HUT76348A - Microorganism permitting the intracellular polyhydroxy alkanoate synthesis with simultaneous extracellular polysaccharide synthesis and process for producing the same - Google Patents

Description

Microorganisms capable of synthesizing extracellular polysaccharide simultaneously with intracellular polyhydroxyalkanoate synthesis and their preparation

Heé c U'H Le Lun.

JNSTLKJT FÜR GENBIOLOCISCHE FORSCHUNC BERLIN GMBH BERLIN, DE

Date of filing: 06.06.1995

Priority: 06.06.1994. P 44 20 223.7 DE International Application Number: PCT / EP95 / 02165

International Publication Number: WO 95/33838

The present invention relates to microorganisms capable of intracellular synthesis of polyhydroxyalkanoate (PHA) and polyhydroxybutyrate (PHB) and extracellular synthesis of at least one polysaccharide, and their use in the production of PHB and PHA as raw materials for biodegradable packaging materials. .

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DISCLOSURE

COPIES

63 068 / BE

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Microorganisms capable of synthesizing extracellular polysaccharide simultaneously with intracellular polyhydroxyalkanoate synthesis and their preparation

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HNSTITUT FÜR ^ ENBtO ^ GGtSCHE FORSClIUNG-BERfetN) GMBH, BERLIN, DE

inventors:

KOSSMANN Jens, BÜTTCHER Volker, WELSH Thomas,

GOLM, DE

LAUENFÖRDE, DE BERLIN, DE

Date of filing: 06.06.1995

Priority: 06.06.1994. P 44 20 223.7 DE

International Application Number: PCT / EP95 / 02165

International Publication Number: WO 95/33838 · ······ ····· · · · · · · · ·

The present invention relates to microorganisms capable of intracellular synthesis of polyhydroxyalkanoate and expression of extracellular enzymes that catalyze the synthesis of various polysaccharides thereby cleaving the di-, oligo- and polysaccharides present in the culture medium. These enzymes are mainly hexosyltransferases. The invention also relates to processes for the preparation of such microorganisms.

Microorganisms are nowadays used in biotechnological production processes for the synthesis of various materials on an industrial scale. Synthesis of complex biopolymers such as polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) is, among other things, of great importance. Biopolymers are extremely commercially important because of their thermoplastic (thermoplastic) properties. These properties are comparable to synthetically produced plastics such as polypropylene.

PHA and PHB biopolymers partially replace conventional industrially produced polymers. These biopolymers have potential benefits for the packaging industry as they have many advantages over conventional plastics such as 100% biodegradability, high environmental compatibility and the advantage of being renewable as a starting material materials can be used for production. So they can contribute to reducing the amount of plastic waste that is difficult to recycle. · · · · · · · · · · · · · · · · · · · · · · · · · ·

However, the wholesale distribution of these biopolymers has so far not been possible due to their high production costs, due, among other things, to the high complexity of the production technology, high costs of production plants, costly processing of products and high raw material prices.

The production of synthetic petrochemical plastics, such as polypropylene, is less expensive, which results in these products being generally inexpensive and therefore producing large quantities of these products.

PHB is a polyester of D (-) - 3-hydroxybutyric acid. The term polyhydroxyalkanoate (PHA) as used in the present invention refers to polymers of 3-hydroxybutyric acid, polymers of related hydroxyalkanoates such as 3-hydroxyvalerate, 3-hydroxyhexanoate and 3-hydroxy decanoate, and in addition to these hydroxy includes copolymers and mixtures of alkanoates.

So far, PHA and PHB have only been detected in prokaryotes. These materials are used by many bacterial species for intracellular carbon and energy storage. These substances accumulate in cells in the form of granules and dry cells

90% of them.

Currently, Imperial Chemical Industries PLC is manufacturing on a large scale a copolymer of PHB and polyhydroxy valerate, which it markets as BIOPOL. Alcaligenes eutrophus is used in the production process [Byrom, FEMS Microbiol. Rev. 103: 247-250 (1992). Said microorganism is cultured in a glucose and salt medium in a reactor by batch digestion under nutrient conditions that allow cell growth only for the first 60 hours and PHB synthesis for the next 48 hours. As a result, the pHB is greatly increased in the cells. The PHB is isolated from the cells by separating the latter from the medium, the PHB is usually extracted from the cells with a solvent (methanol, chloroform / methylene chloride), then precipitated and dried in vacuo.

