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WO2016030373A1 - Microorganisme modifié pour la production améliorée de produits chimiques fins sur du saccharose - Google Patents

Microorganisme modifié pour la production améliorée de produits chimiques fins sur du saccharose Download PDF

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Publication number
WO2016030373A1
WO2016030373A1 PCT/EP2015/069445 EP2015069445W WO2016030373A1 WO 2016030373 A1 WO2016030373 A1 WO 2016030373A1 EP 2015069445 W EP2015069445 W EP 2015069445W WO 2016030373 A1 WO2016030373 A1 WO 2016030373A1
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Prior art keywords
gene
deletion
nucleic acids
wcaj
seq
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Inventor
Joanna Martyna KRAWCZYK
Stefan Haefner
Hartwig Schröder
Esther DANTAS COSTA
Oskar Zelder
Gregory Von Abendroth
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BASF SE
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BASF SE
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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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase

Definitions

  • the present invention relates to a modified microorganism, to a method for producing organic compounds and to the use of modified microorganisms.
  • Organic compounds such as small dicarboxylic acids having 6 or fewer carbons are commercially significant chemicals with many uses.
  • the small diacids include 1 ,4-diacids, such as succinic acid, malic acid and tartaric acid, and the 5-carbon molecule itaconic acid.
  • Other diacids include the two carbon oxalic acid, three carbon malonic acid, five carbon glutaric acid and the 6 carbon adipic acid and there are many derivatives of such diacids as well.
  • the small diacids have some chemical similarity and their uses in polymer production can provide specialized properties to the resin. Such versatility enables them to fit into the downstream chemical infrastructure markets easily.
  • the 1 ,4-diacid molecules fulfill many of the uses of the large scale chemical maleic anhydride in that they are converted to a variety of industrial chemicals (tetrahydrofuran, butyrolactone, 1 ,4-butanediol, 2-pyrrolidone) and the succinate derivatives succindiamide, succinonitrile, diaminobutane and esters of succinate.
  • Tartaric acid has a number of uses in the food, leather, metal and printing industries. Itaconic acid forms the starting material for production of 3-methylpyrrolidone, methyl-BDO, me- thyl-THF and others.
  • succinic acid or succinate - these terms are used interchangeably herein - has drawn considerable interest because it has been used as a precursor of many industrially im- portant chemicals in the food, chemical and pharmaceutical industries.
  • succinic acid is one of 12 top chemical building blocks manufactured from biomass.
  • the ability to make diacids in bacteria would be of significant commercial importance.
  • WO-A-2009/024294 discloses a succinic acid producing bacterial strain, being a member of the family of Pasteurellaceae, originally isolated from rumen, and capable of utilizing glycerol as a carbon source and variant and mutant strains derived there from retaining said capability, in particular, a bacterial strain designated DD1 as deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) having the deposit number DSM 18541 (ID 06-614) and having the ability to produce succinic acid.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany
  • the DD1 -strain belongs to the species Basfia succiniciproducens and the family of Pasteurellaceae as classified by Kuhnert et a/., 2010. Mutations of these strains, in which the Idh- gene and/or the pfID- or the pflA-gene have been disrupted, are disclosed in WO-A- 2010/092155, these mutant strains being characterized by a significantly increased production of succinic acid from carbon sources such as glycerol or mixtures of glycerol and carbohydrates such as maltose, under anaerobic conditions compared to the DD1 -wildtype disclosed in WO-A- 2009/024294.
  • Sucrose (commonly known as sugar) is a disaccharide consisting of glucose and fructose, and it is a carbon source that is very abundant in nature and is produced from all plants having photosynthesis ability.
  • sugarcane and sugar beet contain large amounts of sucrose, and more than 60% of the world's sucrose is currently being produced from sugarcane.
  • sucrose is produced at a very low cost, because it can be industrially produced through a simple process of evaporating/concentrating extracts obtained by mechanical pressing of sug- arcanes.
  • Sucrose as a raw material for producing chemical compounds through microbial fermentation is thus inexpensive and it also functions to protect the cell membrane from an exter- nal environment containing large amounts of desired metabolites, thus producing high- concentrations of desired metabolites as shown by Kilimann et al. (Biochimica et Biophysica Acta, 1764, 2006).
  • sucrose is an excellent raw material having the above-described advantages, in- eluding low price and a function to protect microorganisms from an external environment
  • the disadvantage of this carbon source can be seen in the fact a large number of microorganisms do not have a complete mechanism of transporting sucrose into cell, degrading the transported sucrose and linking the degraded products to glycolysis, and thus cannot use sucrose as a carbon source.
  • microorganisms having a mechanism capable of using sucrose they cannot efficiently produce desired metabolites, because the rate of ingestion and degradation of sucrose as a carbon source is very low.
  • microorganisms which can be used for the fermentative production of organic compounds such as succinic acid and that can efficiently utilize sucrose as a predominant carbon source without sacrificing growth rates or yields.
