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WO1998038313A1 - Amas d'acarbose acb issu d'actinoplanes espece se 50/110 - Google Patents

Amas d'acarbose acb issu d'actinoplanes espece se 50/110 Download PDF

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Publication number
WO1998038313A1
WO1998038313A1 PCT/EP1998/000862 EP9800862W WO9838313A1 WO 1998038313 A1 WO1998038313 A1 WO 1998038313A1 EP 9800862 W EP9800862 W EP 9800862W WO 9838313 A1 WO9838313 A1 WO 9838313A1
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Prior art keywords
fragment
dna
pas5
plasmid
acarbose
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PCT/EP1998/000862
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German (de)
English (en)
Inventor
Anneliese Crueger
Heiner Apeler
Werner Schröder
Hermann Pape
Klaus Goeke
Wolfgang Piepersberg
Jürgen Distler
Paz Marta Diaz-Guardamino Uribe
Martin Jarling
Ansgar Stratmann
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Bayer Aktiengesellschaft
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Application filed by Bayer Aktiengesellschaft filed Critical Bayer Aktiengesellschaft
Priority to AU62968/98A priority Critical patent/AU6296898A/en
Priority to CA002282735A priority patent/CA2282735A1/fr
Priority to PL98335369A priority patent/PL335369A1/xx
Priority to BR9807640A priority patent/BR9807640A/pt
Priority to EP98906953A priority patent/EP0968294A1/fr
Priority to IL13143398A priority patent/IL131433A0/xx
Publication of WO1998038313A1 publication Critical patent/WO1998038313A1/fr
Priority to BG103672A priority patent/BG103672A/xx
Priority to NO994164A priority patent/NO994164L/no

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/365—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinoplanes (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces

