Brevibacterium ammoniagenes (Brevibacterium ammoniagenes) fatty acid synthetase, B.W.Zha et al., J.Am Chem.Soc.126:4534 (2004).Under the situation of enzyme, do not observe the formation of Phloroglucinol with the naturally occurring or sudden change of any of these.The possible precursor of another selectable Phloroglucinol 1a is an activated 3,5-diketone pimelate (diketoheptanedioate) 12 (just, activated 3,5-diketone pimelate (diketopimelate)).
Biosynthetic the detecting of Phloroglucinol 1a pointed to Pseudomonas fluorescens Pf-5 and 2, the biosynthesizing of 4-diacetyl Phloroglucinol 7 (diagram 1).B.Novak-Thompson et al.，Can.J.Microbiol.40：1064(1994)。The biosynthesizing of ethanoyl Phloroglucinol by contain phlACBD, by the albumen and the discrete gene cluster coding of transcribing the regulon of phlE coding of the product output of phlE coding.PhlD is believed to comprise in activated 3,5, in the formation and cyclisation of 7-triketone sad 5 (diagram 2).The intermediate 2-ethanoyl Phloroglucinol 6 of supposition gained is acetylation and forms 2,4-diacetyl Phloroglucinol 7 (diagram 2).Referring to M.G.Bangera﹠amp; L.S.Thomashow, J Bacteriol.181:3155 (1999).The biosynthesizing of Phloroglucinol 1a is not the activity owing to PhlD.
The product that gathers in the culture supernatant of analysis of fluorescence pseudomonas Pf-5/pME6031.Except (table 1 clauses and subclauses 1) 2, outside 4-diacetyl Phloroglucinol 7 and the 2-ethanoyl Phloroglucinol 6,, found the formation of Phloroglucinol.For improving the concentration of biosynthetic Phloroglucinol, Pseudomonas fluorescens PF-5 transforms with pJA2.232, and pJA2.232 is derived from the plasmid that the phlABCDE gene cluster is inserted into pME6031.Purpose is the adjusting of avoiding the PhlF by genome encoding to be undertaken by the multiple copied that presents biological synthesis gene cluster.This method has caused Pseudomonas fluorescens PF-5/pJA2.232 (project 2, table 1) synthetic Phloroglucinol compounds 1a, and 6,7 concentration improves greatly with respect to Pseudomonas fluorescens PF-5/pME6031 (table 1 clauses and subclauses 1).
Table 1. is biosynthetic Phloroglucinol 1a, 2- ethanoyl Phloroglucinol 6 and 2 by the construct of expressing the phlACBDE gene, the peak concentration of 6-diacetyl Phloroglucinol 7.
Project Host/plasmid The plasmid inset Phloroglucinol (mg/L)
1a 6 7
1 2 3 4 5 6 7 8 P.fluorescens PF-5/pME6031 aP.fluorescens PF-5/pJA2.232 aE.coli BL21(DE3)/pJA3.085 bE.coli BL21(DE3)/pJA3.156 bE.coli BL21(DE3)/pJA2.042 bE.coli JWF1(DE3)/pJA3.131A cE.coli BL21(DE3)/pJA3.169 bE.coli BL21(DE3)/pJA3.169 b，d No phlACBDE phlACBDE phlACBD phlD phlD phlACB phlACB 10 470 32 22 720 780 0 39 23 500 14 13 0 0 0 17 35 790 0 0 0 0 0 2
aCell is incubated in the YM substratum under the condition of jolting flask.
bCell is incubated in the TB substratum under the condition of jolting flask and results.
After being resuspended in the minimum base status of M9, cell is cultivated under the condition of jolting flask.
cCell is incubated under the condition of control fermentor tank in the minimum base status of M9.
dCell adds Phloroglucinol (50mg/L) after being resuspended in the minimum base status of M9.
Then further analyzed the heterogenous expression (table 1 clauses and subclauses 3～8) of the T7 promotor of phlACBDE gene among the E.coli.All E.coli constructs also carry the chromogene 1 that has inserted the coding t7 rna polymerase.Carry E.coli BL21 (the DE3)/pJA3.085 method for synthesizing phloroglucinol and the 2-ethanoyl Phloroglucinol of plasmid phlACBDE inset, and Synthetic 2 not, 4-diacetyl Phloroglucinol (table 1 clauses and subclauses 3).The shortage of the product output body of phlE coding does not have adverse influence (table 1 clauses and subclauses 4) to the concentration of biosynthetic Phloroglucinol 1a and 2-ethanoyl Phloroglucinol 2 among E.coli BL21 (DE3)/pJA3.156.The product of using E.coli BL21 (DE3)/pJA2.042 to estimate then to be accompanied by the heterogenous expression that phlD is only arranged forms (table 1 clauses and subclauses 5).Only observed the formation of Phloroglucinol 1a.The concentration of E.coliBL21 (DE3)/biosynthetic Phloroglucinol 1a of pJA2.042 has reflected the degree of approach of phlD and T7 promotor probably with respect to the difference (4 pairs of projects 5 of project, table 1) of the concentration of the Phloroglucinol 1a of E.coliBL21 (DE3)/pJA3.156.With E.coli JWF1 (DE3)/pJA3.131A check and analysis synthetic (table 1 clauses and subclauses 6) of the Phloroglucinol that in minimum base status, begins from glucose under the condition of control fermentor tank.