  • said microorganisms would be able to use a number of low cost carbon sources and produce excellent yields of organic compounds such as succinic acid.
  • the microorganisms of the present invention should be characterized by an increased succinic acid yield and an increased carbon yield during growth of the cells on sucrose as the predominant carbon source.
  • a contribution to achieving the above mentioned aims is provided by a modified microorganism having, compared to its wildtype, a reduced activity of the enzyme that is encoded by the ptsG- gene, and wherein the modified microorganism expresses an enzyme having the activity of sucrose hydrolase and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pasteurellaceae.
  • a contribution to achieving the above mentioned aims is in particular provided by a modified microorganism in which the pfsG-gene or parts thereof have been deleted, wherein the modified microorganism expresses an enzyme encoded by a preferably heterologous cscA-gene or expresses an enzyme encoded by at least a fragment of a preferably heterologous cscA-gene or expression a fusion enzyme encoded by a fusion gene comprising at least a fragment of a preferably heterologous cscA-gene, and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pasteurellaceae.
  • heterologous refers to a structural gene not found in wild type microorganism and to polypeptide sequences not produced by such microorganism.
  • the term "wildtype" refers to a microorganism in which the activity of the enzyme that is encoded by the x-gene has not been decreased, i. e.
  • the expression "wildtype” refers to a mi- croorganism whose genome, in particular whose x-gene, is present in a state as generated naturally as the result of evolution.
  • the term may be used both for the entire microorganism but preferably for individual genes, e.g.
  • modified microorganism thus includes a microorganism which has been genetically altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype and/or phe- notype (e. g., when the genetic modification affects coding nucleic acid sequences of the micro- organism) as compared to the naturally-occurring wildtype microorganism from which it was derived.
  • the modified microorganism is a recombinant microorganism, which means that the microorganism has been obtained using recombinant DNA.
  • recombinant DNA refers to DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.
  • An example of such a recombinant DNA is a plasmid into which a heterologous DNA-sequence has been inserted.
  • a heterologous DNA may be the combination of a gene with a non-natural promoter.
  • the wildtype from which the microorganisms according to the present invention are derived belongs to the family of Pasteurellaceae.
  • Pas- teurellaceae comprise a large family of Gram-negative Proteobacteria with members ranging from bacteria such as Haemophilus influenzae to commensals of the animal and human muco- sa. Most members live as commensals on mucosal surfaces of birds and mammals, especially in the upper respiratory tract.
  • Pasteurellaceae are typically rod-shaped, and are a notable group of facultative anaerobes.
  • Pasteurellaceae can be distinguished from the related Enterobacteriaceae by the presence of oxidase, and from most other similar bacteria by the absence of flagella.
  • Bacteria in the family Pasteurellaceae have been classified into a number of genera based on metabolic properties and there sequences of the 16S RNA and 23S RNA.
  • Many of the Pasteurellaceae contain pyruvate-formate-lyase genes and are capable of anaerobically fermenting carbon sources to organic acids.
  • the wildtype from which the modified microorganism has been derived belongs to the genus Basfia and it is particularly preferred that the wildtype from which the modified microorganism has been derived belongs to the species Basfia succiniciproducens.
  • the wildtype from which the modified microorganism according to the present invention as been derived is Basfia succiniciproducens-straln DD1 deposited under the Budapest Treaty with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenst ⁇ e 7B, D-38124 Braunschweig, Germany), having the deposit number DSM 18541 and being deposited on August 1 1 , 2006.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenst ⁇ e 7B, D-38124 Braunschweig, Germany
  • This strain has been originally isolated from the rumen of a cow of German origin.
  • Pasteurella bacteria can be isolated from the gastro-intestinal tract of animals and, preferably, mammals.
  • the bacterial strain DD1 in particular, can be isolated from bovine rumen and is capable of utilizing glycerol (including crude glycerol) as a carbon source.
  • Further strains of the genus Basfia that can be used for preparing the modified microor- ganism according to the present invention are the Sasf/a-strain that has been deposited under the deposit number DSM 22022 with DSZM or the Sasf/a-strains that have been deposited with the Culture Collection of the University of Goteborg (CCUG), Sweden, having the deposit numbers CCUG 57335, CCUG 57762, CCUG 57763, CCUG 57764, CCUG 57765 or CCUG 57766. Said strains have been originally isolated from the rumen of cows of German or Swiss origin.
  • the wildtype from which the modified microorganism according to the present invention has been derived has a 16S rDNA of SEQ ID NO: 1 or a sequence, which shows a sequence homology of at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99,5%, at least 99,6%, at least 99.7%, at least 99.8% or at least 99.9 % with SEQ ID NO: 1.
  • the wildtype from which the modified microorganism according to the present invention has been derived has a 23S rDNA of SEQ ID NO: 2 or a sequence, which shows a sequence homology of at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99,5%, at least 99,6%, at least 99.7%, at least 99.8% or at least 99.9 % with SEQ ID NO: 2.
