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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters
  • C12P7/625—Polyesters of hydroxy carboxylic acids
• • 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
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• • 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
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
  • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

• Microorganisms permitting the intracellular polyhydroxy al anoate synthesis with simultaneous extracellular polysaccharide synthesis and processes for producing the same
• the present invention relates to microorganisms which are capable of intracellularly synthesizing polyhydroxy alkanoate and of expressing extracellular enzymes which catalyze the synthesis of different polysaccharides whereby di-, oligo- and polysaccharides that are present in the culture medium are cleaved. These enzymes are in particular hexosyltransferases. Furthermore, the present invention relates to processes for preparing such microorganisms.
• biopolymers such as for instance polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) , inter alia, is of great importance.
• PHB polyhydroxybutyrate
• PHA polyhydroxyalkanoate
• the biopolymers PHA and PHB can, in part, replace conven ⁇ tional, industrially produced polymers. They are of potential interest for the packaging industry, as they possess several advantages compared to conventional plastics such as for instance 100% biodegradability and superior environmental compatibility and the advantage of allowing renewable resources to be used as starting materials for the production. Thus, they can contribute to a reduction of plastic waste difficult to recycle.
• PHB is a polyester of D(-) -3-hydroxy butyric acid.
• polyhydroxy alkanoate (PHA) as used hereinafter comprises the polymers of 3-hydroxy butyric acid, polymers of related hydroxyalkanoates, such as 3-hydroxy valerate, 3-hydroxy hexanoate and 3-hydroxy decanoate, and moreover copolymers and mixtures of these hydroxy alkanoates.
• PHB and PHA have only been found in prokaryots and are used by many bacteria species as substances for the intracellular storage of carbon and energy. They are stored in the cells in granules and can amount to 90% of the dry weight of the cells.
• a copolymer of PHB and polyhydroxy valerate is prepared on an industrial scale by Imperial Chemical Industries PLC and marketed under the name of BIOPOL.
• the microorganism Alcaligenes eutrophus is used (Byrom, 1992, FENS Microbiol. Rev. 103:247-250).
• Said microorganism is cultured in a glucose salt medium in a fed batch reactor under nutrient conditions permitting cell growth during the first 60 hours only and PHB synthesis during the subsequent 48 hour phase. This leads to a high PHB accumulation in the cells.
• the latter are separated from the culture medium, and the PHB is, as a rule, extracted from the cells by means of a solvent ( ethanol; chloroform/methylene chloride) , and subsequently precipitated and dried in vacuo.
• a solvent ethanol; chloroform/methylene chloride
• the excessive costs incurred in the production of PHA via Alcaligenes eutrophus are attributable in part to the small hydrocarbon substrate spectrum of this microorganism.
• the wild type strain only utilizes fructose as a sugar and the sugar acid gluconate (Wilde, 1962, Arch. Mikrobiol. 43:109- 137; Gottschalk et al., 1964, Arch. Mikrobiol. 48:95-108) . Mutants derived from the wild type strain can also grow on glucose (Schlegel and Gottschalk, 1965, Biochem. Z. 341:249- 259) . These strains are ' preferably used for technical fermentation, because glucose is less expensive than fructose.
• Suitable glucose sources include disaccharides, such as sucrose (glucose-fructose) , maltose (glucose- glucose) or oligosaccharides, such as dextran or dextrin, some of which form during the processing of agricultural products as secondary or waste products.
• the disadvantage in using these less expensive substrates is that the microorganisms used for the PHA production, including Alcaligenes eutrophus, are unable to import said di- or oligosaccharides, because of the absence of corresponding transport systems for the import into the cells.
• these substrates must be hydrolyzed prior to being used and must thus be converted to the corresponding hexose monomers. Again, such treatments are time-consuming and expensive.
• the present invention addresses the problem of providing microorganisms and processes permitting a less expensive PHA production in microorganisms.
• the present invention thus relates to microorganisms intracellularly producing PHB or PHA and permitting the extracellular synthesis of at least one polysaccharide on account of the expression of at least one extracellular protein having the enzymatic activity of a hexosyl- transferase.