For further studying the function of PhlD in the Phloroglucinol biosynthesizing, PhlD is purified to homogeneity and has analyzed its external zymologic property.When acetyl-CoA is used alone as substrate, do not observe activity (table 1).The situation that malonyl CoA and acetyl-CoA are hatched with PhlD together with only compare with the situation that PhlD is hatched with malonyl CoA, observed the specific activity that approximately equates.Measured the K of PhlD for malonyl CoA m=37 μ M, and k Cat=4.7min -1As a comparison,, use activated 3 as PhlD, the 2-pyrone synthetic enzyme of 5-diketone hexanoate 2 (diagram 1) is purified to homogeneous.2-pyrone synthetic enzyme can not utilize acetyl-CoA as substrate.Yet the specific activity that 2-pyrone synthetic enzyme is hatched generation with malonyl CoA and acetyl-CoA is the twice that the ratio when hatching altogether with malonyl CoA when not having acetyl-CoA is lived.The kinetic parameter of 2-pyrone synthetic enzyme comprises the K to malonyl CoA m=XX μ M is to the K of acetyl-CoA m=2.2 μ M, and k Cat=3.3min -1
The product that heterogenous expression by PhlACBDE and PhlD forms has improved activated 3; the cyclisation possibility of 5-diketone hexanoate 2 (diagram 1); and the progressively acetylize of Phloroglucinol 1a afterwards may be a biosynthesizing 2- ethanoyl Phloroglucinol 6 and 2, the basis of 4-diacetyl Phloroglucinol 7.For further studying this possibility, E.coli BL21 (the DE3)/pJA3.169 that has plasmid-localized phlACB is fabricated.M.G.Bangera & L.S.Thomashow，J Bacteriol.181：3155(1999)。The cultivation (table 1 clauses and subclauses 7) of the construct by this shortage plasmid-localized phlD does not have Phloroglucinol to be synthesized.Yet, in E.coli BL21 (DE3)/pJA3.169 substratum, add Phloroglucinol 1a and caused 2 of 2-ethanoyl Phloroglucinol 6 and a small amount of, the formation of 4-diacetyl Phloroglucinol 7 (project 8, table 1) really.
PhlD also particularly important in setting up the new synthetic draft of Phloroglucinol, Phloroglucinol is to use Na by comprising now 2Cr 2O 7The approach of oxidation is from 28 synthetic (diagram 2, i.e. Fig. 2).G.Leston, In Kirk-Othmer Encyclopedia of Chemical Technology, Vol.19, p 778 (J.I.Kroschwitz﹠amp; M.Howe-Grant, eds.) (the 4th edition, 1996) (Wiley:NewYork).Except the danger of blast, the building-up process of the Phloroglucinol 1a that begins from 28, produced the chromate waste liquid that causes environmental problem.Recently, the approach of an alternate method for synthesizing phloroglucinol 1a (diagram 2) is illustrated, and this approach comprises that the microorganism catalysis of nitrilotriacetic lactone 3a is synthetic.W.Zha et al.，J.Am.Chem.Soc.126：4534(2004)。Need a plurality of chemical steps that nitrilotriacetic lactone 3a is converted into Phloroglucinol 1a needs.C.A.Hansen & J.W.Frost，J.Am.Chem.Soc.124：5926(2002)。Form with these chemistry and the approach of enzyme method for synthesizing phloroglucinol and to contrast, the heterogenous expression of the PhlD among the E.coli can make Phloroglucinol 1a (diagram 2) with one microorganism catalysis step by glucose.
Resorcinol 9 synthetic replacement schemes also become possibility now.Resorcinol is at present by 1,3-phenyl disulfonic acid 10 with alkali fusion or 1, the hydroperoxidation of 3-diisopropylbenzene(DIPB) 11 is made (diagram 2).The alkali fusion method needs high temperature, and produces a large amount of bisulfate waste liquors.The acetone hydroperoxide that produce in the hydroperoxidation process have explosion hazard.Referring to L.Krumenacker et al., In Kirk-OthmerEncyclopedia of Chemical Technology, Vol.13, p 996 (J.I.Kroschwitz ﹠amp; M.Howe-Grant, eds.) (the 4th edition, 1996) (Wiley:New York).In addition, 1,3-benzene disulfonic acid 10 and 1,3-diisopropylbenzene(DIPB) 11 all make (diagram 2) from the carcinogenic benzene of petroleum derivation.The new way of synthetic Resorcinol 9 is on the microorganism synthetic basis of Phloroglucinol 1a, then the hydrogenization (diagram 2) of this intermediate under rhodium catalysis.C.A.Hansen&J.W.Frost，J.Am.Chem.Soc.124：5926(2002)。Since Phloroglucinol 1a now can be synthetic from glucose, Resorcinol and catechol become with Resorcinol should be from glucose synthetic dihydroxyaromatic compound nontoxic, plant origin (diagram 2).Respectively referring to K.D.Draths ﹠amp; J.W.Frost, J.Am.Chem.Soc.117:2395 (1995); With N.Ran et al., J.Am.Chem.Soc.123:10927 (2001).
The expression of PhlD and the Phloroglucinol that is caused is synthetic among the embodiment 2.E.coli
Plasmid pJA3.131 (Kan R, lacI Q, P T7-phlD serA) is colored the transfection of body ground to serA -E.coli bacterial strain BL21 (DE3), W3110 (DE3), and JWF1 (DE3) [RB791serA just, -(DE3)] [AB2834 (serA::aroB) just] (E.coli bacterial strain RB791 and AB2834 can obtain from the E.coli GeneticStock Center of U.S. Kang Naitige state New Haven) and among the bacterial strain KL3 (DE3).All DE3 bacterial strains are by obtaining λ DE3 prophage integration in cell chromosome.