Definitions

  • the present invention relates to the isolation of further genes of the
  • the most potent inhibitor of this group is the compound O-4,6-ddedeoxy-4 - [[IS- ( l S, 4R, 5S, 6S) -4,5,6-t ⁇ hydroxy-l- (hydroxymethyl) -2-cyclohexen-l-yl] -arruno] - ⁇ -D-glucopyranosyl- (l - »4) -O - ⁇ -D-glucopyranosyl- (l ⁇ 4) -D-glucopyranose described as acarbose [DE 23 47 782]
  • Acarbose is a potent ⁇ -glucosidase inhibitor which is used as an oral antidiabetic under the brand name Glucobay ® for the therapy of diabetes melhtus.
  • the secondary metabohten acarbose is formed by Actinoplanes sp SE 50
  • the deoxyglucose part of the acarbose molecule is formed in accordance with the biosynthesis of 6-deoxy sugar residues from various antibiotics (eg aminoglycosides such as streptomycin, kasugamycin, macrolides such as erythromycin, tylosin, polyenes such as amphote ⁇ cin A, B, nystatin, anthracycles, such as daunorubicin, glycopeptides, such as vancomycin) Therefore, a gene probe and heterologous PCR polymer pairs were derived from highly conserved protein regions of known dTDP-glucose-dehydratase enzymes
  • antibiotics eg aminoglycosides such as streptomycin, kasugamycin, macrolides such as erythromycin, tylosin, polyenes such as amphote ⁇ cin A, B, nystatin, anthracycles, such as daunorubicin, glycopeptides,
  • Acarbose 7-phosphotransferase (acarbose kinase) could be involved in producing a form of transport of acarbose from the cell.
  • acarbose 7-phosphotransferase is seen as part of a self-protection mechanism
  • Acarbose strongly inhibits the cytoplasmic ⁇ -glucosidase of the production strain, but the compound phorphorylated by acarbose 7-phosphotransferase does not, so that the cell's own substrate metabolism is not disturbed.
  • Such protective mechanisms have been described for many producers of aminocychtol antibiotics
  • the cluster of the biosynthesis genes and other genes of the metabolism of acarbose from Actinoplanes sp SE 50/1 10 is described in a section of 18 kb (see FIGS. 1-3).
  • This finding is for the biotechnological production of
  • Acarbose biosynthesis genes are essential for a targeted improvement of the production process e.g.
  • a recombinant DNA molecule that contains genes for the biosynthesis of acarbose and acarbose hsmologists, which are arranged in the gene cluster according to Figure 2.
  • genes acbA and acbB with high probability encode enzymes of acarbose biosynthesis, since they have high sequence identity of AcbA and AcbB to be knew bacterial dTDP-glucose synthases or dTDP-glucose 4,6-dehydratases.
  • the similarity of the protein sequences to the next known representative of the two enzyme families is far above that of any other pairs of functionally identical proteins from both groups. It is surprising that AcbA has its closest relatives among Streptomycete proteins, while AcbB is more closely related to various RfbB proteins of Gram-negative bacteria.
  • the acbC gene encodes a probable enzyme of acarbose biosynthesis, since the enzyme AcbC is related to AroB proteins, dehydroquinic acid synthases and, after overexpression in Streptomyces lividans, shows an enzyme activity which - as expected - heptulose phosphates (e.g. sedoheptulose-7 -phosphate) to products which have properties similar to those of Va enon and Va olon (possible precursors of acarbose biosynthesis), but not identical to these compounds
  • genes acbK (acarbose-7-K ⁇ nase), acbL (keto sugar or sugar alcohol oxidoreductase) and acbM (unknown function) as well as the acbN gene (direct connection to the reading frame acbL and acbC is indicated by overlapping start / stop codons) probably encode enzymes Acarbose biosynthesis, since they all form a probable operon (transcription unit) together with acbC and possibly also acbQ and are likely to be read translationally coupled.
  • the acbHFG genes encode a probably extracellular sugar-binding protein, AcbH, and two typical membrane components, AcbF and AcbG, a bacterial sugar transporter of the ABC-Importer type. They are probably involved in or metabolize the metabolism of acarbose by ingestion of oligo-maltodextins
  • Acarbose as a transport vehicle for short ohgo- ⁇ -1,4-glucans (higher homologues of acarbose) by being taken into the cell
  • the amylomaltase-like gene product (AcbQ) of the acbO gene could also be involved in such a process
  • the invention further discloses
  • the 2.2 kb Ba Hl fragment which by means of A gene probe, which was obtained by PCR polymer and was derived from highly conserved protein regions of known dTDP-glucose dehydratase enzymes, is described in the patent application [EP A 0 730 029 / DE 19507214]
  • a method for isolating biosynthetic genes from acarbose-related natural substances in actinomycetes e.g. for vahdamycin, ohgostatine (trestatin), adipo- Process for increasing the acarbose synthesis performance in actinoplanes by increasing the gene dose for rate-determining biosynthetic enzymes, more effective promoters for rate-determining biosynthetic enzymes,
  • a method for changing transport mechanisms with a view to an improved transport of substrates into the cell or a more effective removal of acarbose from the cell is described.
