WO2000022139A2 - Dna sequences for enzymatic synthesis of polyketide or heteropolyketide compounds - Google Patents
Dna sequences for enzymatic synthesis of polyketide or heteropolyketide compounds Download PDFInfo
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- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Definitions
- the present invention relates to DNA sequences for enzymatic synthesis of polyketide or heteropolyketide compounds produced by the bacterium Sorangi um cellulosum.
- PKS Polyketide synthases
- NRPS non-ribosomal peptide syn- thetases
- PKS condenses activated carbonic acids (usually acetate and propi- onate) and reduce the resulting 2-keto acid intermediates step- wise in a fatty acid biosynthesis-like fashion.
- responsible for each reaction step is a specific domain that recognizes, acti- vates, condenses and reduces the carbonic acid.
- every reduction stage can occur in the final product (Rawlings, Nat. Prod. Reports 14, 523-556 [1997]; for a review, see Chem. Rev. 97, 2463-2760 [1997]).
- a typical example for the biosyn- thesis of a polyketide is the macrolide antibiotic erythromycin
- NRPSs are also modular enzymes and condense via peptide bonds amino acids to low molecular weight bioactive substances like bacitracin or tyrocidin. Typical domains of these systems acti- vate the amino acid and condense it with the growing peptide chain. Methylations, epimerisations and modifications via additional protein domains are possible (Stachelhaus and Marahiel, FEMS Microbiol Lett. 125, 3-14 [1995]). Both types of enzymes (NRPS and PKS) share the modular organization of the proteins in which specific catalytic domains are responsible for recognition, activation, condensation and modification of the single elongation units. The growing chain of amino acids and/or carbonic acids is extended through the action of one module adding one unit.
- each module carry the active centers responsible for the enzymatic steps of the biosynthesis. Little is known about the biosynthesis of biologically active polyketides and polypeptides from myxobacte ⁇ a . Fragments of the biosynthetic gene clusters of soraphen and saframycm have been described from Sorangium cellulosum So ce26 and Myxo- coccus xanthus, respectively (Schupp et al . , J. Bacte ⁇ ol . 177 , 3673-3679 [1995] and Pospiech et al . , Microbiology 141, 1793- 1803 [1995] ) .
- a subject of the present invention is therefore to provide DNA sequences according to claim 1 the expression products of which perform or are involved m the enzymatic biosynthesis, mutasynthesis or partial synthesis of polyketide or heteropolyketide compounds.
- the DNA sequences may be inserted into well known and optimized expression vectors by commmon tech- niques of molecular biology, thus allowing transformation, selection and cloning of cells, which cells are then capable of synthezis g polyketide or heteropolyketide compounds by fermentation. Using an overproducing clone allows the desired polyketide or heteropolyketide compounds be easily produced and recovered m high amounts.
- the invention thus further relates to a recomb ant expression vector according to claim 16, cells transformed therewith according to claim 17 and to a process for enzymatic biosynthesis, mutasynthesis or partial synthesis of polyketide or heteropolyketide compounds according to claim 23.
- the invention consists of (1) cloned Sorangium cellulosum polyketide synthase (PKS) and/or peptide synthetase (PS) biosynthetic cluster DNA and (2) the nucleotide sequence and predicted protein coding sequences of the cloned DNA.
- PKS Sorangium cellulosum polyketide synthase
- PS peptide synthetase
- the invention can be used for, but not limited to, (a) increasing yields of PKS product Sorangium cellulosum (e.g., by amplification or genetic modification of the epothilone gene cluster or its component parts) , (b) increasing yields of polyketide and/or peptide synthetase product a heterologous system by transfer of the corresponding gene cluster or its component parts, which may De followed by amplification or genetic modification of the PKS and/or PS gene cluster or its component parts, (c) modification of the polyketide and/or peptide synthetase product chemical structure either Sorangium cellulosum or a heterologous host (e.g., by genetic modification of the corresponding gene cluster or its component parts) and (d) for the detection of genes and gene products involved making polyketides or related molecules m other organisms (e.g., by hybridization or complementation assays) .
- Figure 1 is a restriction map of one of the DNA sequences of the present invention (cosmid A2 insert) indicating also the localization of regulatory DNA segments and the individual structural genes ("open reading frames" or ORFs) 1 to 16.
- Figure 2 shows the open reading frames found on pEPOcos ⁇ region
- DNA sequence data from A2 cosmid are as defined in claim 6.
- Table 1 correlates ORFs 1 to 16 found on A2 cosmid with the respective biological function (Regulators, Enzymes) .