Cell cultures adds in the mineral salt and limited glucose of formula pattern in stream.Though the bacterial strain of all conversions is the Phloroglucinol of expressed in abundance content all, BL21 and W3110 bacterial strain produce 3.0 and the higher titre of 3.1g/L Phloroglucinol respectively; And with respect to the amount that supplies to the glucose in the culture, these bacterial strains produce the higher Phloroglucinol productive rate of per 100 moles of glucose 4.4 and 3.1mol Phloroglucinol (%mol/mol).
These tests have also been compared the same phlD of using at Ptac or P T5Control under the BL21 bacterial strain that transforms of plasmid in the Phloroglucinol expression level; Find P T7Higher result's (data do not provide) is provided.In these trials Phloroglucinol tired poly-in the stationary phase (or maintenance phase) bacterial strain uses therefor all stop to increase.Concerning BL21 and W3110, the highest Phloroglucinol concentration appears at respectively induces initial just the adding for the first time behind the IPTG about 6 hours and about 12 hours.Also observed the end product inhibition.Further test explanation concentration is more than or equal to about 2g/L the time, and Phloroglucinol becomes the reason (data do not provide) of inhibition.
The Phloroglucinol fermentation of embodiment 3. extractions
Anion-exchange resin column class extractive fermentation is used to remove Phloroglucinol and reduces or eliminates synthetic inhibition of Phloroglucinol in its cytotoxicity and the fermenting process.Vibration case reactor is equipped with the pipeline of introducing anion-exchange column and getting back to the vibration case; This pipeline is equipped with peristaltic pump to make matrix cycle through this post.Bio-Rad Econo post (25 * 200mm) KH with 15 column volumes of 80mL (column volume) AG1-X8 resin are housed 2PO 4(0.8M) wash, before the extraction tertiary amine salt is transformed into phos-phate forms in position.Each fermentation 3 to 5 posts altogether; Each post used before being replaced by another post about 6～12 hours, was lower than about 1.5g/L to keep the Phloroglucinol concentration in the substratum.All posts are all with the fluid bed mode running, and its circulation velocity is about 8～12mL/min.
In order to reclaim the Phloroglucinol that is adsorbed onto on the AG1-X8 resin, wash this post to remove remaining cell with fluid bed mode with the distillation deionized water of 10 column volumes; This has also reclaimed about 15% Phloroglucinol in the aqueous solution from this resin.Then, with acidifying ethanol (acetate, 10% (v/v) of 15 column volumes; Ethanol, 75% (v/v); Water, 15% (v/v)) washing this post with the fixed bed pattern is recovered in the acidifying ethanolic soln with the Phloroglucinol that will residue in the resin.After Phloroglucinol reclaimed, this post can be by further using the KH of 15 column volumes respectively 2PO 4(0.8M), the aseptic distillation deionized water rinsing of ethanol of 2 column volumes (70%) and 5 column volumes is regenerated.
Be the Phloroglucinol that purifying reclaims, the cell in the obtained aqueous solution is by centrifugal removal; This solution is concentrated to 1/10 of about initial volume then.Individually, the acidifying ethanolic soln is concentrated into drying.Residuum dissolves with the above-mentioned spissated aqueous solution again.The ethyl acetate extraction of the water usefulness equal volume that obtains then three times.Merge organic phase, and at MgSO 4Last dry, mix with the silicon gel then, be concentrated into drying, and reprint on the flashing tower.By using hexane: acetic ester (1: 1) rinsing, separate Phloroglucinol and other brown impurity, and identify with TCL.The component that contains Phloroglucinol then is concentrated into drying, and dry under high vacuum condition, obtains Phloroglucinol canescence crystal.
The optimization of embodiment 4. Phloroglucinols fermentation
Multiple two temperature fermentation diagram is used for the extraction and the non-extractive fermentation of the W3110 bacterial strain of above-mentioned conversion.Glucose passes through pO 2Tandem control supply, and the CO that consumes with being stabilized 2Amount is maintained at maintenance level, up to the terminal point of fermentation.In this fermentation of two types, find that the initial 36 ℃ of temperature that reduce have during the fermentation significantly improved the titre and the productive rate of Phloroglucinol, the titre of gained and productive rate are higher in extractive fermentation.Divide other fermentation 12 hours (for the first time with the IPTG inductive time), 15 hours (beginning that keeps the phase) or 30 hours with temperature transition to 30 ℃.Temperature transition has obtained result preferably when occurring in 15 hours, and this extractive fermentation has carried out 60 hours altogether.Under these conditions, W3110serA -(DE3)/pJA3.131A is with the Phloroglucinol of the synthetic 15g/L of the productive rate of 11% (mol/mol).Compare with non-extractive fermentation, extractive fermentation can provide the nondecreasing Phloroglucinol output that runs through fermenting process, stable PhlD specific activity, the cell viability of maintenance and longer maximum fermentation time.
Under same extractive fermentation condition, same fermentation diagram also is used to check above-mentioned BL21serA -(DE3)/production of the Phloroglucinol of pJA3.131A bacterial strain.Obtained and the suitable result of W3110 fermentation.Another two temperature curve, wherein 36 ℃ of initial temperature are changed into 33 ℃ in the time of 15 hours, find further to have improved from the rate of recovery of BL21 method for synthesizing phloroglucinol, have obtained the productive rate of the titre and 12.3% (mol/mol) of 17.3g/L.