  • a method for expression in heterologous host strains eg pseudo-oligosaccharide-forming Streptomycetes as well as in other Streptomycetes such as Streptomyces lividans, in fast-growing bacteria such as E. coli or in yeasts and fungi
  • heterologous host strains eg pseudo-oligosaccharide-forming Streptomycetes as well as in other Streptomycetes such as Streptomyces lividans, in fast-growing bacteria such as E. coli or in yeasts and fungi
  • the plasmid pAS2 was prepared from E coli DH5 ⁇ using the "boiling method" or by alkaline lysis and using the BamHl residual endonuclease hydrolyzed The resulting 2.2 kb Ba HI fragment was isolated and labeled with j2 P-labeled deoxynucleotides by so-called "nick translation”.
  • This radioactively labeled fragment was used as a gene probe for the isolation of acarbose biosynthesis genes and is hereinafter referred to as acb -Sonde-II designated
  • the second gene probe was isolated from the plasmid p AS 5/7 3
  • the Sphl-Sstl fragment was isolated and radioactively labeled as described above.
  • this gene probe is referred to as acb-Probe-III isolated from plasmid pAS6 / 3.
  • the BamHl fragment was isolated and radioactively labeled as described above. This probe was given the name acb-probe-I V
  • Acarbose biosynthesis genes were isolated in two ways as follows 1) Chromosomal DNA from Actinoplanes sp was hydrolyzed with the restriction enzymes Ssf, Bglll and Pstl, separated by gel chromatography and by "Southern" hybridization with acb probe II (Sstl and BgHl hydrolysis) and acb probe III (P hydrolysis) after a homologous DNA sequence.
  • the S tl fragment hybridizing with the gene probe has a size of approx. 10.7 kb and the 5 g // I fragment of approx. 10 2 kb.
  • the 10.7 kb Etl fragment and the 12 kb .Sg ZI fragment were eluted from the gel, ligated into the vector pUC18 or pBluescript II KS and cloned into E.co/i DH5 ⁇ .
  • the resulting plasmids were given the names pAS5 (SstI fragment) and pAS6 (5g / I fragment) one with the Etl fragment overlapping 2.8 kb Rs II fragment, which hybridized with the gene probe acb probe III, was cloned into the vector pUC18 and designated pMJl
  • a GEM 12 phage gene bank of genomic DNA from Actinoplanes sp was screened with the probes acb probe III and acb probe IV by means of plaque hybridization. A total of 15 phages were isolated with the acb probe III, two phages with the acb probe IV, which total approx. 38 5 kb colinear Actinoplanes sp. Containing DNA with acarbose biosynthesis genes. The phages that were characterized in more detail bear the designations 10/3 and 5/4
  • pAS5 / l 7 0.46 kb PCR fragment
  • pAS5 / 18 0.26 kb PCR fragment
  • pAS5 / 19 0.27 kb PCR fragment
  • Plasmids constructed for the determination of the DNA sequence which contain DNA fragments with acarbose biosynthesis genes from Actinoplanes sp from plasmid pAS6 (cf. Example 6).
  • the DNA cloned in plasmid pAS6 has a 6.2 kb acarbose biosynthesis gene contained ßg / II / SstI fragment together with the plasmid pAS5
  • the following recombinant plasmids were constructed in the vector pUC 18.
  • pAS6 / 3 2.8 kb BamHl fragment from the plasmid pAS6 pAS6 / 3.1 1, 1 kb Hincll fragment from the plasmid pAS6 / pAS6 / 3.2 1, 2 kb SalI fragment from the plasmid pAS6 / 3 pAS6 / 3.3 1.45 kb Pstl Fragment from the plasmid pAS6 / 3
  • Plasmid pAS5 / l 7 Primer name sequence acbD / El 5 'GGCGGCGATTCGGCCTGCGCGG 3 1 acbD / E2 5' GCGGCGATGGCATGCCTGGCG 3 '
  • Plasmid pAS5 / l 8 primer name sequence acbD3 5 'ACCAGGCCGAGGACGGCGCCC 3 1 acbD4 5' AGCGGCATGTGCTTGACGGCG 3 '
  • Plasmid pAS5 / 19 Primer name sequence acbD5 5 'ACCGGCTCGAACGGGCTGGCACC 3' acbD6 5 'CCCTCGACGGTGACGGTGGCG 3' Primer for the amplification of the acbC gene:
  • Primer for the sequence reaction Primer name sequence universal primer 5 1 GTAAAACGACGGCCAGT 3 'reverse primer 5' GAAACAGCTATGACCATG 3 '
  • the N-terminal sequence analysis of the Acb ⁇ protein was carried out using the gas phase protein sequencer 473 A from Applied Biosystems (Forster City, CA., USA). The standard Fastblott protein sequencing program was used. The protein sequencer, the various programs, the breakdown cycles and the
  • PTH identification systems are described in the manual of the sequencer (User's manual protein sequencing system model 473 A (1989); Applied Biosystems Forster City, CA 94404, USA).
  • the detection of the PTH amino acids was carried out on-line with an RP-18 column (220 mm x 2 mm, 5 ⁇ material) from Applied Biosystems.
  • the PTH amino acids were identified and quantified using a 50 pmol standard.
  • the data was processed with Applied Biosystems 610A sequencer data system.
  • E coli DH5 ⁇ was incubated in LB medium at 37 ° C. Plasmid-bearing bacteria were kept under selection pressure (ampicillin, 100 ⁇ g / ml). The cultivation was carried out on a rotary duster at 270 rpm. Approaches were described as overnight culture (UK) which incubated for at least 16 h were
  • plasmid DNA For the preparation of plasmid DNA, the cells from 1.5 ml of a UK incubated under selection pressure were used. The plasmids were isolated by the alkaline SDS lysis method [Birnboim, HC, and J Doly (1979)]