- CA-2 agar is prepared by autoclaving 18 g Bacto-agar (Difco Laboratories, Detroit, MI) m 800 ml dH 2 0 for 20 min at 121°C and cooling to 50-55°C in a water bath. The following filter-sterilized solutions are added to the agar: 20% (w/v) glucose, 50 ml; Solution A (7.5%
- G51t consists of 0.5% starch (Cerestar) , 0.2% tryptone, 0.1% yeast extract, 0.05% CaCl 2 , 0.05% MgSO 4 -7H 2 0, 1.2% 4 - (2 -hydroxyethyl ) - 1-piperazine- ethanesulfonic acid (HEPES) , 0.2% glucose, pH 7.6.
- the flasks were shaken at 30°C, 160 rpm until a dense orange bacterial growth was obtained (ca. 5-7 d.) .
- the cells were pelleted by centrifugation at 6,000 x g and used immediately or stored fro- zen at -20°C.
- genomic DNA was isolated from S . cellulosum cells cultured as described m section A.l using the Midi Qiagen Blood & Cell Culture DNA purification Kit (Qiagen, Hilden, Germany) following the Qiagen Genomic DNA Handbook protocol for bacterial DNA isolation (1997, Qiagen, Hilden, Ger- many, p. 29 ff.) .
- the precipitated DNA was recovered with a bent pasteur pipette as described m section A.l.
- pFD666 is a bifunctional E. coli -Streptomyces cosmid cloning vector (see Denis and Brzezmski, Gene 111 , 115-118 [1992]) . To maintain stability of large inserts, it is present low-medium copy number when replicated E . coli . For this reason, isolation of sufficient pure DNA to carry out cloning experiments was difficult using commercial kits with standard protocols. A modified procedure was therefore used to obtain pFD666 DNA.
- a 10 ml culture of DH10B (pFD666) was grown for 16- 20 hr at 37°C m LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl , pH 7.0) medium containing 50 ⁇ g/ml kanamycm sulfate.
- Fifty ml of LB + kanamycm was inoculated to a starting OD 60 o of ca 0.25 and shaken at 300 rpm, 37°C, until the OD 60 o reached ca . 0.6.
- Five hundred ml of LB + kanamycm medium a 2 1 flask was inoculated with 25 ml of this culture and incubated under the same conditions for 2.5 hr .
- Chloramphenicol ( 2.5 ml of a 34 mg/ml solution 100% EtOH) was added and the incubation continued for an additional 16-20 hr .
- the previous steps were performed according to Maniatis et al . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
- Cells were pelleted for 10 mm, 16,000 x g . They were resuspended in 9 ml of 50 mM glucose/25 mM Tris-HCl (pH 8.0) /10 mM EDTA and transferred to a 50 ml disposable centrifuge tube.
- the tube was centrifuged at 6,000 x g for 10 mm at 4°C and the supernatant poured though 2 layers of sterile cheese cloth into a fresh 50 ml disposable tube. Isopropanol to a final concentration of 0.6% was added and the contents of the tube mixed several times. The precipitated nucleic acid was centrifuged at 6,000 x g for 10 mm at 4°C. The pellet was washed with 70% EtOH and any excess EtOH was aspirated from the pellet, which was allowed to air dry for 5 mm.
- S . cellulosum chromosomal DNA prepared as described m section A.l. a was partially cleaved with restriction endonuclease Sau3Al m a 1000 ⁇ l reaction volume consisting of 50 ⁇ g chromo- so al DNA, 5 units enzyme (Pro ega, Madison , WI), 0.006 M Tris-HCl, 0.006 M MgCl 2/ 3.10 M NaCl , and 0.001 M dithiothrei- tol (pH 7.5) for 5 mm at 37°C.
- the reaction mixture was extracted once with an equal volume of 1:1 phenol : chloroform. After centrifugation, tr.e upper aqueous phase was saved, to which 0.1 vol.
- DNA was pelleted by centrifugation for 5 mm at 16,000 x g m a microfuge and washed once with 0.5 ml 70% EtOH. After drying m a SpeeaVac (Savant Instruments, Farmmgdale, NY) for 5 mm, the pellet was resuspended m 0.1 ml TE buffer.
- the DNA was layered ontcp of a 12 ml 10-40% sucrose gradient prepared m TE buffer ana centrifuged at 113,600 x g for 16 hr, 10°C using a Beckman SW40T ⁇ rotor (Beckman Instruments, Palo Alto, CA) .
- Sucrose was diluted from the corresponding 0.5 ml fraction by addition of 0.5 vol. TE . Subsequently, DNA was precipitated by addition of 0.1 vol. 3 M sodium acetate and 0.6 vol. isopropanol . DNA was pelleted by centrifugation at 16,000 x g for 10 mm m a microfuge. DNA was washed with 0.5 ml 70% EtOH and dried m a SpeedVac with moderate heat for 10 mm. Finally, the DNA was resuspended m distilled H 2 0 at a concentration of 0.5 mg/ml.