  • the identity in percentage values referred to in connection with the various polypeptides or polynucleotides to be used for the modified microorganism according to the present invention is, preferably, calculated as identity of the residues over the complete length of the aligned se- quences, such as, for example, the identity calculated (for rather similar sequences) with the aid of the program needle from the bioinformatics software package EMBOSS (Version 5.0.0, http://emboss.source-forge.net/what ) with the default parameters which are, i.e. gap open (penalty to open a gap): 10.0, gap extend (penalty to extend a gap): 0.5, and data file (scoring matrix file included in package): EDNAFUL.
  • the modified microorganism according to the present invention can not only be derived from the above mentioned wildtype-microorganisms being present in a state as generated naturally as the result of evolution, especially from Basfia succiniciproducens-straln DD1 , but also from variants of these strains.
  • the expression "a variant of a strain” comprises every strain having the same or essentially the same characteristics as the wildtype- strain.
  • the 16 S rDNA of the variant has an identity of at least 90 %, preferably at least 95 %, more preferably at least 99 %, more preferably at least 99.5 %, more preferably at least 99.6 %, more preferably at least 99.7 %, more preferably at least 99.8 % and most preferably at least 99.9 % with the wildtype from which the variant has been derived.
  • the 23 S rDNA of the variant has an identity of at least 90 %, preferably at least 95 %, more preferably at least 99 %, more preferably at least 99.5 %, more preferably at least 99.6 %, more preferably at least 99.7 %, more preferably at least 99.8 % and most preferably at least 99.9 % with the wildtype from which the variant has been derived.
  • a variant of a strain in the sense of this definition can, for example, be obtained by treating the wildtype-strain with a mutagenizing chemical agent, X-rays, or UV light.
  • the modified microorganism according to the present invention is characterized in that, compared to its wildtype, the activity of the enzyme that is encoded by the pfsG-gene is reduced.
  • the reduction of the enzyme activity (A ac tivit y ) is preferably defined as follows: activity of the modified microorganism
  • the reduced activity of the enzymes disclosed herein in particular the reduced activity of the enzyme encoded by the pfsG-gene, the lactate dehydrogenase, the pyruvate formate lyase, the enzyme encoded by the wcaJ-gene and/or the enzyme encoded by the fruA-gene, can be a reduction of the enzymatic activity by at least 50%, compared to the activity of said enzyme in the wildtype of the microorganism, or a reduction of the enzymatic activity by at least 90%, or more preferably a reduction of the enzymatic activity by at least 95%, or more preferably a reduction of the enzymatic activity by at least 98%, or even more preferably a reduction of the enzymatic activity by at least 99% or even more preferably a reduction of the enzymatic activity by at least 99.9%.
  • the reduced activity can be a reduction a reduction of the activity of the enzyme encoded by the pykA-gene in the range of 15 to 99 %, more preferably in the range of 50 to 95 % and even more preferably in the range of 90 to 99 %.
  • reduced activity of the enzyme that is encoded by the ptsG-gene or - as described below - "a reduced lactate dehydrogenase activity, "a reduced pyruvate formate lyase activity, "a reduced activity of the enzyme encoded by the wcaJ-gene", “a reduced activity of the enzyme encoded by the pykA-gene” and/or "a reduced activity of the enzyme encoded by the fruA-gene”a ⁇ so encompasses a modified microorganism which has no detectable activity of these enzymes.
  • reduced activity of an enzyme includes, for example, the expression of the enzyme by said genetically modified (e.g., genetically engineered) microorganism at a lower level than that expressed by the wildtype of said microorganism.
  • Genetic manipulations for reducing the expression of an enzyme can include, but are not limited to, altering or modifying regulatory sequences or sites associated with expression of the gene encoding the enzyme (e.g., by removing strong promoters or repressible promoters), modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of the gene encoding the enzyme and/or the translation of the gene product, or any other conventional means of decreasing expression of a particular gene routine in the art (including, but not limited to, the use of antisense nucleic acid molecules or other methods to knock-out or block expression of the target protein).
  • RNA Ribonucleic acid
  • RBS ribosomal binding sites
  • a reduced activity of an enzyme can also be obtained by introducing one or more gene mutations which lead to a reduced activity of the enzyme.
  • a reduction of the activity of an enzyme may also include an inactivation (or the reduced expression) of activating enzymes which are necessary in order to convert an enzyme in its active form.
  • the enzyme the activity of which is to be reduced is preferably kept in an inactivated state.
  • Microorganisms having a reduced activity of the enzyme encoded by the ptsG-gene may occur naturally, i.e. due to spontaneous mutations.
  • a microorganism can be modified to lack or to have significantly reduced activity of the enzyme that is encoded by the pfsG-gene by various techniques, such as chemical treatment or radiation.
  • microorganisms will be treated by, e.g., a mutagenizing chemical agent, X-rays, or UV light.
  • those microorganisms which have a reduced activity of the enzyme that is encoded by the pfsG-gene will be selected.