  • restriction endonucleases Only restriction endonucleases according to the manufacturer's instructions (Gibco BRL, Eggenstein, Germany) were used for the targeted hydrolysis of vector DNA. 5 U of the respective restriction endonuclease were used for the restriction of 10 ⁇ g plasmid DNA and incubated at 37 ° C. for 2 hours To ensure complete hydrolysis, the same amount of residual endonuclease was added a second time and incubated again for at least 1 h
  • the cleaved DNA was electrophoretically separated on 0.5-1.2% horizontal agarose gels.
  • the gel piece which contained the DNA fragment, was cut out with a sterile scalpel and weighed DNA fragments from the agarose were carried out according to the instructions with the ETsorb kit (Genomed, Bad Oeynhausen, Germany) 2.
  • Actinoplanes sp SE50 / 1 10 was incubated at 30 ° C. in TSB medium on a rotary debris for 3 d.
  • the preculture (5 ml) was carried out at 240 rpm in culture tubes
  • the total DNA was prepared with 1.5-2 mg cells (fresh weight) according to the phenol / chloroform extraction method [Hopwood, D A, et al (1985)]
  • the cleaved DNA was electrophoresed on 0.6% horizontal agarose gels
  • the DNA fragments were again eluted using the JETsorb kit (see Example 1).
  • DNA fragments from agarose gels were transferred to membranes by the "Southern Transfer” method [Southern, EM (1975)].
  • the agarose gels obtained according to Example 2 were swirled in 0.25 M HC1 for 20 minutes 3MM absorbent Whatman paper (Whatmann, Maidstone,
  • nylon filters were then shaken at least 2 h at 68 ° C in 50-100 ml prahyb ⁇ disisation solution in a water bath. The solution was changed at least twice. The hybridization took place in the hybridization cabinet for at least 12 h. 15 ml hybridization solution, which the acb -Sonde-II contained (s example
  • the nylon filters were then washed for 15 minutes with 6x postwash and 1 x postwash.
  • the nylon filters were then covered with cling film while still moist.
  • the autoradiography was carried out with Hyperfilm-MP
  • the ligation took place in a volume of 20 ⁇ l, the ratio of fragment to vector being 3 1 with 0.01-0.1 ⁇ g of DNA in the batch.
  • 1 U of the T4 DNA ligase with the appropriate buffer Gibco BRL , Eggenstein, Germany
  • the eluted _9g / ⁇ fragments were ligated into the Z-mHI-hydrolyzed vector plasmid pBluescript II KS.
  • the ligation was carried out as with the Sstl and Pstl
  • Transformation-competent cells from E.coh DH5 ⁇ were transformed with complete ligation approaches [according to Hanahan, D (1983)] Ampicil n-resistant transformants were transferred to LB-Amp selective plates (100 ⁇ g / ml)
  • Ampicillin-resistant transformants were examined for the presence of the 10.7 kb SstI fragment and 12 kb P / II fragment which hybridized with the acb probe II. Ten of these clones were streaked on a selective plate, incubated overnight and washed from the plate with 3 ml LB medium. The plasmid DNA was then isolated from 20 such pools of ten [according to Birnboim, HC, uJ
  • the recombined phage 10/3 was hydrolyzed with Pstl, the DNA on a horizontal agarose gel and the 2.8 kb Pvtl fragment eluted from the matrix (see example 1) and ligated into the vector pUC18.
  • the recombinant plasmid was given the name pMH and was ⁇ n Transformed £ coli DH5 ⁇
  • the recombinant phage 5/4 was hydrolyzed with Sstl, the DNA separated on a horizontal agarose gel and the 6.3 kb Sstl fragment eluted from the matrix (see Example 1) and ligated into the vector pUC18.
  • the recombinant plasmid was given the name pMJ9 and was transformed into E coli DH5 ⁇
  • the phages with acarbose biosynthesis genes were identified by plaque hybridization (according to Sambrook et al 1989) with the gene probes acb-HI and acb-IV.
  • the phage DNA which contained acarbose biosynthesis genes was made from phages which were propagated on E. coli LE392 according to Sambrook et al. (1989)
  • the PCR is used for the in vitro multiplication of selected DNA areas [Mullis, KB, u FA Falloona (1987)].
  • the Taq DNA polymerase according to the manufacturer (Gibco BRL, Eggenstein) was used in 25 reaction cycles.
  • the batches contained 5% formamide to suppress possible secondary structures in GC-rich DNA.
  • the volume was 100 ⁇ l with 50 pmol per polymer and 200 uM dNTP's were used
  • an initial five minute denaturation of DNA at 95 ° C 2.5 U of thermostable DNA polymerase were mixed in a "hot-start" added to the lugs primer extension was carried out at 72 ° C and the denaturation of DNA at the beginning of each cycle was carried out at 95 ° C. for 1 min.
  • the reactions were carried out in a Biometra thermal cycler (Gottingen)
  • the plasmid pAS5 was hydrolysed with the restriction enzyme Pstl, gel electrophoresis (0.7% agarose gel), the 5.4 kb Pstl fragment eluted from the gel and in pUC 18 (hydrolyzed with Pstl) in E. coli DH5 ⁇ cloned
  • pAS5 / 3. pAS5 / 4. pAS5 / 13. pAS5 / 16 The plasmid pAS5 was hydrolyzed with the restriction enzyme BamHl and gel-electrophoretically separated. The fragments had the following size