In addition, the expression of reorganization phlD in yeast, S.cerevisiae also succeedd, though productive rate is 0.5 to about 1.5mg/L (data do not provide) under the condition of test.
In the description to the four corner of the compositions and methods of the invention, these embodiment described here and other embodiment are exemplary rather than restrictive.Being equal to replacement, changing and changing and to carry out within the scope of the invention of embodiment, material, composition and method with similar substantially result.
Sequence table
<110>Michigan State University
Frost，John W.
＜120〉biosynthesizing of Phloroglucinol and by its preparation 1 ,-dihydroxy-benzene
<130>6550-000110POA
<150>US 60/618,024
<151>2004-10-12
<150>US 60/617,959
<151>2004-10-12
<160>2
<170>PatentIn version 3.3
<210>1
<211>1050
<212>DNA
<213>Pseudomonas fluorescens Pf-5
<220>
<221>CDS
<222>(1)..(1050)
＜223〉Phloroglucinol synthetic enzyme ORF
<400>1
atg tct aca ctt tgc ctt cca cac gtc atg ttt ccg caa cac aag atc 48
Met Ser Thr Leu Cys Leu Pro His Val Met Phe Pro Gln His Lys Ile
1 5 10 15
acc cag caa cag atg gtc gat cac ctg gaa aac ctg cac gcc gac cat 96
Thr Gln Gln Gln Met Val Asp His Leu Glu Asn Leu His Ala Asp His
20 25 30
cea cgc atg gcc ctg gcc aag cgc atg atc gcc aac acc gaa gtc aac 144
Pro Arg Met Ala Leu Ala Lys Arg Met Ile Ala Asn Thr Glu Val Asn
35 40 45
gag cgc cac ctg gtg ttg ccg atc gac gaa ctg gca gtg cac acc ggt 192
Glu Arg His Leu Val Leu Pro Ile Asp Glu Leu Ala Val His Thr Gly
50 55 60
ttc acc cac cgc agc atc gtc tac gag cgt gaa gcc cgg cag atg tcg 240
Phe Thr His Arg Ser Ile Val Tyr Glu Arg Glu Ala Arg Gln Met Ser
65 70 75 80
tcg gcc gcg gcg cgc cag gcc atc gag aat gcc ggg ctg cag atc agc 288
Ser Ala Ala Ala Arg Gln Ala Ile Glu Asn Ala Gly Leu Gln Ile Ser
85 90 95
gac att cgc atg gtg atc gtc act tcc tgc acc ggc ttc atg atg ccg 336
Asp Ile Arg Met Val Ile Val Thr Ser Cys Thr Gly Phe Met Met Pro
100 105 110
tcg ctg acc gcg cac ctg atc aac gac ctg gcc ctg cca acc tcc acc 384
Ser Leu Thr Ala His Leu Ile Asn Asp Leu Ala Leu Pro Thr Ser Thr
115 120 125
gtg cag ttg ccg atc gcc cag ctg ggc tgc gtg gcc ggt gcc gcg gcc 432
Val Gln Leu Pro Ile Ala Gln Leu Gly Cys Val Ala Gly Ala Ala Ala
130 135 140
atc aac cgc gcc aac gac ttc gcc cgg ctc gat gcc cgc aac cac gta 480
Ile Asn Arg Ala Asn Asp Phe Ala Arg Leu Asp Ala Arg Asn His Val
145 150 155 160
ctg atc gtg tcc ctg gaa ttc tcc tcg ctg tgc tac cag ccg gac gac 528
Leu Ile Val Ser Leu Glu Phe Ser Ser Leu Cys Tyr Gln Pro Asp Asp
165 170 175
acc aag ctg cac gcc ttc atc tcc gcg gcg ctg ttc ggc gat gcg gta 576
Thr Lys Leu His Ala Phe Ile Ser Ala Ala Leu Phe Gly Asp Ala Val
180 185 190
tcc gcc tgc gtg ctg cgc gcc gat gac cag gcc ggc ggc ttc aag atc 624
Ser Ala Cys Val Leu Arg Ala Asp Asp Gln Ala Gly Gly Phe Lys Ile
195 200 205
aag aag acc gag tcg tac ttc ctg ccc aag agc gag cac tac atc aag 672
Lys Lys Thr Glu Ser Tyr Phe Leu Pro Lys Ser Glu His Tyr Ile Lys
210 215 220
tac gac gtg aag gac acc ggc ttt cac ttc acc ctc gac aag gcg gtg 720
Tyr Asp Val Lys Asp Thr Gly Phe His Phe Thr Leu Asp Lys Ala Val
225 230 235 240
atg aac tcc atc aag gac gtg gca ccg gtc atg gag cgg ctc aac tac 768
Met Asn Ser Ile Lys Asp Val Ala Pro Val Met Glu Arg Leu Asn Tyr
245 250 255
gag agc ttc gaa cag aac tgt gcg cac aac gac ttc ttc atc ttc cac 816
Glu Ser Phe Glu Gln Asn Cys Ala His Asn Asp Phe Phe Ile Phe His
260 265 270
acc ggt ggt cgc aag atc ctc gac gag ctg gtg atg cac ctg gac ctg 864
Thr Gly Gly Arg Lys Ile Leu Asp Glu Leu Val Met His Leu Asp Leu
275 280 285
gca tcc aac cgg gtc tcg caa tcg cgc agc agc ctg tcg gaa gcc ggc 912
Ala Ser Asn Arg Val Ser Gln Ser Arg Ser Ser Leu Ser Glu Ala Gly
290 295 300
aac att gcc agc gtg gtg gtg ttc gac gta ctc aag cgg cag ttc gat 960
Asn Ile Ala Ser Val Val Val Phe Asp Val Leu Lys Arg Gln Phe Asp
305 310 315 320
tcc aac ctc aat cgc ggc gac atc ggc ctg ctg gca gcc ttc ggc ccg 1008