• microorganisms of the invention are preferably microorganisms belonging to the genus Alcaligenes, in particular Alcaligenes eutrophus or the microorganism E. coli. which is capable of intracellularly synthesizing PHA on account of the introduction of genes encoding enzymes for the PHA synthesis.
• the invention relates to processes for preparing such microorganisms wherein DNA sequences coding for at least one extracellular protein having the enzymatic activity of a hexosyltransferase are introduced into a microorganism which synthesizes PHB or PHA intracellularly. Said DNA sequences are linked to regulatory DNA sequences for controlling the transcription and contain a signal sequence ensuring the secretion of the synthesized proteins.
• the invention relates in particular to such a process comprising the following steps:
• step (b) transformation of a microorganism with the expression cassette constructed in step (a) ;
• any microorganism capable of synthesizing PHB or PHA intracellularly may be used as the abovementioned microorganism; bacteria belonging to the genus Alcaligenes and bacteria of the species E. coli, incorporating DNA sequences encoding enzymes for the PHA synthesis are preferred.
• An expression cassette as indicated in process step (a) which permits the expression of an extracellular protein possessing hexosyltransferase activity as a rule contains the following DNA sequences:
• Such an expression cassette is preferably located on a vector molecule which apart from the expression cassette contains the following DNA sequences:
• DNA sequence mentioned in item (iii) above and the selection marker gene mentioned in item (v) is not necessary in all cases.
• the DNA sequences, permitting the expression of a protein possessing hexosyltransferase activity do not have to be located on a vector in each case, but may be integrated into the genome of the host organism via a homologous or non-homologous recombination in one or more copies at one or more loci.
• the DNA sequence mentioned in item (vi) does not have to be present.
• it is possible that not only one but several DNA sequences coding for hexosyltransferases of different types are expressed in an organism.
• the DNA sequence naturally controlling the transcription of the hexosyltransferase gene selected can be used as the promoter sequence, if it is active in the organism selected. However, this sequence may also be exchanged for other promoter sequences. It is possible to use promoters which cause a constitutive expression of the gene as well as inducible promoters allowing a downstream DNA sequence to be controlled by external factors. Bacterial and viral promoter sequences possessing these properties have been described in the literature in detail.
• Promoters permitting a particularly strong expression of downstream DNA sequences are for instance the T7 promoter (Studier et al., 1990, in Methods in Enzymology 185:60-89), lacuvS, trp-lacUV5 (DeBoer et al, in Rodriguez, R.L. and Chamberlin, M.J. , (Eds.), Promoters, Structure and Function; Praeger, New York, 1982, pp. 462-481; DeBoer et al, 1983, Proc. Natl. Acad. Sci. USA 80:21-25), lp lf rac (Boros et al., 1986, Gene 42:97-100) or the ompF-promoter.
• promoters include those which are inducible by sucrose, for instance from Bacillus amyloliquefaciens.
• the DNA sequence mentioned in item (ii) coding for a protein with the enzymatic activity of a hexosyltransferase may have different origins.
• Such enzymes and DNA sequences encoding them are for instance known from different microorganisms.
• the enzymes or DNA sequences encoding them described in the following are used according to a preferred embodiment of the invention.
• hexosyltransferases is to mean enzymes catalysing reactions whose mechanism is distinguished by the fact that a hexose is directly transferred from a di-, oligo- or polysaccharide to an acceptor, which as a rule is a growing polysaccharide chain.
• catalysis requires neither activated glucose derivatives, such as occurring in the polysaccharide synthesis in plants and animals, nor cofactors.
• the energy necessary for the polymerization of the hexose residues is directly obtained through the cleavage of the glycosidic bond in the corresponding di-, oligo- or polysaccharide.
• the hexosyltransferases using sucrose as a substrate are differentiated on the basis of whether they transfer the glucose residue (glucosyltransferases) or the fructose residue (fructosyltransferases) from the sucrose molecule to a growing polysaccharide chain.