  • Modified microorganisms are also obtainable by homologous recombination tech- niques which aim to mutate, disrupt or excise the pfsG-gene in the genome of the microorganism or to substitute the gene with a corresponding gene that encodes for an enzyme which, compared to the enzyme encoded by the wildtype-gene, has a reduced activity.
  • a reduction of the activity of the enzyme encoded by the pfsG-gene is achieved by a modification of the pfsG-gene, wherein this gene modification is preferably realized by a deletion of the pfsG-gene or at least a part thereof, a deletion of a regulatory element of the pfsG- gene or at least a part thereof, such as a promotor sequence, by replacing the regulatory element of the pfsG-gene with a different regulatory element leading to a reduced transcription or translation of the pfsG gene, by antisense technologies, by RNAi-technologies, by an introduction of at least one mutation into the pfsG-gene or by substitution of the pfsG-gene by an inactive pfs-gene.
  • a suitable technique for recombination in particular for introducing a mutation or for deleting sequences, is described.
  • Particularly preferred is the at least partial substitution of the pfsG-gene by a preferably heterologous cscA-gene, but it is also possible to delete the ptsG-gene or at least a part thereof in a different manner and to introduce the preferably heterologous cscA-gene in a different location of the genome of the microorganism.
  • Campbell in refers to a transformant of an original host cell in which an entire circular double stranded DNA molecule (for example a plasmid) has integrated into a chromosome by a single homologous recombination event (a cross in event), and that effectively results in the insertion of a linearized version of said circular DNA molecule into a first DNA sequence of the chromosome that is homologous to a first DNA sequence of the said circular DNA molecule.
  • an entire circular double stranded DNA molecule for example a plasmid
  • a cross in event a single homologous recombination event
  • “Campbelled in” refers to the linearized DNA sequence that has been integrated into the chromosome of a “Campbell in” transformant.
  • a “Campbell in” contains a duplication of the first homologous DNA sequence, each copy of which includes and surrounds a copy of the homologous recombination crossover point.
  • “Campbell out”, as used herein, refers to a cell descending from a "Campbell in” transformant, in which a second homologous recombination event (a cross out event) has occurred between a second DNA sequence that is contained on the linearized inserted DNA of the "Campbelled in” DNA, and a second DNA sequence of chromosomal origin, which is homologous to the second DNA sequence of said linearized insert, the second recombination event resulting in the deletion (jettisoning) of a portion of the integrated DNA sequence, but, importantly, also resulting in a portion (this can be as little as a single base) of the integrated Campbelled in DNA remaining in the chromosome, such that compared to the original host cell, the "Campbell out” cell contains one or more intentional changes in the chromosome (for example, a single base substitution, multiple base substitutions, insertion of a heterologous gene or DNA sequence, insertion of an additional copy or copies of a homologous gene or a modified homologous
  • a "Campbell out” cell is, preferably, obtained by a counter-selection against a gene that is contained in a portion (the portion that is desired to be jettisoned) of the "Campbelled in” DNA sequence, for example the Bacillus subtilis sacB gene, which is lethal when expressed in a cell that is grown in the presence of about 5% to 10% sucrose.
  • a desired "Campbell out” cell can be obtained or identified by screening for the de- sired cell, using any screenable phenotype, such as, but not limited to, colony morphology, colony color, presence or absence of antibiotic resistance, presence or absence of a given DNA sequence by polymerase chain reaction, presence or absence of an auxotrophy, presence or absence of an enzyme, colony nucleic acid hybridization, antibody screening, etc.
  • the term "Campbell in” and “Campbell out” can also be used as verbs in various tenses to refer to the method or process described above.
  • the homologous recombination events that leads to a "Campbell in” or “Campbell out” can occur over a range of DNA bases within the homologous DNA sequence, and since the homologous sequences will be identical to each other for at least part of this range, it is not usually possible to specify exactly where the crossover event occurred. In other words, it is not possible to specify precisely which sequence was originally from the inserted DNA, and which was originally from the chromosomal DNA.
  • the first homologous DNA sequence and the second homologous DNA sequence are usually separated by a region of partial non-homology, and it is this region of non-homology that remains deposited in a chro- mosome of the "Campbell out" cell.
  • first and second homologous DNA sequence are at least about 200 base pairs in length, and can be up to several thousand base pairs in length.
  • the procedure can be made to work with shorter or longer sequences.
  • a length for the first and second homologous sequences can range from about 500 to 2000 bases, and the obtaining of a "Campbell out" from a "Campbell in” is facilitated by arranging the first and second homologous sequences to be approximately the same length, preferably with a difference of less than 200 base pairs and most preferably with the shorter of the two being at least 70% of the length of the longer in base pairs.