Ser Asn Leu Asn Arg Gly Asp Ile Gly Leu Leu Ala Ala Phe Gly Pro
325 330 335
ggg ttc acc gcg gaa atg gcg gtg ggc gag tgg acc gcc tga 1050
Gly Phe Thr Ala Glu Met Ala Val Gly Glu Trp Thr Ala
340 345
<210>2
<211>349
<212>PRT
<213>Pseudomonas fluorescens Pf-5
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Met Ser Thr Leu Cys Leu Pro His Val Met Phe Pro Gln His Lys Ile
1 5 10 15
Thr Gln Gln Gln Met Val Asp His Leu Glu Asn Leu His Ala Asp His
20 25 30
Pro Arg Met Ala Leu Ala Lys Arg Met Ile Ala Asn Thr Glu Val Asn
35 40 45
Glu Arg His Leu Val Leu Pro Ile Asp Glu Leu Ala Val His Thr Gly
50 55 60
Phe Thr His Arg Ser Ile Val Tyr Glu Arg Glu Ala Arg Gln Met Ser
65 70 75 80
Ser Ala Ala Ala Arg Gln Ala Ile Glu Asn Ala Gly Leu Gln Ile Ser
85 90 95
Asp Ile Arg Met Val Ile Val Thr Ser Cys Thr Gly Phe Met Met Pro
100 105 110
Ser Leu Thr Ala His Leu Ile Asn Asp Leu Ala Leu Pro Thr Ser Thr
115 120 125
Val Gln Leu Pro Ile Ala Gln Leu Gly Cys Val Ala Gly Ala Ala Ala
130 135 140
Ile Asn Arg Ala Asn Asp Phe Ala Arg Leu Asp Ala Arg Asn His Val
145 150 155 160
Leu Ile Val Ser Leu Glu Phe Ser Ser Leu Cys Tyr Gln Pro Asp Asp
165 170 175
Thr Lys Leu His Ala Phe Ile Ser Ala Ala Leu Phe Gly Asp Ala Val
180 185 190
Ser Ala Cys Val Leu Arg Ala Asp Asp Gln Ala Gly Gly Phe Lys Ile
195 200 205
Lys Lys Thr Glu Ser Tyr Phe Leu Pro Lys Ser Glu His Tyr Ile Lys
210 215 220
Tyr Asp Val Lys Asp Thr Gly Phe His Phe Thr Leu Asp Lys Ala Val
225 230 235 240
Met Asn Ser Ile Lys Asp Val Ala Pro Val Met Glu Arg Leu Asn Tyr
245 250 255
Glu Ser Phe Glu Gln Asn Cys Ala His Asn Asp Phe Phe Ile Phe His
260 265 270
Thr Gly Gly Arg Lys Ile Leu Asp Glu Leu Val Met His Leu Asp Leu
275 280 285
Ala Ser Asn Arg Val Ser Gln Ser Arg Ser Ser Leu Ser Glu Ala Gly
290 295 300
Asn Ile Ala Ser Val Val Val Phe Asp Val Leu Lys Arg Gln Phe Asp
305 310 315 320
Ser Asn Leu Asn Arg Gly Asp Ile Gly Leu Leu Ala Ala Phe Gly Pro
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Gly Phe Thr Ala Glu Met Ala Val Gly Glu Trp Thr Ala
340 345

Claims (63)

1, a kind of isolating or the reorganization PhlD +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one; Or PhlD +Reconstitution cell, described PhlD +Reconstitution cell PhlA -, PhlB -Or PhlC -In at least one; Or genetic design has improved the wherein PhlD of PhlD expression +Reconstitution cell, wherein said enzyme system and reconstitution cell can be converted into Phloroglucinol with malonyl CoA.
2, enzyme system according to claim 1 or cell, wherein said enzyme system or cell are PhlA -, PhlB -And PhlC -
3, enzyme system according to claim 1, wherein said system further comprises the enzyme of at least a synthetic malonyl CoA.
4, enzyme system according to claim 3, the enzyme of wherein said synthetic malonyl CoA are any one in malonyl CoA synthetic enzyme, malonyl CoA decarboxylase and the acetyl-CoA carboxylase.
5, a kind of method of producing anabolic Phloroglucinol comprises the following steps:
(A) provide
(1) (a) isolating or the reorganization PhlD +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or
(b) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one, or
(c) genetic design improves the wherein PhlD of the expression of PhlD +Reconstitution cell,
Described enzyme system or reconstitution cell can be converted into Phloroglucinol with carbon source; With
(2) at least a in malonyl CoA or other carbon source, wherein said other carbon source can be converted into malonyl CoA by described enzyme system or reconstitution cell; With
(B) contact
(1) malonyl CoA and described enzyme system or reconstitution cell, under can reaction conditions by its method for synthesizing phloroglucinol, or
(2) described other carbon source and described enzyme system or reconstitution cell can be converted into described carbon source the third two
Under single acyl coenzyme A of acid and the reaction conditions by its method for synthesizing phloroglucinol;
Thereby obtain anabolic Phloroglucinol.