• the reaction products formed are fructose and glucans in the first case and glucose and fructans in the second case.
• Glucosyltransferases using sucrose as the substrate generally catalyze reactions of the following type:
• glucans may occur as reaction products.
• Extracellular glucosyltransferases from Streptococcus species which catalyze the synthesis of glucans possessing different properties are known. These enzymes are divided into three groups:
• glucosyltransferases which synthesize a combination of water soluble and water insoluble glucans (GTF-SI type) .
• a gene has been described coding for a dextransucrase (sucrose: 1, 6- ⁇ -D-glucane 6- ⁇ -D-glucosyltrans- ferase, E.C. 2.4.1.5.) from Leuconostoc mesenteroides (WO 89/12386) .
• This transferase is likewise a glucosyl ⁇ transferase which uses sucrose as a substrate.
• the resulting glucan, i.e. dextran consists predominantly of ⁇ -1,6 linked glucose molecules, with the parallel chains being cross- linked among each other.
• DNA sequences coding for dextranmaltases or dextran dextrina ⁇ es may also be used.
• amylosucrase also designated: sucrose: 1,4- ⁇ -D-glucan 4- ⁇ -glucosyltransferase, E.C. 2.4.1.4.
• This enzyme catalyses the reaction: sucrose + ( ⁇ -1 , 4-D-glucan) n —> fructose + ( ⁇ -l,4-D- glucan) n+1
• amylosucrase has been found only in a few bacteria species, mainly including Neisseria species (MacKenzie et al., 1978, Can. J. Microbiol. 24:357-362).
• a DNA sequence containing a region coding for amylosucrase activity has been isolated from a genomic DNA library of Neisseria poly ⁇ accharea. Said DNA sequence is contained in the plasmid pNB2 (DSM 9196) .
• Fructosyltransferases using sucrose as the substrate have also been described. They catalyze reactions of the following type:
• sucrose + (fructose)_ > glucose + (fructose) n+1
• the products produced in this reaction are fructans, apart from glucose. They contain one sucrose molecule to which fructose polymers are added and which acts as a starter molecule of the polymerization reaction. Depending on the type of linkage of the fructose molecules, the synthesized fructans can be divided into two groups:
• fructosyltransferases are likewise divided into two types which are known by the common names of levansucrase (sucrose: ⁇ -D-fructosy1- transferase, E.C. 2.4.1.10.) and inulosucrase (E.C. 2.4.1.9.), respectively.
• DNA sequences coding for levansucrases and inulosucrases, respectively, have so far been isolated from different microorganisms. They include DNA sequences from Bacillus a yloliquefaciens (Tang et al, 1990, Gene 96:89-93) , Bacillus subtilis (Stein etz et al, 1985, Mol. Gen. Genetics 200: 220-228 and Erwinia amylovora (Geier and Geider, 1993, Phys. Mol. Plant Pathology 42:387-404; DE 42 27 061.8 and WO 94/04692) . They code for levansucrases which catalyze the synthesis of polyfructans of the levan type.
• hexosyltransferases which use sucrose as the substrate
• maltose a ⁇ the substrate are known.
• an amylomaltase also designated: ⁇ -1,4-glucan:D-glucose 4-glucosyltransferase, E.C. 2.4.1.3.
• Escherichia coli has been described for which the following reaction mechanism has been proposed:
• DNA sequence coding for a protein possessing hexosyl ⁇ transferase activity which has been selected for use, does not possess in its 5 ' region a DNA sequence coding for a signal peptide sequence ensuring the secretion of the hexosyltransferase, then a DNA sequence coding for such a signal peptide sequence can be inserted between the promoter and the coding DNA sequence.
• the sequence to be used must, in each case, be in the same reading frame as the DNA sequence coding for the enzyme.
• signal peptide sequences are for instance found in the gene coding for levansucrase from bacteria of the Bacillus genus (Borchert and Nagarajan, 1991, J. Bacteriol. 173:276-282) .