  • the pfsG-gene the activity of which is reduced in the modified microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: nucleic acids having the nucleotide sequence of SEQ ID NO: 3; b1 ) nucleic acids encoding the amino acid sequence of SEQ ID NO: 4; c1 ) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identi- cal to the nucleic acid of a1 ) or b1 ), the identity being the identity over the total length of the nucleic acids of a1 ) or b1 ); d
  • nucleic acids of a1 ) or b1 are identical to the amino acid sequence encoded by the nucleic acid of a1 ) or b1 ), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a1 ) or b1 ); e1 ) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a1 ) or b1 ); and f1 ) nucleic acids encoding the same protein as any of the nucleic acids of a1 ) or b1 ), but differing from the nucleic acids of a1 ) or b1 ) above due to the degeneracy of the genetic code.
  • hybridization includes "any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing" (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules.
  • Tm is used in reference to the "melting temperature".
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • stringency conditions refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 x SSC, 0.1 % SDS at 50°C or 65°C, preferably at 65°C, with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA).
  • SDS sodium dodecyl sulfate
  • the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 x SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C, more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1 * SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C.
  • the complementary nucleotides hybridize with a fragment or the whole pykA nucleic acids.
  • preferred hybridization conditions encompass hybridisation at 65°C in 1 ⁇ SSC or at 42°C in 1 ⁇ SSC and 50% formamide, followed by washing at 65°C in 0.3 * SSC or hybridisation at 50°C in 4 x SSC or at 40°C in 6 * SSC and 50% formamide, followed by washing at 50°C in 2 x SSC.
  • Further preferred hybridization conditions are 0.1 % SDS, 0.1 SSD and 65°C.
  • the pfsG-gene or parts of which that may be deleted by the above mentioned “Campbell recombination” or in which at least one mutation is introduced by the above mentioned “Campbell recombination” preferably comprises a nucleic acid as defined above.
  • Nucleic acid having the nucleotide sequence of SEQ ID NO: 3 corresponds to the pfsG-gene of Basfia succiniciproducens-stra ' m DDL
  • the modified microorganism according to the present invention is also characterized in that it expresses an enzyme having the activity of sucrose hydrolase, preferably having the activity of sucrose hydrolase encoded by the cscA-gene.
  • the modified microorganism according to the present invention can be characterized in that the wildtype of the microorganism al- ready expresses an enzyme having the activity of sucrose hydrolase (in this case the reduced activity of the enzyme encoded by the pfsG-gene can be the sole genetic modification), or in that the wildtype of the microorganism does not express an enzyme having the activity of sucrose hydrolase (in this case the reduced activity of the enzyme encoded by the pfsG-gene is not the sole genetic modification and the microorganism is furthermore characterized by an expression of a heterologous enzyme having the activity of sucrose hydrolase).
  • the wildtype of the microorganism does not express an enzyme having the activity of sucrose hydrolase, in particular an enzyme having the activity of sucrose hydrolase encoded by the cscA-gene, and an expression of an enzyme having the activity of a sucrose hydrolase (EC:3.2.1 .26) is ensured by the introduction of a heterologous cscA-gene or at least a fragment thereof.
  • the modified microorganism therefore has, compared to its wildtype, an increased activity of sucrose hydrolase and it is particularly preferred that the increased activity of sucrose hydrolase is accomplished by the expression of an enzyme encoded by a cscA-gene, by the expression of an enzyme encoded by a fragment of a cscA-gene or by the expression of a fusion enzyme encoded by a fusion gene comprising at least a fragment of a cscA-gene, provided that the enzyme encoded by the fragment of the cscA-gene and the fusion enzyme encoded by the fusion gene comprising at least a fragment of a cscA-gene have the activity of sucrose hydrolase.
  • the ptsG- gene or at least a part of this gene is replaced by the heterologous cscA-gene or by at the fragment of the cscA-gene, which leads to a reduced activity of the enzyme encoded by the ptsG- gene (preferably to a complete inactivation of this enzyme) and, at the same time, to the expression of a heterologous enzyme encoded by the cscA-gene or to the expression of at least a part of a heterologous enzyme encoded by the cscA-gene.
  • the increase of the enzyme activity (A ac tivit y ) is - in case of a wildtype which already has a certain activity of sucrose hydrolase - preferably defined as follows: activity of the modified microorganism
  • the increased activity of activity of sucrose hydrolase can be an increase of the enzymatic activity by 1 to 10000%, compared to the activity of said enzyme in the wildtype of the microorganism, or an increase of the enzymatic activity by at least 50 %, or at least 100 %, or at least 200 %, or at least 300 %, or at least 400 %, or at least 500 %, or at least 600 % or at least 700 %, or at least 800 %, or at least 900 %, or at least 1000 %, or at least 5000 %.
  • the increase of the activity of an enzyme is in the range of 10 to 1000 %, more preferably in the range of 100 to 500 %.
  • the cscA-gene encoding for the sucrose hydrolase that is expressed by the recombinant microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of:
  • SEQ ID NO: 5 corresponds to the cscA-gene of E. co// ' W (ATCC 9637).