6, method according to claim 5, wherein said enzyme system (1) (a) or cell (1) be PhlA (b) -, PhlB -And PhlC -
7, method according to claim 5, wherein said enzyme system or reconstitution cell comprise the enzyme of synthetic malonyl CoA.
8, method according to claim 5, the enzyme of wherein said synthetic malonyl CoA are any one in malonyl CoA synthetic enzyme, malonyl CoA decarboxylase or the acetyl-CoA carboxylase.
9, according to the Phloroglucinol of the method for claim 5 preparation.
10, a kind of enzyme of method for synthesizing phloroglucinol of isolating or reorganization, described endonuclease capable is converted into Phloroglucinol with malonyl CoA.
11, enzyme according to claim 10, wherein said enzyme comprises the aminoacid sequence of SEQ ID NO:2.
12, enzyme according to claim 10, wherein said enzyme comprise the aminoacid sequence that at least 70% homology is arranged with SEQ ID NO:2.
13, enzyme according to claim 12, wherein said enzyme comprise the conservative replacement variant of the aminoacid sequence of SEQ ID NO:2.
14, a kind of isolating or recombinant nucleotide, described Nucleotide comprise the open reading frame of at least one coding PhlD enzyme, and are phlA -, phlB -Or phlC -In at least one.
15, Nucleotide according to claim 14, wherein said Nucleotide further comprise the open reading frame of the enzyme of the synthetic malonyl CoA of at least one coding.
16, Nucleotide according to claim 14, wherein said Nucleotide comprise the base sequence of SEQ ID NO:1, RNA base sequence correspondingly or with its redundant codon sequence.
17, Nucleotide according to claim 14, wherein said Nucleotide comprise at least 80% homologous base sequence with SEQ ID NO:1.
18, a kind of phlD +Reconstitution cell, wherein said cell transform with Nucleotide according to claim 14, and described cell can be expressed the Phloroglucinol synthetic enzyme by described Nucleotide.
19, cell according to claim 18, wherein said phlD +Reconstitution cell is phlA -, phlB -Or phlC -In at least a.
20, cell according to claim 18, wherein said phlD +Reconstitution cell is phlA -, phlB -And phlC -
21, a kind of method for preparing Resorcinol comprises
(A) provide
(1) passes through the PhlD that (a) is isolating or recombinate +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or (b) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one, or (c) genetic design improve the wherein PhlD of the expression of PhlD +Reconstitution cell comes biosynthetic anabolic Phloroglucinol; With
(2) hydrogen and rhodium catalyst; With
(B) be hydrogenated under the condition that forms Resorcinol at described Phloroglucinol, described Phloroglucinol contacted with described rhodium catalyst with described hydrogen,
Thereby preparation Resorcinol.
22, anabolic Phloroglucinol, or Resorcinol prepared therefrom, application in drug manufacture, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, matrix material or laminated or bonded material, wherein said anabolic Phloroglucinol are the PhlD of or reorganization isolating by (a) +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or (b) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one, or (c) genetic design improve the wherein PhlD of the expression of PhlD +Reconstitution cell comes biosynthetic.
23, a kind of anabolic Phloroglucinol or Resorcinol prepared therefrom of containing, or the medicine, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, matrix material or the composition laminated or the bonded material that obtain by the chemically modified of anabolic Phloroglucinol or Resorcinol prepared therefrom, wherein said anabolic Phloroglucinol is the PhlD of or reorganization isolating by (a) +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or (b) PhlD +Reconstitution cell, described is PhlD +Reconstitution cell PhlA -, PhlB -Or PhlC -In at least one, or (c) genetic design improve the PhlD that PhlD wherein expresses +Reconstitution cell comes biosyntheticly, and described composition is used for or as medicine, makeup, dyestuff, fluoropolymer resin, rubber, tackiness agent, sealing agent, coating, matrix material or laminated or bonded material.
24, a kind of can comprising from the preparation method of the reconstitution cell of malonyl CoA biosynthesizing Phloroglucinol
(A) through the following steps with the coding PhlD the Nucleotide transformant:
(1) provide Nucleotide isolating or reorganization, described Nucleotide is phlA -, phlB -Or phlC -In at least one, and comprise open reading frame of at least one coding PhlD enzyme, or comprise the open reading frame of the enzyme of the open reading frame of at least one coding PhlD enzyme and the synthetic malonyl CoA of at least one coding simultaneously,
(2) (a) energy PhlD that enough described Nucleotide transforms is provided -Cell, and described PhlD -Cell is phlA -, phlB -Or phlC -In at least one, described Nucleotide also has described phlA -, phlB -Or phlC -Feature, or (b) can be enough the PhlD that transforms of described Nucleotide +Cell; With
(3) under the condition that the enough described Nucleotide of described cell energy transforms, described cell is contacted with described Nucleotide,
Described Nucleotide can be by described cell expressing, thus the cell that obtains recombinating; Or
(B) by following steps deactivation phlD +Gene in the cell,
(1) provides phlD +Cell, described phlD +Cell is phlA +, phlB +Or phlC +In at least one; And
(2) at least one in the wherein said phlA of deactivation, phlB or the phlC gene, thereby the cell that obtains recombinating.