- Vector pFD666 was cleaved with restriction endonuclease BamHI m a 0.02 ml reaction volume consisting of 2 ⁇ g plasmid DNA, 10 units of BamHI (Promega), 0.006 M Tris-HCl, 0.006 M MgCl 2 , 0.05 M NaCl , and 0.001 M dithiothreitol (pH 7.5) for 90 mm at 37°C.
- 0.3 ml of stop buffer (0.01 M Tris-HCl [pH 7.5], 0.001 M EDTA, 0.2 M NaCl , 0.5% SDS) and 0.35 ml of 1 : 1 phenol; CHCl 3 was added to the reaction.
- the sample was mixed gently several times by inversion and centrifuged at 16,000 x g for 3 mm to separate the phases.
- the aqueous layer was removed to a new microfuge tube.
- 0.1 vol. 3 M sodium acetate and 2 vol. 100% EtOH were added and the precipitated DNA pelleted by centrifugation at 16,000 x g for 10 mm. Liquid was removed by aspiration and the pellet washed once with 0.5 ml 70% EtOH.
- the DNA was dried m a SpeedVac and resuspended m TE buffer to 0.5 mg/ml .
- the entire packaging reaction (0.5 ml) was diluted with 4.5 ml SM buffer (per liter: 5.8 g NaCl, 2 g MgSO 4 .7H 2 0, 1 M T ⁇ s- HCl [pH 7.5] , 5 ml 2% gelatin solution) .
- Transfection was performed by adding 10 ml cf an overnight culture of E. coli DH5 ⁇ that had been grown m LB medium with 0.01 M MgS0 4 and 0.2% maltose to the diluted pnage and incubating at 37°C for 20 mm.
- 0.8 ml of LB was added and the cells shaken at 225 rpm for 1 hr at 37°C.
- DNA was digested with restriction endonuclease Pstl and samples electrophoresed on a 0.8% TAE agarose gel for 1.5 hr at 100 V.
- a unique restriction pattern was noted m eac sample and the total size of the insert was calculated to be between 40 and 45 kilobases.
- digeste ⁇ vector DNA was ligated with 10 ⁇ g partially hydrolyzed genomic DNA from S . cellulosum (as described m section A.3.b) m a final volume of 20 ⁇ l using 2 U T4 DNA ligase and the appropriate reaction buffer (Gibco BRL, Eggen- stem, Germany) . The reaction was carried out at 16°C overnight. The reaction mixture was packaged into phage particles using the Gigapack III XL packaging extract kit (Stratagene) according to the manufacture's protocol. Treatment of packaging reaction mixture and transfection of E . coli SURE (Stratagene) was performed as described m 4. a.
- Transfected cells were concentrated by centrifugation, resuspended m fresh LB medium and distributed on LB agar plates containing 50 ⁇ g/ml 1 kanamycm. The plates were incubated overnight at 30°C. 1600 recombmant clones were transferred into 96 well microtiter plates filled with 80 ⁇ l LB medium containing 50 ⁇ g/ml kanamycm per well and propagated overnight at 30°C. The following day the microtiter plates were used to inoculate a second set of microtiter plates m order to obtain a duplicate of the recombmant clones.
- Plasmid pZero2.1 (Invitrogen, Carlsbad, CA) was cleaved with restriction endonuclease BamHI m a 0.02 ml reaction volume consisting of 1 ⁇ g plasmid DNA, 10 units of BamHI (Promega) , 0.006 M Tris-HCl, 0.006 M MgCl 2 , 0.05 M NaCl, and 0.001 M dl- thiothreitol ( pH 7.5) for 20 mm at 37°C. 0.08 ml of dH 2 0 and 0.1 ml of 1:1 phenol :CHC13 was added. The sample was briefly vortexed and centrifuged at 16,000 x g for 2 mm.
- the aqueous layer was removed to a new microfuge tube. 0.1 vol. 3 M sodium acetate and 2 vol. 100% EtOH were added and the precipitated DNA pelleted by centrifugation at 16,000 x g for 10 mm. Liquid was removed by aspiration and the pellet washed once with 0.5 ml 70% EtOH. The DNA was dried m a SpeedVac and resuspended m TE buffer to 0.004 ⁇ g/ml. Digested pZero2.1 was ligated to the partially-cleaved chromosomal DNA m a 0.01 ml reaction consisting of 0.004 ⁇ g pZero2.1 , 0.05 ⁇ g S .