  • this part comprises at least 500 nucleotides (e. g. nucleotide 935 to 1434 of SEQ ID NO: 5) of the cscA-gene, preferably at least 600 nucleotides (e. g. nucleotide 835 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably at least 700 nucleo- tides (e. g. nucleotide 735 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably at least 800 nucleotides (e. g.
  • nucleotide 635 to 1434 of SEQ ID NO: 5 of the cscA-gene and most preferably at least 900 nucleotides (e. g. nucleotide 535 to 1434 of SEQ ID NO: 5) of the cscA- gene.
  • this fragment of the cscA-gene is fused with a fragment of the argD-gene (encoding for N-acetylornithin-aminotransferase; EC:2.6.1 .1 1 and/or EC 2.6.1 .17) such that the modified microorganism expresses a fusion protein consisting of two parts, a first part being a part of the N-acetylornithin-aminotransferase encoded by the argD- gene and a second part being a part of the sucrose hydrolase encoded by the cscA-gene.
  • the argD-gene encoding for the N-acetylornithin-aminotransferase a part of which may be the first part of a fusion protein preferably comprises a nucleic acid selected from the group consisting of: a3) nucleic acids having the nucleotide sequence of SEQ ID NO: 7;
  • SEQ ID NO: 7 corresponds to the argD-gene of Basfia succiniciproducens-straln DDL
  • the part being the first part of the fusion protein is preferably encoded by at least 500 nucleotides (e. g. nucleotide 1 to 500 of SEQ ID NO: 7) of the argD-gene, preferably by at least 600 nucleotides (e. g. nucleotide 1 to 600 of SEQ ID NO: 7) of the argD-gene, more preferably by at least 700 nucleotides (e. g. nucleotide 1 to 700 of SEQ ID NO: 7) of the argD-gene, more preferably by at least 800 nucleotides (e.
  • nucleotide 835 to 1434 of SEQ ID NO: 5) of the cscA-gene more preferably by at least 700 nucleotides (e. g. nucleotide 735 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably by at least 800 nucleotides (e. g. nucleotide 635 to 1434 of SEQ ID NO: 5) of the cscA-gene and most preferably by at least 900 nucleotides (e. g. nucleotide 535 to 1434 of SEQ ID NO: 5) of the cscA-gene.
  • the expression of such an enzyme in particular by introducing a heterologous cscA-gene or a part thereof can be ensured by recombinant methods known to the person skilled in the art, for example by transformation, transduction, conjugation, or a combination of these methods with a vector containing the desired gene or the desired part of the gene, an allele of this gene or parts thereof and ensuring an expression of the gene in the microorganism.
  • Heterologous expression can be achieved in particular by integration of the gene or of a part thereof into the chromosome of the cell or an extrachromosomally replicating vector.
  • the heterologous cscA-gene can be integrated into the vector that is used for this recombination or can be introduced before or after the "Campbell recombination" in a separate vector.
  • the modified microorganism according to the present invention is not characterized by an increased activity of sucrose permease (EC 2.7.1.69) encoded by the cscS-gene and/or an increased activity of fructokinase (EC 2.7.1 .4) encoded by the cscK-gene.
  • sucrose permease EC 2.7.1.69
  • fructokinase EC 2.7.1 .4
  • neither sucrose permease encoded by the cscS-gene nor fructokinase encoded by the cscK-gene are expressed in an increased amount compared to the wildtype.
  • this microorganism is not only characterized by a reduced activity of the enzyme encoded by the pfsG-gene and by the expression of an enzyme having the activity of sucrose hy- drolase, but also, compared to its wildtype, i) by a reduced pyruvate formate lyase activity, and/or ii) by a reduced lactate dehydrogenase activity, and/or iii) by a reduced activity of an enzyme encoded by the wcaJ-gene, and/or iv) by a reduced activity of an enzyme encoded by the pykA-gene, and/or v) by a reduced activity of an enzyme encoded by the fruA-gene.
  • Modified microorganisms being deficient in lactate dehydrogenase and/or being deficient in pyruvate formate lyase activity are disclosed in WO-A-2010/092155, US 2010/0159543 and WO- A-2005/052135, the disclosure of which with respect to the different approaches of reducing the activity of lactate dehydrogenase and/or pyruvate formate lyase in a microorganism, preferably in a bacterial cell of the genus Pasteurella, particular preferred in Basfia succiniciproducens strain DD1 , is incorporated herein by reference.
  • Methods for determining the pyruvate formate lyase activity are, for example, disclosed by Asanum N. and Hino T. in "Effects of pH and Energy Supply on Activity and Amount of Pyruvate-Formate-Lyase in Streptococcus bovis", Appl. Environ. Microbiol. (2000), Vol. 66, pages 3773-3777 and methods for determining the lactate dehydrogenase activity are, for example, disclosed by Bergmeyer, H.U., Bergmeyer J. and Grassl, M. (1983-1986) in "Methods of Enzymatic Analysis", 3 rd Edition, Volume III, pages 126- 133, Verlag Chemie, Weinheim.