  • the fragments intended for subcloning with a size of 1.4 kb and 0.5 kb were eluted from the gel (see example 1).
  • the vector pUC 18 was prepared using the restriction enzyme BamHl as described in example 1. The ligations were carried out The 0.5 kb fragment was ligated into the prepared pUC 18 and the subclone pAS5 / 16 was formed.
  • the subclone pAS5 / 3 was formed after the ligation of the 1.4 kb fragment with the prepared pUC 18 the subclone pAS5 / 4 was created after ligation of the 1.2 kb fragment with the vector pUC18.
  • the subclone pAS5 / 13 was created by rehabilitation of the 7.5 kb BamHl fragment
  • PAS5 / 5 PAS5 / 7 pAS5 / U. PAS5 / 12
  • the plasmid pAS5 was with the restriction enzymes BamHl u Sstl, Pstl u. Sstl, Bglll u Pstl and Bglll u Hindill hydro- The remaining fragments were separated in a 1.2% agarose gel. The corresponding fragments were eluted from the agarose gel and cloned in E.
  • Subclone pAS5 / 5 contains the 0.48 kb Sstl BamHl fragment
  • subclone pAS5 / 12 contains the 0.63 kb BglW Pstl fragment
  • subclone pAS5 / l 1 contains the 0.68 kb BgllllHin ⁇ lll fragment
  • Example 1 and in the vector pUCBM21 (Boeh ⁇ nger, Mannheim) (hydrolyzed with Ncol Kpnl) in E. coli DH5 ⁇ , and the subclones pAS5 / 15 12 (0.9 kb fragment) and pAS5 / 15 1 1 (1, 1 kb fragment)
  • the plasmid pAS6 was hvdrolysed with the residual endonuclease Sst1 (using the residual interface of the vector) and a 5.9 kb Sstl fragment was eluted from the agarose gel and ligated with the plasmid pUC 18.
  • L coli DH5 ⁇ was transformed with the recombinant plasmid
  • the plasmid pMI6 / 6 was hydrolyzed with the residual endonucleases BamHl and Pstl, and a 0.36 kb BamHl Pstl and a 0.5 kb ⁇ wHI / Pstl fragment were obtained from the Agarose gel eluted and ligated with the plasmid pUC18 E. coli DH5 ⁇ was transformed with the recombinant plasmids
  • pMI6 / 6 2 2. pMI6 / 6 2 3. pMI6 / 6 2 4, pMJ6 / 6 2 5, pMJ6 / 6 2 6 pMJ6 / 6 2 7, pMI6 / 6 2 8 the plasmid pMI6 / 6 was used with the residual endonuclease Sall hydrolyzed and a 3.3 kb fragment, a 1.2 kb fragment, a 1.0 kb fragment, a 07 kb fragment, a 0.14 kb fragment and a 0.13 kb fragment were made from eluted the agarose gel and ligated with the plasmid pUC18. E coli DH5 ⁇ was with the recombinant plasmids transformed The plasmid pMJ6 / 6 2 2 was obtained by hydrolysis and the following reg
  • pMI6 / 8 1 the plasmid pMI6 / 6 was hydrolyzed with the restriction enzymes C / al and BamHl and a 1.1 kb fragment was eluted from an agarose gel and with the restriction enzymes C / al and BamHl and a 1.1 kb fragment was eluted from an agarose gel and with the restriction enzymes C / al and BamHl and a 1.1 kb fragment was eluted from an agarose gel and with the
  • Plasmid pBluesc ⁇ pt II KS ligated E coli DH5 ⁇ was transformed with the recombinant plasmid
  • the plasmid pMI6 / 6 was hydrolyzed with the restriction enzymes Pstl and Sall and a 1.5 kb fragment was eluted from an agarose gel and with the restriction enzymes Pstl and Sall and a 1.5 kb fragment was eluted from an agarose gel and with the restriction enzymes Pstl and Sall and a 1.5 kb fragment was eluted from an agarose gel and with the
  • Plasmid pUC18 ligated E. coli DH5 ⁇ was transformed with the recombinant plasmid
  • the plasmid pAS6 was hvdrolyzed with the residual endonuclease BamHl and a 2.8 kb BamHl fragment was eluted from the agarose gel, ligated to the plasmid pUC 18 and transformed into E coli DH5 ⁇
  • pAS6 / 3 1 The plasmid pAS6 / 3 was hvdrolyzed with the residual endonuclease Hi cll and a 1.1 kb fragment in pUC18, hydrolyzed with Hincll, ligated and clomerized in L coli DH5 ⁇
  • the plasmid pAS6 / 3 was hvdrolvated with the residual endonuclease Sall, a 1.2 kb fragment in pUC18, hydrolyzed with Sall, ligated and clomerized in L coli DH5 ⁇
  • the plasmid pAS6 was hydrolysed with the residual endonuclease Pstl, a 1.45 kb fragment was eluted from the gel and ligated with pUC18. L coli DH5 ⁇ was transformed with the recombinant plasmid 11. Subcloning of the plasmid pMJl
  • the plasmid pMJl was hydrolyzed with the residual endonuclease Sphl and a 3.3 kb S M fragment (0.6 kb Sphl / Pstl fragment ligated m ⁇ tpUC 18) eluted from the agarose gel. This fragment was re gated and clomerized in E coli DH5 ⁇
  • the plasmid pMJ1 was hydrolyzed with the residual endonuclease Sal and a 3.9 kb Sal fragment (1.2 kb Sal / Pstl fragment ligated with pUC18) eluted from the agarose gel. This fragment was rehabilitated and clomerized in E coli DH5 ⁇
  • the plasmid pM l was hydrolyzed with the residual endonuclease Sal and a 4.1 kb Sstl / Pstl fragment (1.4 kb SstllPstl fragment ligated to pUC 18) eluted from the agarose gel. This fragment was rehabilitated and clomerized in E coli DH5 ⁇
  • the plasmid pMJl was hvdrolyzed with the residual endonuclease Sall and a 0.9 kb SaWSmal fragment was eluted from the agarose gel and ligated with the plasmid pUC 18.
  • the recombinant plasmid was transformed into E coli DH5 ⁇