- the ligated DNA was resuspended m 0.005 ml dH 2 0 and mixed with 0.04 ml of electro- competent Escherichia coli DH10B cells (GIBCO/BRL, Gaithers- burg, MD) .
- the sample was placed into a pre-chilled 0.2 mm-gap electroporation cuvette and transformed into the bacteria by electroporation using a BioRad Gene Pulser II unit (BioRad, Hercules, CA) at 25 ⁇ F and 200 ⁇ .
- a 20 x 20 cm sheet of Duralon UN membrane (Stratagene) was placed on top of a 24.5 x 24.5 cm square bioassay dish containing 250 ml LB agar - kanamycm. An aliquot of the frozen cosmid library m 1 ml L ⁇ medium was spread on the filter. The plate was incubated at 37"C for 24 hr . Colonies were replicated onto two fresh filters which were placed onto LB + kanamycm agar medium and incubated at 28°C for 18 hr. Lysis of cells and neutralization of released D ⁇ A was performed according to di- rections that were provided with the filters.
- the D ⁇ A was crosslmked to the filters using a UV Stratalmker 2400 unit (Stratagene) m the autc crosslink mode.
- Cell debris was removed by placing the filters m a container with a solution of 3 X SSC (20 X SSC contains, per liter, 173.5 g ⁇ aCl , 88.2 g so- dium citrate, pH adjuste ⁇ to 7.0 with 10 ⁇ ⁇ aOH) , 0.1% SDS and rubbing the lysed colonies with a Kimwipe .
- the filters were then incubated at least 3 hr with the same wash solution for at least 3 hr at 65°C.
- the plasmid library was treated similarly except cells were sprea ⁇ onto a 137 mm circular Duralon UN mem- brane placed on top of a 150 mm petri dish containing 80 ml LB agar + kanamycm.
- a probe consisting of a 650-base pair (bp) polymerase chain C?CR) fragment representing a portion of a S . cell ulosum polyketide synthase gene was used.
- the fragment was amplified using primers to consensus regions of Type I (macrolide) polyketide synthase (PKS) genes (Swan et al . , Mol . Gen. Genetics 242 , 358-362 [1994]).
- PKS polyketide synthase
- Reactions were carried out m a Perkm-Elmer Model 480 Thermocycler (Perkm- Elmer Corporation, Foster City, CA) under the following conditions: 94°C, 1 mm; 50°C, 1 mm, 72°C, 1.5 mm for a total of 30 cycles. Each possible combination of sense and anti-sense p ⁇ m- ers were tried.
- a 650-bp and 350-bp fragment was amplified using oligos 120 + 124 and 123 +124, respectively.
- the sequence of the fragments were determined using the ALFexpress AutoRead kit to fluorescently label the DNA, which was analyzed on an ALFexpress sequencing apparatus (Pharmacia) .
- the 650-bp fragment was chosen for hybridization experiments.
- the fragment was labeled with 32 P-dCTP using the NEBlot kit (New England Biolabs, Beverly, MA) and purified on a Bio-Spm 6 column (BioRad, Hercules, CA. ) .
- Duplicate blots were pre- hybndized 3 X SSC (1 X SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0) , 4 X Denhardt ' s solution
- Plasmid DNA was prepared using the standard alkaline lysis method and digested with restriction endonuclease Pstl. The digested DNA was electrophoresed on a 0.8% aga- rose gel m TAE for 3 hr at 100 V. Fragments were transferred to Duralon UV using the VacuGene XL vacuum blotting unit (Pharmacia) and the recommended alkaline denaturation protocol . Hybridization with radioactively-labeled PCR fragment and washing were carried out as described above.
- a 137 -mm circle of Duralon UN membrane was placed on top of a 150-mm containing 75 ml LB agar + kanamycm.
- An aliquot of the plasmid library (representing ca . 2,000 recombmant colonies) m 0.5 ml LB medium was spread on the filter.
- the plate was incubated at 37°C for 20 hr .
- Colonies were replicated onto two fresh filters which were placed onto LB + kanamycm agar medium and incubated at 37°C for 6 hr .
- the filters were proc- essed for hybridization as described Section C.l.
- This plasmid, called Sau4 was characterized in more detail.
- the recombmant E . coli clones from the microtiter plates were used to produce two identical sets of hybridization filters order to identify cosmids carrying PKS and PS genes .
- the recomc ant clones were spotted onto 2 sets of 22 x 22 cm LB agar plates containing 50 ⁇ g/ml kanamycm. Each plate contained 384 clones therefore representing 4 microtiter plates.
- the clones were incubated at 30°C overnight. After pre-coolmg for approximately 3 h at 4°C, 20 x 20 cm Hybond ⁇ + Nylon membranes (A ⁇ ersham, Braunschweig, Germany) were placed onto the agar sur aces. After 2 mm.