• the process according to the invention allows microorganisms that are employed for the PHA production by fermentation processes to be cultured using inexpensive substrates.
• the hexosyltransferases secreted into the medium lead to the cleavage of suitable di-, oligo- or polysaccharides present in the medium. This cleavage results in the release of hexoses which are imported by the microorganisms and can be used for the cell growth or the synthesis of intracellular products.
• Preferred embodiments of the microorganisms and the process of the invention are those embodiments in which the hexosyl ⁇ transferases use disaccharides, in particular sucrose or maltose as substrates.
• disaccharides in particular sucrose or maltose
• sucrose as a substrate in the culturing medium and expres ⁇ ion of a secreted gluco ⁇ yltran ⁇ fera ⁇ e causes fructose which can be imported by the microorganism ⁇ to be released in the medium.
• fructose i ⁇ a particularly suitable substrate for the intracellular PHA synthesis and compared to other carbon sources leads to an especially high PHA portion of the dry weight of the cells.
• the process of the invention therefore also provides the possibility of producing the advantageous, though relatively expensive substrate fructose from the considerably less expensive sub ⁇ trate sucrose and of lowering the costs.
• the secreted hexosyltransferases enable the extracellular synthesis of different polysaccharides as has been described above.
• the majority of the ⁇ e poly ⁇ accharides are of considerable commercial importance.
• dextran for instance in the food sector, pharmaceutical sector, e.g. as blood plasma substitutes or for increasing the viscosity of aqueous solution ⁇ , and in the chemical industry, e.g. as bases for dextran gels.
• the ⁇ -1,4 glucans formed by amylosucrase are of particular commercial interest, a ⁇ their chemical ⁇ tructure corresponds to the amylose portion of plant ⁇ tarch.
• Amylo ⁇ e is widely used inter alia in the food, paper and textile industries and in the production of cyclodextrin ⁇ .
• Starch it ⁇ elf, which so far has been the sole source for obtaining ⁇ -1,4 glucans, however, consist ⁇ of two components. Apart from the amylose which i ⁇ an unbranched chain of ⁇ -1,4 linked glucose units, starch contains another component, the amylopectin.
• Thi ⁇ is a highly branched polymer of glucose units, which, apart from the ⁇ -1,4 links, shows branches of the gluco ⁇ e chain ⁇ through ⁇ -1,6 link ⁇ .
• the two components also offer quite different possibilities of use. In order to benefit from the individual components directly, it is neces ⁇ ary to obtain them in pure form. Both components can be obtained from starch, which, however, requires several purification steps and is time consuming and involves costs.
• polyfructose i ⁇ an inexpen ⁇ ive fructo ⁇ e ⁇ ource, because it is stable, non-hygroscopic, and therefore possesses good storage properties. Given its visco ⁇ ity properties, polyfructose would al ⁇ o ⁇ eem to be a suitable thickening agent. In this connection it is also of importance that fructose can only insufficiently (i.e by microorganisms) be utilized, and therefore polyfructose is ideally suited as an additive to low calorie foodstuffs. Moreover, polyfructose is suitable for encapsulating flavours, colorants and other additives, as it cannot absorb water and therefore permits storage under atmospheric conditions.
• polyfructose is also of interest as a replacement for chemically produced linear polymers that are biologically not degradable.
• ⁇ -1,4 glucans which are synthe ⁇ ized by amylomaltase and which under ⁇ uitable condition ⁇ can achieve chain length ⁇ similar to those of amylose, are used for corresponding purpo ⁇ es as has already been described above for the glucans synthesized by amylosucra ⁇ e.
• the extracellularly ⁇ ynthe ⁇ ized polysaccharide ⁇ can be i ⁇ olated directly from the culture medium.
• the intracellularly formed polyhydroxy alkanoates can be isolated from the cells after separation of the cells from the culture medium. Therefore, the simultaneous extracellular synthesis of these polysaccharides in conjunction with the intracellular PHA synthesi ⁇ can entail a further reduction of the production costs and can thus contribute to an increa ⁇ e in the rentability of the whole PHA production process.