  • the pAS5 / 6 subclones were produced using the “double-stranded nested deletion kit” (Pharmacia, Freiburg, Germany). 10 ⁇ g pAS5 / 6 DNA was prepared as described in Example 1 and hydrolyzed with 10 U each Xhol and Ss / I The subsequent incubation with exonuclease III was carried out according to the manufacturer's instructions for a total of 20 min. At intervals of 5 min, portions were removed from the reaction mixture which corresponded to a DNA amount of approx. 2.5 ⁇ g DNA. Treatment with S 1 nuclease to produce non-overlapping DNA -Ends took place for 30 min at 20 ° C according to the manufacturer's instructions. These DNA molecules were rehabilitated with T4-L ⁇ gase and clomerized in E coli DH5 ⁇ 13. DNA sequencing of acarbose biosynthesis genes from Actinoplanes sp.
  • the plasmids described in Examples 8 to 11 were sequenced. 6-8 ⁇ g of plasmid DNA from a preparation (see Example 1) were used in the sequencing reaction. The sequencing reaction was carried out using the auto-read sequencing kit (Pharmacia, Freiburg , Germany) The standard protocol for the sequencing of dsDNA was used. In order to enable the nucleotide sequence to be evaluated with the ALF (automated laser fluorescence (DNA) sequencer), the starter molecules for the sequence reaction were the
  • Fluorescein-labeled universal and reverse sequencing polymer was used (see Table 2). 8 ml of Hydro Link Long Ranger (Serva Heidelberg), 33.6 g of urea, 8 ml of 10X TBE buffer, ad 80 ml with H 2 were used to prepare the gel 0 mixed, filtered and degassed for 1 minute The polymerization was initiated by adding 350 ⁇ l 10% (w / v) ammonium persulfate and 40 ⁇ l N, N, N ', N', tetramethylethylenediamine. The solution was dissolved in a gel form (50x50x0, 05 cm) cast The electrophoresis was carried out at 38 W and a constant temperature of 45 ° C.
  • the Tx buffer was used as the running buffer.
  • the measured fluorescence was processed into a DNA sequence using a connected computer (Compaq 386 / 20e), which was also used for Control of the electrophoresis unit was used (program A. LF Manager 2 5
  • the DNA sequence of the acbC gene shows two possible translation starting points for
  • starting point 1 represents the probable start of AcbC due to a more significant ribosome binding site, both possible AcbC proteins were overexposed.
  • the plasmids pETl la and pET16b were used for expression in E coli in order to ensure an optimal start of the expression If the ATG start codon, with a suitable distance to an E coli-like RBS, the pET vectors should be used, it is necessary to construct a ⁇ Wel recognition sequence at the start codon of acbC.
  • the O gonucleotides AS7 (sequence position 6617) and AS8 (sequence position 6638) are used for the synthesis of a elWel recognition site at the two possible start codons.
  • the gonucleotide AS9 binds 66 bp downstream of a BamHl recognition sequence at sequence position 6877 to the DNA using the PCR method (see example 8) two DNA fragments were amplified, which are necessary for expression of the two possible AcbC Prot one was used.
  • the addition of the polymers took place at 40 ° C. for 40 seconds, and the extension of the polymer took place in 30 seconds.
  • the two amplified DNA fragments were hydrolyzed with the residual endonucleases Ndel and BamHl and correspondingly into the vectors pET11a and pET16b ligated
  • the 2.2 kb ⁇ wHI fragment was isolated from the recombinant plasmid pAS2 [EP A 0 730 029 / DE 19507214] and fused to the monied PCR fragments via the _5_7mHI recognition site after the orientation of the 2.2 kb P ⁇ mHI fragment was checked, the complete acbC gene was present in the expression vectors.
  • the expression vectors were given the name pAS8 / l - pAS8 / 4 (FIG. 4).
  • the AcbC protein was expressed in S lividans 1326 by means of the plasmid vector pIJ6021 [Takano, e, et al (1995)].
  • a fragment from chromosomal DNA was amplified using the PCR method [Mulhs u Falloona, (1987)], which only the encoding region of the acbC gene
  • the OHgonucleotide AS-Cl and AS-C2 were used, a Ndel recognition sequence at the start codon 2 of the acbC gene being constructed with the aid of the polymer AS-C1 (sequence position 6089)
  • Ogonucleotide AS-C2 binds at sequence position 7882 and was used to construct an EcoRI recognition sequence.
  • the addition of the polymer took place in 20 seconds at 50 ° C., and the polymer was extended in 40 seconds.
  • the acbC DNA fragment thus obtained was first blom-ended in the vector pUC18 and the correctness of the DNA sequence was checked after the PCR.
  • This recombinant plasmid with the cloned acbC gene was called pAS8 / 5 1.
  • the plasmid pAS8 / 5 1 was labeled with the residual functions endonucleases Ndel and EcoRI hydrolyzed, the DNA separated on an agarose gel and eluted from the matrix.
  • the “cAC fragment thus prepared was ligated into the vector pIJ6021.
  • the acbE gene could be obtained from the plasmid pAS5 / 6.9-6 by means of hydrolysis with the restriction endonucleases EcoRI and Hmdlll. After separation of the DNA on an agarose gel and elution of the 3.8 kb EcoRI / HwdIII fragment from the matrix, this acbE fragment was ligated accordingly into the vector pUWL219 [J. Wehmeier, U.F .. (1995)]. For a later expression of AcbE in S. lividans, a possible promoter sequence on the 200 bp "upstream" region should be used in these vectors (see Table 5). The recombinant plasmid was named pASl 1 (FIG. 7).