The extremely high costs of producing PHA using Alcaligenes eutrophus are partly attributable to the low hydrocarbon spectrum of this microorganism. The wild-type strain uses only fructose and sugars gluconate as sugars [Wilde, Arch. Microbiol., 43,

109-137 (1962); Gottschalk et al., Arch, Microbiol., 4,8,95-108 (1964)]. Mutants from the wild-type strain can also grow on glucose [Schlegel, Gottschalk, Biochem. Z. 341: 249-259 (1965)]. These strains are preferably used for industrial fermentation because glucose is cheaper than fructose. Suitable sources of glucose include disaccharides such as sucrose (glucose-fructose), maltose (glucose-glucose) or oligosaccharides such as dextran or dextrin. Some of these are produced as by-products or waste during the processing of agricultural products.

However, the disadvantage of using less expensive substrates is that the microorganisms used to produce PHA, including Alcaligenes eutrophus, are unable to utilize these di- or oligosaccharides because they do not have the appropriate transport systems for delivery to the cells. These t

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Substrates should therefore be hydrolyzed prior to use and converted to the corresponding hexose monomers. These operations are time consuming and also costly.

In order to reduce the production costs of PHA, by increasing the substrate spectrum of Eutrophus, we have attempted to produce strains that, unlike wild type, can use other, less expensive substrates, and have attempted to produce heterologous genes. introduce into the microorganism Alcaligenes eutrophus, which allow cells to use alternative substrates such as sucrose. However, no attempt was successful. A large number of clones had to be screened for mutagenesis in order to obtain a suitable clone. The introduction of heterogeneous genes encoding the transport system of alternative substrates often presents difficulties, since such transport systems require many genes. Therefore, each of the essential genes must be transferred and provided with promoters that are accepted by the host cell to be used. Here we often face the difficulty that the coordinated expression of these genes in the new host cell is not progressing properly and therefore the expression of the transport system will not be effective. Therefore, in order to transfer transport systems for alternative substrates to other microorganisms, we generally need to know the precise regulation of gene expression.

Despite the many efforts made, the

To broaden the spectrum of substrates for PHA producing microorganisms to include inexpensive substrates that would have contributed to reduce the cost of producing PHA. Thus, there is still an urgent need for the development of economical PHB and PHA production processes that allow the wholesale distribution of these biopolymers.

It is therefore an object of the present invention to provide a solution to this problem, that is, to provide microorganisms and methods which are suitable for the less expensive production of PHA in microorganisms.

Claims (1)

The object is solved by the embodiments described in the claims. The invention thus relates to microorganisms which produce PHB or PHA intracellularly and are capable of extracellular synthesis of at least one polysaccharide by expression of at least one extracellular protein having hexosyltransferase enzyme activity. The microorganisms of the invention preferably belong to the genus Alcaligenes, namely the Alcaligenes eutrophus or the E. coli microorganism which is capable of intracellular synthesis of PHA by introducing genes encoding PHA synthesizing enzymes into this bacterium. The invention further relates to methods for producing microorganisms comprising introducing DNA sequences into a microorganism which synthesizes PHB or PHA intracellularly which encode at least one protein having hexosyltransferase enzyme activity. Said DNA sequences are linked to regulatory DNA sequences to direct transcription. These sequences also contain signal sequences that provide secretion of the synthesized proteins. More particularly, the invention relates to a process comprising the steps of: (a) constructing an expression cassette that expresses an extracellular protein having hexosyltransferase activity; (b) transforming a microorganism with the expression cassette constructed in step (a); and (c) culturing the transformed microorganism under conditions that allow expression of said protein. In principle, any microorganism capable of synthesizing PHB or PHA intracellularly may be used in the same manner as the aforementioned microorganism; we prefer the bacteria of the genus Alcaligenes and those of the E. coli species which contain DNA sequences encoding enzymes for PHA synthesis. The expression cassette referred to in step (a) of the method, which expresses an extracellular protein having hexosyltransferase activity, generally contains the following DNA sequences: (i) a promoter sequence which is a 3 'DNA sequence in the selected host microorganism; · · · · · · · · · - controls expression of 8 cia; (ii) a DNA sequence comprising at least one hexosyl-