  • the reduction of the activity of lactate dehydrogenase is achieved by an inactivation of the IdhA-gene (which encodes the lactate dehydrogenase LdhA; EC 1 .1 .1.27 or EC 1.1 .1 .28) and the reduction of the pyruvate formate lyase is achieved by an inactivation of the pflA-gene (which encodes for an activator of pyruvate formate lyase PfIA; EC 1.97.1.4) or the pflD-gene (which encodes the pyruvate formate lyase PfID; EC 2.3.1 .54), wherein the inactivation of these genes (i.
  • IdhA, pfIA and pfID is preferably achieved by a deletion of theses genes or parts thereof, by a deletion of a regulatory element of these genes or at least a part thereof or by an introduction of at least one mutation into these genes, particu- lar preferred by means of the "Campbell recombination" as described above.
  • the IdhA-gene the activity of which may be reduced in the modified microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: a1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 9; a2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 10; a3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least
  • nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of a1 ) or a2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a1 ) or a2);
  • Nucleic acid having the nucleotide sequence of SEQ ID NO: 9 corresponds to the IdhA-gene of Basfia succiniciproducens-straln DD1 .
  • the pf/A-gene the activity of which may be reduced in the modified microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: ⁇ 1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 11 ; ⁇ 2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 12; ⁇ 3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably
  • Nucleic acid having the nucleotide sequence of SEQ ID NO: 11 corresponds to the pf/A-gene of Basfia succiniciproducens-stra ' m DDL
  • the pf/D-gene the activity of which may be reduced in the modified microroganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: ⁇ 1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 13; ⁇ 2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 14; ⁇ 3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of ⁇ 1 ) or y2), the identity being the identity over the total length of the nucleic acids of ⁇ 1 ) or y2); nucleic acids encoding
  • Nucleic acid having the nucleotide sequence of SEQ ID NO: 13 corresponds to the pflD-gene of Basfia succiniciproducens-stra ' m DDL
  • the wcaJ-gene the activity of which may be reduced in the modified microorganism according to the present invention presumably encodes for an enzyme being a glucose transferase, whereas the pykA-gene encodes for a pyruvate kinase catalyzing the conversion of phosphoe- nolpyruvate (PEP) to pyruvate (EC 2.7.1.40).
  • the fruA-gene the activity of which may be reduced in the modified microorganism according to the present invention presumably encodes for a fructose-specific phosphotransferase.
  • Microorganisms having a reduced activity of the enzyme encoded by the wcaJ-gene or the enzyme encoded by the pykA-gene may occur naturally, i.e. due to spontaneous mutations.
  • a microorganism can be modified to lack or to have significantly reduced activity of the enzyme that is encoded by the wcaJ-gene or the pykA-gene by various techniques, such as chemical treat- ment or radiation. To this end, microorganisms will be treated by, e.g., a mutagenizing chemical agent, X-rays, or UV light.
  • microorganisms which have a reduced activity of the enzyme that is encoded by the wcaJ-gene or the pykA-gene will be selected.
  • Modified microorganisms are also obtainable by homologous recombination techniques which aim to mutate, disrupt or excise the wcaJ-gene or the pykA-gene in the genome of the microor- ganism or to substitute the gene with a corresponding gene that encodes for an enzyme which, compared to the enzyme encoded by the wildtype-gene, has a reduced activity.
  • a reduction of the activity of the enzyme encoded by the wcaJ-gene or a reduction of the activity of the enzyme encoded by the fruA-gene is preferably achieved by a modification of the wcaJ-gene and the fruA-gene, respectively, wherein this gene modification is preferably realized by a deletion of the wcaJ-gene and/or the fruA-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ- gene and/or the fruA-gene or at least a part thereof, such as a promotor sequence, or by an introduction of at least one mutation into the wcaJ-gene and/or into the fruA-gene.
  • Particularly preferred in this context is a deletion of the wcaJ-gene and/or the fruA-gene by "Campbell recombination" as described above. Also particularly preferred is an introduction of at least one mutation into the wcaJ-gene, wherein this mutation preferably leads to the expression of a truncated enzyme encoded by the wcaJ-gene. In this context it is furthermore preferred that in the truncated enzyme at least 100 amino acids, preferably at least 125 amino acids, more preferred at least 150 amino acids and most preferred at least 160 amino acids of the wildtyp enzyme encoded by the wcaJ-gene are deleted from the C-terminal end.
  • Such a truncated enzyme en- coded the wcaJ-gene can, for example, be obtained by inserting or deleting nucleotides at appropriate positions within the wcaJ-gene gene which leads to a frame shift mutation, wherein by means of this frame shift mutation a stop codon introduced.
  • a stop codon introduced as shown in SEQ ID NO: 17.
  • Such mutations of the wcaJ-gene can be introduced, for example, by site- directed or random mutagenesis, followed by an introduction of the modified gene into the genome of the microorganism by recombination.