  • Tab. 5 The intercistronic area between the genes acbE and acbD.
  • Inverted repeat (IR) and direct repeat (DR) sequences that could be involved in regulation are underlined.
  • TAC TTAAAG CTC TGC GCA AGC TTA GGG TTG AAG TGG CGG TGA TGC ATC CAT CAC TGT ATG ATG AAT TTC GAG ACG CGT TCG AAT CCC AAC TTC ACC GCC ACT ACG TAG GTA GTG ACA TAC
  • Protoplasts from S. lividans TK23 were transformed with the plasmid pASl 1.
  • both in the S. lividans TK23 / pASl 1 and in the Actinoplanes sp. Approaches an extracellular protein the size of
  • Electrophoresis was carried out either with the SERVA Blue-Vertical 100 / C apparatus (gel form, 80 x 100 x 0.75 mm) or with the Renner Twin Vertical apparatus (gel form, 180 x 170 x 1 mm)
  • the protein concentration of the samples to be analyzed was determined using the protein assay (BioRad, Kunststoff), a calibration line being established using BSA
  • the "VILL Dalton marker” (14.2 kDa - 66 kDa) and the "High Molecular Weight Standard” (29 kDA - 210 kDa) from Sigma (Deisenhofen) served as the standard for the molecular weights of the separated proteins.
  • the N-terminal amino acid sequence of the AcbE protein was compared by comparing Actinoplanes sp and the clone S. lividans TK23 / pASl 1. 50 ml cultures were incubated in MD 50 medium for three days. The cells were removed by centrifugation and the culture supernatants for 12 h against buffer (5 mM
  • Tris / HCl pH 7.5, 1 mM CaCl 2 dialyzed at 4 ° C.
  • the supernatants were then freeze-dried in 48 h and taken up in 1.5 ml of sample buffer.
  • the culture supernatants prepared in this way were analyzed in an SDS-PAGE using the Renner-Twin-Vertical - Equipment (gel form, 180 x 170 x 3 mm) separated In order to ensure the best possible separation of the AcbE protein from other extracellular proteins, a gradient gel (5% - »10%) was used.
  • the AcbC extract required for the enzyme test was prepared by dialysis (12 h) against 2.5 liters of digestion buffer at 4 ° C. This extract could be kept at -20 ° C. for two months be stored without noticeably losing activity.
  • the protein content of the extract was determined with the protein assay (BioRad, Kunststoff) and 15 ⁇ g analyzed in an SDS-PAGE (FIG. 6)
  • the enzyme test was carried out in a 20 mM P buffer (pH 7.5) with 40 ⁇ M CoCl 2 at RT for 2 h. 20 ⁇ g total protein from the AcbC extract and 8 mM sedoheptulose-7-phosphate in the enzyme - test used In addition, the reaction mixture contained 2 mM NaF, to unspecific
  • Inhibit phosphatases in the extract were 100 ⁇ l.
  • the evaluation was carried out by TLC on silica gel foils with butanol / ethanol H 2 O (9 7 4) as the eluent, 25 ⁇ l being analyzed from a reaction batch.
  • cerium -Reagent s buffer and solutions
  • After a 15-minute incubation at 90 ° C in the drying cabinet the organic compounds were made visible.
  • a mixture of Valienon and Valiolon (Prof. HG Floss, Seattle) served as reference substance.
  • Sedoheptulose-7-phosphate could be implemented specifically by the AcbC protein expressed in S. lividans (FIG. 9). However, the reaction product showed a slightly different running behavior in the DC than the Valienon-Naliolon standard. A reduction in the migration distance of the reaction product on the silica gel Foil could be excluded by the reaction buffer (Fig. 9, lane 5)
  • the cultures of S lividans TK23 / pAS l 1 were grown in TSB medium and MD 50 medium in the presence of 25 ⁇ g / ml thiostrepton. After 3-4 days of incubation, the cultures were harvested by centrifugation (3500 g) at 4 ° C for 10
  • the cells were removed for min.
  • the supernatants were dialyzed against buffer (25 mM Tris HCl pH 7.5, 1 mM CaCl) for 12 h at 4 ° C. From the supernatants prepared in this way, 500 ⁇ l were dried in vacuo, in sample buffer (s buffer and Solutions) were added, the proteins in the supernatants were separated on an SDS-PAGE (FIG. 8).
  • Sample buffer s buffer and Solutions
  • the ⁇ -amylase activity was determined by measuring the decrease in turbidity of a 1% starch suspension.
  • the solutions are sterile filtered after mixing
  • the acb probe is placed in 15 ml prehybridization solution.
  • Genome of Actinoplanes sp SE50 / 110 (see Fig. 2)
  • the black bar indicates the area claimed in the original patent, which overlaps the genes acbCBA (order from left to right as indicated) z T
  • Fig. 2 gene map of the acarbose biosynthesis cluster
  • FIG. 4 The recombinant plasmids which - starting from the plasmids pETl la and pETl ⁇ b - were constructed for the expression of AcbC in E. coli
  • Fig. 5 The recombinant plasmid pAS8 / 7 2, which - starting from the
  • Plasmid pIJ6021 - was constructed for the expression of AcbC in S lividans 1326
  • Plasmid pUWL219 - was constructed for the expression of AcBE in S lividans rK 23
  • Track 5 and track 6 can be seen Detection of the AcbC enzyme activity by thin layer chromatography on silica gel films (see Example 19 1)