- the membranes were removed and placed for 15 mm. on Whatman 3 MM paper (Whatman paper Ltd., Maidstone, England) soaked with denaturation solution (0.5 N NaOH, 1,5 V NaCl) before they were transfered onto Whatman 3 MM paper saturated with neutralization solution (1 M Tris-HCl, pH 7.5, 1.5 iv NaCl) . Subsequently the membranes were placed onto Whatman 3 MM paper soaked with 2 X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.2) for 10 min. The membranes were baked for 40 min at 85°C.
- each membrane was overlayed with 5 ml Proteinase K solution (2 mg/ml Proteinase K in 2 x SSC) and incubated at 37 °C for 90 min. Finally, cell debris was removed by wiping the membranes with a Kimwipe pre-wetted with 2 X SSC.
- PCR fragments were used as gene probes to detect recombinant cosmids of the S . cellulosum cosmid library.
- Oligonucleotides based on conserved amino acid sequences of ketosynthase domains from various type I PKS were optimized for myxobacterial DNA by comparison to a known myxobacterial biosynthetic gene cluster (Schupp et al . , J. Bacteriol . 177, 3673-3679 [1995]) resulting in primer KSlUp (5 ' -
- KSD1 (5'-GG(A/G)TCICCIA(A/G) I (G/C) (T/A) IGTICCIGTICC (A/G) TG-3 ' ) .
- PCR-primers TGD (5'- T(A/T) (C/T) CGIACIGGIGA(C/T) (C/T) (G/T) IG(G/T) ICG-3') and LGG ( 5 ' -
- A(A/T) IGA(A/G) (G/T) (G/C) ICCICCI (A/G) (A/G) (G/C) I (A/C) (A/G) AA (A/G )AA-3') directed to genes encoding adenylation modules have been de- scribed by Turgay et al . (Pept. Res. 7, 238-241 [1994]).
- PCR reaction mixtures with a final volume of 25 ⁇ l contained 0.1 ⁇ g template DNA, 0.2 U Taq DNA-polymerase (Gibco BRL, Eggenstem, Germany), 5 ⁇ mol dNTP, 5% dimethyl sulfoxide (Sigma), 1.5 mM MgCl 2 , 25 pmol of each primer and the appropriate reaction buffer supplied by Gibco BRL. Chromosomal DNA of S . cellulosum was used as template. Additionally, chromosomal DNA of Myxococ - cus fulvus was used with PS primers.
- KS fragments Three different KS fragments, designated pM008.4, pM008.6, pM008.7, one PS fragment (pAPsl) corresponding to S . cellulosum and one PS fragment (pDPsl) obtained with chromosomal DNA of M. fulvus .
- the PCR fragments were re- isolated by digestion with EcoRI from the plasmids pM008.4, pM008.6, and pM008.7, laoeled, pooled and used as gene probes m hybridization experiments as described below. The same procedure was performed with the PS fragments of pAPsl and pDPsl.
- Hybridization with PKS and PS specific DNA probes was carried out using the DIG nonradioactive labeling and detection kit (Boehrmger Mannheim, Germany) and performed according to the supplier's manual using buffer containing 50% formamide .
- the membranes were hybridized m plastic bags containing approx. 10 ml of hybridization solution at 39°C overnight. Unspecific binding of probes was removed by 2 wash steps with 2 x SSC, 0.1% SDS at room temperature for 20 mm. and one stringent wash step with 0.5 x SSC, 0.1% SDS at 60°C for 20 mm. Detection of hybridizing DNA fragments was performed with the above mentioned system according to the manufacturer's protocol using CSPD as chemilummescent substrate. The signals were recorded by exposure of the treated membrane to Hyperfilm ECL (Amersham Life Science, Little Chalfont, England) which was developed m appropriate time intervals.
- Randomly sheared l oraries were constructed for cosmids and plasmids of interest using a protocol similar to that of of Fleischmann et al . , 19S ⁇ (Science 269, 496) and modified m Fraser et al . , 1995 (Science 370 , 397). Briefly, Qiagen-column purified cosmid DNA ( ⁇ 1C ⁇ g) was sheared to a size of approximately 2 kb and the DNA end-repaired using BAL31 nuclease.
- the DNA was gel -purified after electrophoresis through a 0.75% low- melting temperature agarose gel containing 0.5 ⁇ g/ml ethidium bromide m IX TAE buffer run at ⁇ O V for 2 hours.