• control elements for initiating the transcription promoter
• a DNA sequence coding for a protein with hexo ⁇ yl- tran ⁇ fera ⁇ e activity and being linked to the promoter in sense orientation and a DNA sequence at the 3' end of the DNA sequence specified in (ii) above, serving as a termination signal for transcription
• translatable RNA is constructed in a vector suitable for the host chosen for use.
• the expression cas ⁇ ette can be constructed in a conventional cloning vector, isolated from the cloning vector with the u ⁇ e of ⁇ uitable re ⁇ triction enzyme ⁇ and in ⁇ erted into a vector suitable for the transformation of the host selected for use.
• an expression vector can be prepared by inserting a DNA sequence coding for a protein pos ⁇ es ⁇ ing hexosyl- tran ⁇ fera ⁇ e activity into a vector already containing control elements for the initiation of the transcription and a DNA sequence ⁇ erving a ⁇ a termination ⁇ ignal for transcription.
• a single restriction site or a polylinker into which the DNA sequence to be expressed may be inserted lies between the control elements for the initiation of the transcription and the termination signal.
• cloning vectors which are useful for preparing the DNA sequences mentioned in the proce ⁇ s steps and which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
• examples of such vectors are pBlueskript pla ⁇ mids, pBR322, pUC-series, M13mp-series, pACYC184 etc.
• the desired sequence may be inserted into the vector at a suitable restriction site.
• the re ⁇ ulting pla ⁇ mid i ⁇ used for the tran ⁇ formation of E. coli cells.
• the transformation can be carried out according to ⁇ tandard methods as described in Sambrook et at.
• the tran ⁇ for ed E. coli cells are cultured in a ⁇ uitable medium, harve ⁇ ted and subjected to lysi ⁇ .
• the pla ⁇ mid is then isolated.
• the methods for the characterization of the resulting plasmid DNA are restriction analy ⁇ is, gel electrophoresis, ⁇ equencing reaction ⁇ and other methods used in biochemistry and molecular biology. After each operation, the plasmid DNA can be cleaved with restriction endonucleases and the DNA fragments that are isolated can be linked to other DNA sequences.
• vectors so-called “broad host range” vectors are available, with the aid of which a plurality of gram negative bacteria can be transformed. These contain DNA sequences which ensure replication of the plasmid DNA both in the bacteria such as E. coli and in Alcaligenes eutrophus.
• the ⁇ e vector ⁇ include for instance the plasmids pLAFR3 and pLAFR6 (Staskawicz et al. , 1987, J. Bacteriol. 169:5789-5794; Bonas et al., 1989, Mol. Gen. Genet. 218:127-136) .
• the transformed microorganism is cultured in media which must meet the requirements of the particular ho ⁇ t, in particular con ⁇ idering the pH value, temperature, ventilation etc.
• the polysaccharide ⁇ of the corresponding type are synthesized in the medium with the si ultaneou ⁇ release of hexoses.
• the hexo ⁇ es can be imported by the microorgani ⁇ m that is cultivated.
• the synthesized polysaccharides can be isolated from the culture medium after termination of the fermentation proces ⁇ . The isolation of the intracellularly synthesized PHB from the cells after recovery of the latter is carried out according to methods known in the literature.
• the invention also relates to the microorganisms produced by the process of the invention.
• Another embodiment of the present invention relates to the u ⁇ e of DNA sequences coding for a protein possessing the enzymatic activity of a hexosyltran ⁇ ferase, for preparing microorganism ⁇ which synthesize PHB or PHA intracellularly and which, on account of the expression of at lea ⁇ t one protein po ⁇ e ⁇ ing the enzymatic activity of a hexosyl ⁇ transferase permit the extracellular synthesis of at least one polysaccharide in the culture medium.
• the invention relates to the use of microorganisms prepared according to one of the processes of the present invention, for combining an intracellular PHB or PHA synthesis with a simultaneous extracellular synthesi ⁇ of a poly ⁇ accharide on account of the ⁇ ecretion of a protein having the enzymatic activity of a hexosyltransferase.