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Abstract

L'invention concerne des gènes de biosynthèse issus du groupe de gènes acarbose provenant d'actinoplanes espèce SE 50/110, leur isolement à partir d'actinoplanes d'espèce ou de producteurs de pseudo-oligosaccharides. L'invention concerne un procédé d'isolement de ces gènes de biosynthèse, les protéines codées par lesdits gènes, l'expression des protéines dans des souches-hôtes hétérologues et l'utilisation des gènes de biosynthèse d'acarbose pour optimiser le procédé.
PCT/EP1998/000862 1997-02-28 1998-02-16 Amas d'acarbose acb issu d'actinoplanes espece se 50/110 WO1998038313A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU62968/98A AU6296898A (en) 1997-02-28 1998-02-16 Acarbose (acb) cluster from (actinoplanes) sp. se 50/110
CA002282735A CA2282735A1 (fr) 1997-02-28 1998-02-16 Amas d'acarbose acb issu d'actinoplanes espece se 50/110
PL98335369A PL335369A1 (en) 1997-02-28 1998-02-16 Acarbosic claster from actinoplanes sp. se 50/110
BR9807640A BR9807640A (pt) 1997-02-28 1998-02-16 Aglomerado de acb de acarbose a partir de actinoplanes sp. se 50 / 110
EP98906953A EP0968294A1 (fr) 1997-02-28 1998-02-16 AMAS D'ACARBOSE ACB ISSU $i(D'ACTINOPLANES) ESPECE SE 50/110
IL13143398A IL131433A0 (en) 1997-02-28 1998-02-16 Biosynthesis genes from actinoplanes sp.
BG103672A BG103672A (en) 1997-02-28 1999-08-20 Acarbose acb group, isolation of acarbose biosynthesis and acarbose metabolism from actinoplanes sp. se 50/110 and their application
NO994164A NO994164L (no) 1997-02-28 1999-08-27 Acarbose-(ACB)-klustere fra Actinoplanes sp. SE 50/110

Applications Claiming Priority (2)

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DE19708127A DE19708127A1 (de) 1997-02-28 1997-02-28 Acarbose acb-Cluster: Isolierung von weiteren Genen der Acarbose Biosynthese und des Acarbose-Stoffwechsels aus Actinoplanes sp. SE 50/110 sowie deren Verwendung
DE19708127.4 1997-02-28

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0796915A3 (fr) * 1996-03-22 1999-04-14 Bayer Ag Procédé de préparation et utilisation d'acarviosyl-transferase dans la conversion d'homologues d'acarbose et dans la préparation d'homologues d'acarbose
US9719064B2 (en) 2010-08-04 2017-08-01 Bayer Intellectual Property Gmbh Genomics of actinoplanes utahensis

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106167814B (zh) * 2016-08-31 2019-08-09 河北华荣制药有限公司 一种提高阿卡波糖发酵单位的方法
CN106566796B (zh) * 2016-10-28 2020-11-10 上海交通大学 阿卡波糖生产菌Actinoplanes spp.的遗传操作体系
EP4045520A1 (fr) * 2019-10-16 2022-08-24 Bayer Aktiengesellschaft Procédés pour la formation améliorée d'acarbose
CN112592878B (zh) * 2020-12-25 2022-08-26 上海交通大学 增强正调控蛋白基因表达以提高阿卡波糖发酵水平的方法
CN113444670A (zh) * 2021-07-28 2021-09-28 山东鲁抗医药股份有限公司 一种高活性阿卡波糖产生菌的筛选方法和培养方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0730029A2 (fr) * 1995-03-02 1996-09-04 Bayer Ag Gènes biosynthétiques de l'acarbose obtenus à partir de Actinoplanes sp., procédé d'obtention et utilisation
DE19622783A1 (de) * 1996-06-07 1997-12-11 Hoechst Ag Isolierung der Biosynthesegene für Pseudo-Oligosaccharide aus Streptomyces glaucescens GLA.O und ihre Verwendung

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0730029A2 (fr) * 1995-03-02 1996-09-04 Bayer Ag Gènes biosynthétiques de l'acarbose obtenus à partir de Actinoplanes sp., procédé d'obtention et utilisation
DE19622783A1 (de) * 1996-06-07 1997-12-11 Hoechst Ag Isolierung der Biosynthesegene für Pseudo-Oligosaccharide aus Streptomyces glaucescens GLA.O und ihre Verwendung

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0796915A3 (fr) * 1996-03-22 1999-04-14 Bayer Ag Procédé de préparation et utilisation d'acarviosyl-transferase dans la conversion d'homologues d'acarbose et dans la préparation d'homologues d'acarbose
US5989882A (en) * 1996-03-22 1999-11-23 Bayer Aktiengesellschaft Processes for preparing acarviosyl transferase and for using it in the conversion of acarbose homologues into acarbose, for the preparation of acarbose homologues
US9719064B2 (en) 2010-08-04 2017-08-01 Bayer Intellectual Property Gmbh Genomics of actinoplanes utahensis

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ZA981659B (en) 1998-09-10
CA2282735A1 (fr) 1998-09-03
DE19708127A1 (de) 1998-09-03
CN1249001A (zh) 2000-03-29
BG103672A (en) 2000-05-31
EP0968294A1 (fr) 2000-01-05
PL335369A1 (en) 2000-04-25
BR9807640A (pt) 2000-03-21
CZ9903054A3 (cs) 1999-11-17
AU6296898A (en) 1998-09-18
NO994164D0 (no) 1999-08-27
IL131433A0 (en) 2001-01-28
HUP0000889A2 (en) 2000-07-28
NO994164L (no) 1999-08-27

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