- the volume of the low-melt agarose gel slice was estimated by adding the gel slice to a microfuge tune and weighing, then 0.1 vol. of 3 M sodium acetate (pH 7) was added and the agarose incubated at 60°C. Tne temperature .--as equilibrated to 37°C, and DNA ex- tracted twice using an equal volume of buffered phenol (Life Technologies) .
- aqueous phase was transferred and extracted once with an equal volume of chloroform, then ethanol preci- pated by the addition of 2 vol. cold 100% ethanol.
- DNA was con- centrated by spinning at 16,000 x g m a microcentrifuge. The DNA pellet was washed with 1 ml 70% ethanol and resuspended m 100 ⁇ l of 0. IX TE.
- the DNA was ligated to Smal -digested, phos- phatase-treated pUCl ⁇ vector (Pharmacia) , and single insert re- combmants isolated by gel-pu ⁇ fication of the band containing vector plus a single insert, followed by T4 polymerase polishing, and a final intramolecular ligation of the vector-plus- s gle-msert DNA.
- This final ligation represents a library of highly random ca . 2 kb fragments that was used for shotgun sequencing of the ca . 40 kb cosmids or ca. 10 kb plasmids.
- Cosmid A2 Cosmid DNA with inserts of S . cellulosum was isolated by an alkaline lysis procedure and purified with Ma- cherey Nagel columns (Machery und Nagel GmbH und CoKG, D ⁇ ren, Germany) using manufacturer's recommendation. Purified Cosmid DNA was sonicated, end-repaired using T4 DNA Polymerase (Boe- hrmger Mannheim, Germany) . After gel -purification fragments of a size of approximately 2 kb were ligated into Smal -digested, phcsphatase-treated pTZl ⁇ R vector (Pharmacia) . The ligation represents a library of highly random ca. 2 kb fragments that was used for shotgun sequencing of the ca . 40 kb cosmid.
- DNA (1 ⁇ l of 100 ⁇ l total m the library) was transformed into E . coli by electro- poration (20 ⁇ l of Electromax DH10B cells from Life Technolo- gies) and cells spread onto LB plates containing 50 ⁇ g/ml ampi- cillin. After growth overnight at 37 °C, transformants (ca. 300- 3000 CFU total) were tranfered to 96-well growth blocks and shaken overnight at 37°C in 1.3 ml LB medium with 50 ⁇ g/ml am- picillin.
- coli DH10B by electroporation and cells were spread onto LB agar medium containing 50 mg/ml ampicillm. After growth for l ⁇ hr at 37°C, transformants were transferred to 96-well growth blocks and shaken overnight at 37°C m 1.3 ml 2x YT medium with 50 mg/ml ampicillm. Templates were prepared from these cells by an alkaline lysis procedure (Qiagen Qia- quick Turbo Prep) to yield purified, double-stranded plasmid DNA.
- Cycle-sequencing of the plasmid templates was performed using universal forward and reverse primers and Big Dye Termi- nator sequencing kits (PEBiosystems) or Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham Pharmacia Biotech) using the manufacturer's protocols.
- PBiosystems Universal Forward and reverse primers and Big Dye Termi- nator sequencing kits
- Thermo Sequenase fluorescent labelled primer cycle sequencing kit Analogenase fluorescent labelled primer cycle sequencing kit
- data were collected using Licor and ABI 377 automated sequencers and assembled with the GAP4 program (Bon- field, Smith, Staden, Nucl . Acids Res. 23, 4992-4999 [1995]). Gaps were closed using custom made primers (MWG-Biotech) on plasmid templates or PCR products m combination with dye- terminators .
- ORF Open reading frame identification ORFs were identified the pEPOcos ⁇ region using the OMIGA 1.1.2 (GCG 0.4D) program from Oxford Molecular Limited. Default values were used (Stan ⁇ ard genetic code, all ORFs over 50 bases) to generate ORFs; analysis of these results lead to the list of 14 highest quality ORFs as defined m claim 9. Other ORFs, genes, or genetic elements may be found m the pEPOcos ⁇ insert that have not yet been annotated.
- the MAGPIE automated genome analysis tool : (htt : //genomes . rockefeller . edu/magp e/magpie . html )
- ORFs have been identified within the DNA sequences of A5 (contigs 10, 11, 12) and of A2 using the FramePlot analysis program from Ishikawa and Hotta (FEMS
- BLASTP2 similarity searches were performed using the peptide files from the above ORF identification strategy as query sequences. Searches were performed using the m-house Biomfor- matics BLASTP2 (Version: BLASTP 2.0al9MP-WashU) web page at the Bristol-Myers Squibb Pharmaceutical Research Institute (allows BlastN2, BlastP2, BlastX2 , TblastN, and TBlastX searches). In addition, peptide files generated by the MAGPIE analysis were automatically searched using a FASTA algorithm.