25, according to the method for claim 24, wherein said reconstitution cell is phlA -, phlB -And phlC -
26, reconstitution cell according to claim 1, wherein said reconstitution cell is a microorganism.
27, reconstitution cell according to claim 26, wherein said reconstitution cell are yeast, fungi or bacterium.
28, reconstitution cell according to claim 26, wherein said reconstitution cell is a bacterium.
29, reconstitution cell according to claim 26, wherein said reconstitution cell are any one members among Escherichia Escherichia or the Rhodopseudomonas Pseudomonas.
30, reconstitution cell according to claim 26, wherein said reconstitution cell are any one among the false unicellular P.fluorescens of colon bacillus E.coli or fluorescence.
31, method according to claim 5, wherein said carbon source are simple carbon sources.
32, method according to claim 31, wherein said carbon source comprise carbohydrate, aliphatic polyhydroxy compound or its combination.
33, according to claim 31 described method, wherein said carbon source comprises glucose, wood sugar, pectinose, glycerine or its combination.
34, method according to claim 5 wherein provides reconstitution cell, and described contact procedure (B) is included in the described cell of cultivation in the growth medium that contains described carbon source.
35, method according to claim 34, wherein said cultivation is implemented with the extractive fermentation method.
36, method according to claim 34, wherein said cultivation adopt carries out with the two temperature curve.
37, a kind of method of producing propelling agent or blasting agent cpd comprises
(A) provide anabolic Phloroglucinol, described Phloroglucinol is the PhlD of or reorganization isolating by (1) +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or (2) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one, or (3) genetic design improve the wherein PhlD of the expression of PhlD +Reconstitution cell, and biosynthetic; (B) the described anabolic Phloroglucinol of chemically modified, or chemically modified is produced propelling agent or blasting agent cpd by the Resorcinol of described Phloroglucinol preparation.
38, according to the described method of claim 37, wherein said anabolic Phloroglucinol is biosynthetic by the method that comprises the following steps:
(A) provide
(1) (a) isolating or the reorganization PhlD +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or (b) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one, or (c) genetic design improve the wherein PhlD of the expression of PhlD +Reconstitution cell,
Described enzyme system or reconstitution cell can be converted into Phloroglucinol with carbon source; With
(2) at least one in malonyl CoA or other carbon source, wherein said carbon source can be converted into malonyl CoA by described enzyme system or reconstitution cell; With
(B) contact
(1) described malonyl CoA and described enzyme system or reconstitution cell, under can condition by its method for synthesizing phloroglucinol, or
(2) described other carbon source and described enzyme system or reconstitution cell can be converted into described carbon source under malonyl CoA and the condition by its method for synthesizing phloroglucinol;
Thereby obtain anabolic Phloroglucinol.
39, according to the described method of claim 38, wherein said enzyme system or reconstitution cell comprise the enzyme of synthetic malonyl CoA.
40, according to the described method of claim 39, wherein the enzyme of synthetic malonyl CoA is any one in malonyl CoA synthetic enzyme, malonyl CoA decarboxylase and the acetyl-CoA carboxylase.
41, according to the described method of claim 37, wherein said PhlD +Enzyme system (A) (1) or reconstitution cell (A) (2) are PhlA -, PhlB -And PhlC -
42, according to the described method of claim 37, wherein provide reconstitution cell.
43, according to the described method of claim 42, wherein said reconstitution cell is the phlD+ reconstitution cell, and described phlD+ reconstitution cell is phlA -, phlB -Or phlC -In at least a.
44, according to the described method of claim 43, wherein said phlD +Reconstitution cell is phlA -, phlB -And phlC.
45, according to the described method of claim 43, wherein said reconstitution cell is to provide phlABCD by comprising +Cell, and deactivation wherein the method for at least one in phlA, phlB or the phlC gene produce.
46. according to the described method of claim 44, wherein said reconstitution cell is to provide phlABCD by comprising +Cell, and deactivation wherein the method for all phlA, phlB and phlC gene produce.
47, according to the described method of claim 44, wherein said reconstitution cell is to provide phlABCD by comprising -Cell, and insert therein that the method for phlD gene produces.
48, according to the described method of claim 47, the wherein said phlD assignment of genes gene mapping is in the genomic dna of described cell.
49, according to the described method of claim 47, among the DNA of the wherein said phlD assignment of genes gene mapping outside the genome of described cell.
50, according to the described method of claim 43, wherein said reconstitution cell is that the method by the gene that further comprises the enzyme that inserts the synthetic malonyl CoA of coding prepares, and the enzyme of wherein said synthetic malonyl CoA is any one in malonyl CoA synthetic enzyme, malonyl CoA decarboxylase or the acetyl-CoA carboxylase.
51, according to the described method of claim 42, wherein said reconstitution cell is a microorganism.
52, according to the described method of claim 42, wherein said reconstitution cell is yeast, fungi or bacterium.
53, according to the described method of claim 42, wherein said reconstitution cell is a bacterium.
54, according to the described method of claim 42, wherein said reconstitution cell is any one the member among Escherichia Escherichia or the false unicellular genus Pseudomonas.
55, according to the described method of claim 42, wherein said reconstitution cell is any one among the false unicellular P.fluorescens of colon bacillus E.coli or fluorescence.
56, according to the described method of claim 38, wherein said carbon source is simple carbon source.