• Another embodiment of the invention relates to processes for preparing PHB, PHA or a polysaccharide using a microorganism of the invention.
• FIG. 1 shows colonies of A. eutrophus, which contain the plasmid pGE9 and were cultured on 0.2% sucrose-containing FN-medium at 28°C for 2 days. After exposure to iodine vapour for about 5 minutes the colonie ⁇ are surrounded by a blue-coloured halo.
• Figure 2A shows a microscopic picture of a heat-fixed preparation of A. eutrophus transconjugant ⁇ containing the pGE9 pla ⁇ id and stained with 1% (w/v) of Nile Blue A solution (1000 fold enlargement)
• Figure 2B shows a microscopic picture as de ⁇ cribed in Figure 2A, prepared using light with a wave length of 460 nm. At this wave length the Nile Blue A dye which binds to PHB grana shows fluorescence (1000 fold enlargement) .
• a "broad host" vector permitting the expression of an extracellular amylosucrase in Alcaligene ⁇ eutrophu ⁇ was carried out u ⁇ ing a genomic DNA fragment from Neisseria polysaccharea containing the coding region for an amylosucrase.
• a genomic DNA fragment from Neisseria polysaccharea containing the coding region for an amylosucrase.
• Such a fragment wa ⁇ i ⁇ olated as the PstI fragment from the vector pNB2 (DSM 9196) .
• Said fragment comprises the DNA sequence depicted in SeqID No. 1.
• the fragment was ligated into the vector pGE151 linearized by PstI (a derivative of vector pKM9-6, Kortl ⁇ cke et al. J. Bacteriol. 174 (1992), 6277-6289) .
• the resulting vector pGE9 was transformed into the Escherichia coli strain S17-1 (Simon et al., Bio/Technology 1 (1983), 784-791) and plated onto selection medium (YT-medium containing 15 ⁇ g/ml of tetracyclin) .
• the donor strain E. coli S17-1 with pGE9 prepared according to Example 1 was incubated in 4 ml YT-medium at 37"C overnight.
• the recipient (A. eutrophus H16; Wilde, Arch. Mikrobiol. 43 (1962) , 109-13 ) was likewise incubated in 4 ml of YT-medium at 28 ⁇ overnight.
• the overnight cultures were pelleted, wa ⁇ hed with YT-medium once and then pelleted again.
• the pellet ⁇ were placed in 10 fold concentration into a physiological saline ⁇ olution (0.9% of NaCl solution).
• 0.2 ml each of the donor and recipient concentrates were mixed and plated onto solid YT- medium. This batch was incubated for about 6 hours at 28°C without being disturbed.
• Some of the colonies prepared according to example 2 and grown on the selection medium were transferred to FN-medium containing tetracyclin and 0.2% sucrose in addition, and were incubated at 28°C for 2 days.
• the transconjugant ⁇ were expo ⁇ ed to iodine vapour in order to demonstrate glucans formed by the amylo ⁇ ucrase.
• the exposure to iodine vapor result ⁇ in the formation of a blue-coloured halo in con ⁇ equence of the formation of linear ⁇ -1,4 glucans (see Figure 1) .
• the transconjugants were placed into a defined minimal medium (MM) which additionally contained 1% of ⁇ ucro ⁇ e and were incubated at 28°C for four days (Peoples et al. J. Biol. Chem. 264 (1989), 15298- 15303) .
• MM minimal medium
• Lugol solution 13.1 mM of I 2 39.6 mM of KI dissolved in H-O (distilled twice)
• YT-medium 0.8% of bacto-trypton 0.5% of yeast extract 0.5% of NaCl
• MOLECULE TYPE DNA (genomic)
• GCGTGTGGCG CAATACTTCG CCGATGCTGC CCGCGCATTC CAAAAAATCG GCGCGGAACT 540
• CAAGCAGCAT CCGCATATCG GAATGCAGAC TTGGCACAAG CCTGTCTTTT CTAGTCAGTC 840

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