- a higher probability score indicates a higher liklihood that the probable function corresponds to that of the best match; e.g., the polyketide synthase matches are all above e-100, and given the very high significance scores are presumed to function as polyketide synthases (as are the high scoring peptide synthetases) .
- IS-Scl An IS element
- DNA sequence data are as defined in claim 7.
- pEP0cos6_0RF7 sequences (cf. claim 9): the predicted N-terminus of 0RF7 shows 145 nucleotide overlap with ORF6.
- Table 3 shows ORF data summary. Note: pEPOcos6_ORFi . seq is truncated at its 5' end; correspondingly pEPOcos6_ORFl .pep is truncated at its N-termmus.
- DNA sequence data from contigs are as defmded claim 10. Table 4 shows more data.
- pEPOcos ⁇ protein data are as defined m claim 11, i.e. for selected ORFs (polyketide synthase, peptide synthetases, or ORFs with high similarity to known genes) .
- ORFs polyketide synthase, peptide synthetases, or ORFs with high similarity to known genes.
- DNA sequence data from contigs are as defined in claim 12. Table 5 shows more data.
- Protein sequence data from selected A5 ORFs are as defined in claim 13.
- Insert contains PKS genes on two large contigs - most similar to the soraphen PKS gene from Sorangium.
- DNA sequence data from plasmid Sau4 contigs are as defined m claim 14. Table 6 shows more data.
- Protein sequence data from selected plasmid Sau4 ORFs are as defined m claim 15.
- Heterologous expression of the ORFs shown m Figure 1 is performed by using a derivative of plasmid pSUP102 (Simon, R., Priefer, U. , P ⁇ hler, A., Methods m Enzymology (1986), vol. 118, pp. 643-659) .
- pSUP102 plasmid pSUP102
- the gene for chloramphenicol resistance is changed for a cassette comprising the gene for streptomycin resistance and the promoter element of the Tn5 transposon.
- Short homologous genomic DNA segments from the host organism are ligated with the DNA sequences of Figure 1 and witr efficient regulatory elements into, for example, the EcoRI restriction site of the vector.
- Heterologous expression of the ORFs shown m Figure 1 is performed by using bifunctional Strepomyces-Escherichia coli cosmids pKU206 and pOJ466.
- Heterologous expression of the ORFs shown m Figure 1 is performed by using "bacterial artificial chromosomes", cosmids (for example Supercos, Stratagene GmbH, Heidelberg) and T7 ex- pression systems (Stratagene GmbH, Heidelberg; New England Bio- labs Schwalbach, FRG) .
- Expression of recombinant enzymes occurs m Escherichia coli cells constitutively expressing phosphopan- tethemyl transferase required for the formation of holoenzyme polyketide synthetases and polypeptide synthetases .
- the predicted ORFl gene and gene product is truncated due to cloning of the DNA into the cosmid vector.
- 731 is the last nucleotide of the last amino-acid-encoding codon; 732-735
- Termination codon is TGA (termination codon) . Termination codons have been excluded in the present annotation.
- 11 bp terminal inverted repeat that is similar to 1S1131 from Agrobacteriura tume aciens (TS-66 like element, 2773 bp, 4 ORFs, 11 bp inverted repeat)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP2000576029A JP2002527067A (en) | 1998-10-09 | 1999-10-11 | DNA sequences for enzymatic synthesis of polyketide or heteropolyketide compounds |
CA002346499A CA2346499A1 (en) | 1998-10-09 | 1999-10-11 | Dna sequences for enzymatic synthesis of polyketide or heteropolyketide compounds |
AU65126/99A AU6512699A (en) | 1998-10-09 | 1999-10-11 | Dna sequences for enzymatic synthesis of polyketide or heteropolyketide compounds |
EP99953117A EP1119628A2 (en) | 1998-10-09 | 1999-10-11 | Dna sequences for enzymatic synthesis of polyketide or heteropolyketide compounds |
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DE19846493.2 | 1998-10-09 | ||
DE1998146493 DE19846493A1 (en) | 1998-10-09 | 1998-10-09 | DNA sequence coding for products involved in the biosynthesis of polyketide or heteropolyketide compounds, especially epothilone |
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EP (1) | EP1119628A2 (en) |
JP (1) | JP2002527067A (en) |
AU (1) | AU6512699A (en) |
CA (1) | CA2346499A1 (en) |
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Cited By (4)
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WO2000031247A3 (en) * | 1998-11-20 | 2000-12-07 | Kosan Biosciences Inc | Recombinant methods and materials for producing epothilone and epothilone derivatives |