57, according to the described method of claim 56, wherein said carbon source comprises carbohydrate, aliphatic polyhydroxy compound or its combination.
58, according to the described method of claim 56, wherein said carbon source comprises glucose, wood sugar, pectinose, glycerine or its combination.
59, according to the described method of claim 38, wherein provide reconstitution cell, and described contact procedure (B) is included in the described cell of cultivation in the growth medium that contains described carbon source.
60, according to the described method of claim 59, wherein said cultivation is implemented with the extractive fermentation method.
61, according to the described method of claim 59, wherein said cultivation adopts the two temperature curve to carry out.
62, anabolic Phloroglucinol or Resorcinol prepared therefrom application in producing explosive or propelling agent, wherein said Phloroglucinol are the PhlD of or reorganization isolating by (a) +Enzyme system, described enzyme system is PhlA -, PhlB -Or PhlC -In at least one, or (b) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -Or PhlC -In at least one, or (c) genetic design improve the wherein PhlD of the expression of PhlD +Reconstitution cell comes biosynthetic.
63, a kind ofly comprise anabolic Phloroglucinol or by its synthetic Resorcinol, or by anabolic Phloroglucinol or the explosive or the propellant compositions that are obtained by the chemically modified of its synthetic Resorcinol, wherein said anabolic Phloroglucinol is the PhlD of or reorganization isolating by (a) +Enzyme system, described enzyme system is PhlA -, PhlB -, or PhlC -In at least one, or (b) PhlD +Reconstitution cell, described PhlD +Reconstitution cell is PhlA -, PhlB -, or PhlC -In at least one, or (c) genetic design improve the wherein PhlD of the expression of PhlD +Reconstitution cell, next biosynthetic, and described composition is as explosive or propelling agent.
CN 200580034761 2004-10-12 2005-10-11 Biosynthesis of phloroglucinol and preparation of 1,3-dihydroxybenzene therefrom Pending CN101084311A (en)

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CN101724662B (en) * 2008-10-29 2012-08-15 青岛生物能源与过程研究所 Synthesizing phloroglucinol by microbial catalysis
CN103497081A (en) * 2013-09-21 2014-01-08 安徽省无为县花炮总厂 Formula of radial firework
CN105734003A (en) * 2015-12-20 2016-07-06 寿光富康制药有限公司 Algal symbiotic bacteria and method therewith to produce phloroglucinol
CN109439606A (en) * 2018-11-14 2019-03-08 中国科学院青岛生物能源与过程研究所 A kind of genetic engineering bacterium improving phloroglucin yield and its construction method and application
CN109456927A (en) * 2018-11-14 2019-03-12 中国科学院青岛生物能源与过程研究所 The recombinant bacterium and its construction method of a kind of high yield 2,4- diacetyl phloroglucin and application
CN110896642A (en) * 2018-06-08 2020-03-20 韩国科学技术院 Novel malonyl-CoA biosensor based on type III polyketide synthase and use thereof
CN112410389A (en) * 2019-08-23 2021-02-26 中国科学院微生物研究所 Application of branched-chain alpha-ketoacid dehydrogenase complex in preparation of malonyl-CoA
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CN101724662B (en) * 2008-10-29 2012-08-15 青岛生物能源与过程研究所 Synthesizing phloroglucinol by microbial catalysis
CN103497081A (en) * 2013-09-21 2014-01-08 安徽省无为县花炮总厂 Formula of radial firework
CN105734003B (en) * 2015-12-20 2019-09-13 寿光富康制药有限公司 A kind of seaweed fungal component and its method for preparing phloroglucin
CN105734003A (en) * 2015-12-20 2016-07-06 寿光富康制药有限公司 Algal symbiotic bacteria and method therewith to produce phloroglucinol
CN110896642A (en) * 2018-06-08 2020-03-20 韩国科学技术院 Novel malonyl-CoA biosensor based on type III polyketide synthase and use thereof
CN109439606A (en) * 2018-11-14 2019-03-08 中国科学院青岛生物能源与过程研究所 A kind of genetic engineering bacterium improving phloroglucin yield and its construction method and application
CN109456927A (en) * 2018-11-14 2019-03-12 中国科学院青岛生物能源与过程研究所 The recombinant bacterium and its construction method of a kind of high yield 2,4- diacetyl phloroglucin and application
CN109439606B (en) * 2018-11-14 2022-06-28 中国科学院青岛生物能源与过程研究所 Genetically engineered bacterium for increasing phloroglucinol yield and construction method and application thereof
CN113412329A (en) * 2019-02-05 2021-09-17 米其林集团总公司 Phloroglucinol resistant cells, in particular yeasts
CN113412329B (en) * 2019-02-05 2024-03-01 米其林集团总公司 Phloroglucinol resistant cells, particularly yeasts
CN112410389A (en) * 2019-08-23 2021-02-26 中国科学院微生物研究所 Application of branched-chain alpha-ketoacid dehydrogenase complex in preparation of malonyl-CoA
CN112410389B (en) * 2019-08-23 2023-07-18 中国科学院微生物研究所 Application of branched-chain alpha-keto acid dehydrogenase complex in preparation of malonyl-CoA
CN112961672A (en) * 2021-02-10 2021-06-15 吉林大学 Preparation method of fluorescent bimetallic organic JLUE-MOG-6 aerogel material
CN112961672B (en) * 2021-02-10 2022-03-22 吉林大学 Preparation method of fluorescent bimetallic organic JLUE-MOG-6 aerogel material

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