WO2001083800A2 (en) * | 2000-04-28 | 2001-11-08 | Kosan Biosciences, Inc. | Heterologous production of polyketides |
US6410301B1 (en) | 1998-11-20 | 2002-06-25 | Kosan Biosciences, Inc. | Myxococcus host cells for the production of epothilones |
US6998256B2 (en) | 2000-04-28 | 2006-02-14 | Kosan Biosciences, Inc. | Methods of obtaining epothilone D using crystallization and /or by the culture of cells in the presence of methyl oleate |
Families Citing this family (5)
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US6242469B1 (en) | 1996-12-03 | 2001-06-05 | Sloan-Kettering Institute For Cancer Research | Synthesis of epothilones, intermediates thereto, analogues and uses thereof |
US6121029A (en) * | 1998-06-18 | 2000-09-19 | Novartis Ag | Genes for the biosynthesis of epothilones |
CA2496477C (en) | 2002-08-23 | 2012-10-16 | Sloan-Kettering Institute For Cancer Research | Synthesis of epothilones, intermediates thereto, analogues and uses thereof |
US7649006B2 (en) | 2002-08-23 | 2010-01-19 | Sloan-Kettering Institute For Cancer Research | Synthesis of epothilones, intermediates thereto and analogues thereof |
CN112941002B (en) * | 2021-02-08 | 2023-04-25 | 中国科学院天津工业生物技术研究所 | Recombinant strain of escherichia coli for producing dopamine as well as construction method and application thereof |
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JP2007097595A (en) * | 1998-11-20 | 2007-04-19 | Kosan Biosciences Inc | Recombinant method and material for producing epothilone and epothilone derivatives |
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US6410301B1 (en) | 1998-11-20 | 2002-06-25 | Kosan Biosciences, Inc. | Myxococcus host cells for the production of epothilones |
JP2002530107A (en) * | 1998-11-20 | 2002-09-17 | コーサン バイオサイエンシーズ, インコーポレイテッド | Recombinant methods and materials for producing epothilone and epothilone derivatives |
WO2000031247A3 (en) * | 1998-11-20 | 2000-12-07 | Kosan Biosciences Inc | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US6303342B1 (en) | 1998-11-20 | 2001-10-16 | Kason Biosciences, Inc. | Recombinant methods and materials for producing epothilones C and D |
US6858411B1 (en) | 1998-11-20 | 2005-02-22 | Kosan Biosciences, Inc. | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US7402421B2 (en) | 1998-11-20 | 2008-07-22 | Kosan Biosciences, Inc. | Recombinant methods and materials for producing epothilone and epothilone derivatives |
KR100851418B1 (en) * | 1998-11-20 | 2008-08-08 | 코산 바이오사이언시즈, 인코포레이티드 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US6921650B1 (en) | 1998-11-20 | 2005-07-26 | Kosan Biosciences, Inc. | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US7067286B2 (en) | 1998-11-20 | 2006-06-27 | Kosan Biosciences, Inc. | Cystobacterineae host cells containing heterologous PKS genes for the synthesis of polykedtides |
KR100716272B1 (en) * | 1998-11-20 | 2007-05-09 | 코산 바이오사이언시즈, 인코포레이티드 | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US7129071B1 (en) | 1998-11-20 | 2006-10-31 | Kosan Biosciences, Inc. | Recombinant methods and materials for producing epothilone and epothilone derivatives |
WO2001083800A3 (en) * | 2000-04-28 | 2003-04-10 | Kosan Biosciences Inc | Heterologous production of polyketides |
EP1652926A3 (en) * | 2000-04-28 | 2006-08-09 | Kosan Biosciences, Inc. | Heterologous production of polyketides |
US7323573B2 (en) | 2000-04-28 | 2008-01-29 | Kosan Biosciences, Inc. | Production of polyketides |
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EP1652926A2 (en) * | 2000-04-28 | 2006-05-03 | Kosan Biosciences, Inc. | Heterologous production of polyketides |
US6998256B2 (en) | 2000-04-28 | 2006-02-14 | Kosan Biosciences, Inc. | Methods of obtaining epothilone D using crystallization and /or by the culture of cells in the presence of methyl oleate |
WO2001083800A2 (en) * | 2000-04-28 | 2001-11-08 | Kosan Biosciences, Inc. | Heterologous production of polyketides |
Also Published As
Publication number | Publication date |
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DE19846493A1 (en) | 2000-04-13 |
CA2346499A1 (en) | 2000-04-20 |
EP1119628A2 (en) | 2001-08-01 |
JP2002527067A (en) | 2002-08-27 |
WO2000022139A3 (en) | 2001-01-18 |
AU6512699A (en) | 2000-05-01 |
WO2000022139A9 (en) | 2000-09-08 |
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