  • Variants of the wcaJ -gene can be are generated by mutating the wcaJ-gene sequence SEQ ID NO: 15 by means of PCR.
  • the "Quickchange Site-directed Mutagenesis Kit' (Stratagene) can be used to carry out a directed mutagenesis. A random mutagenesis over the entire coding sequence, or else only part thereof, of
  • SEQ ID NO: 15 can be performed with the aid of the "GeneMorph II Random Mutagenesis Kit' (Stratagene).
  • a reduction of the activity of the enzyme encoded by the pykA-gene is preferably also achieved by a modification of the pykA-gene, wherein this gene modification is preferably realized by a deletion of the pykA-gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof, such as a promotor sequence, or by an introduction of at least one mutation into the pykA-gene. It is, however, particularly preferred that the reduction of the activity of the enzyme encoded by the pykA-gene is achieved by introducing at least one mutation into the wildtype-py/oA-gene.
  • a mutation into the pykA-gene can be introduced, for example, by site- directed or random mutagenesis, followed by an introduction of the modified gene into the genome of the microorganism by recombination.
  • Variants of the pykA-gene can be are generated by mutating the pykA-gene sequence by means of PCR.
  • the "Quickchange Site-directed Muta- genesis Kit' (Stratagene) can be used to carry out a directed mutagenesis.
  • a random mutagenesis over the entire coding sequence, or else only part thereof, of the pykA-gene can be performed with the aid of the "GeneMorph II Random Mutagenesis Kit' (Stratagene).
  • the mutagenesis rate is set to the desired amount of mutations via the amount of the template DNA used. Multiple mutations are generated by the targeted combination of individual mutations or by the sequential performance of several mutagenesis cycles. Introduction of the modified gene into the genome of the microorganism can again be accomplished by "Campbell recombination" as described above.
  • the wcaJ-ge e comprises a nucleic acid selected from the group consisting of: ⁇ 1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 15; ⁇ 2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 16; ⁇ 3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of ⁇ 1 ) or ⁇ 2), the identity being the identity over the total length of the nucleic acids of ⁇ 1 ) or ⁇ 2); ⁇ 4) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80
  • Nucleic acid having the nucleotide sequence of SEQ ID NO: 15 corresponds to the wcaJ-gene of Basfia succiniciproducens-stra ' m DDL
  • the pykA-gene comprises a nucleic acid selected from the group consisting of: ⁇ 1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 18; s2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 19; s3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of ⁇ 1 ) or ⁇ 2), the identity being the identity over the total length of the nucleic acids of ⁇ 1 ) or s2); nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %,
  • modified microorganisms having a reduced activity of the enzyme encoded by the pykA-gene are microorganisms of the genus Basfia and in particular of the species Basfia suc- ciniciproducens, in which at least one mutation has been introduced in the pykA-gene, preferably at least one mutation the results in the substitution of at least one amino acid in the enzyme encoded by the pykA-gene, most preferred a mutation that results at least in a substitution of glycine by cysteine a position 167, or a substitution of cysteine by tyrosine at position 417 or a substitution of alanine by glycine at position 171 , or a substitution glycine by cysteine a position 167 and a substitution of cysteine by tyrosine at position 417, or a substitution of glycine by cysteine a position 167 and a substitution of cysteine by tyrosine at position 417,
  • the fruA-gene comprises a nucleic acid selected from the group consisting of: ⁇ 1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 20; ⁇ 2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 21 ; nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of ⁇ 1 ) or ⁇ 2), the identity being the identity over the total length of the nucleic acids of ⁇ 1 ) or ⁇ 2); ⁇ 4) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at
  • Nucleic acid having the nucleotide sequence of SEQ ID NO: 20 corresponds to the fruA-gene of Basfia succiniciproducens-stra ' m DDL
  • modified microorganism according to the present invention further comprises:
  • modified microorganisms are: modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted and in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E.
  • coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced, and in which, compared to the wildtype, the activity of the lactate dehydrogenase is reduced, preferably by a modification of the IdhA- gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E.
  • coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the pyruvate formate lyase is reduced, preferably by a modification of the pflA-gene or the pflD-gene, in particular by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12 or by a modification of the pflD- gene encoding for PfID having the amino acid sequence according to SEQ ID NO: 14; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E.
  • coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase and the pyruvate formate lyase is reduced, preferably by a modification of the IdhA-gene and the pflA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10 or by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12, or a modification of the IdhA-gene and the pflD- gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10 or by a modification of the pflD-gene encoding for PfID having the amino acid sequence according to SEQ ID NO: 14; modified bacterial cells of the
  • the coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase, the pyruvate formate lyase and the enzyme encoded by the wcaJ-gene is reduced, preferably by a modification of the IdhA-gene, the pflA-gene and the wcaJ-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12 and by a modification of the wcaJ-gene encoding for an enzyme having the amino sequence according to SEQ ID NO: 16, or a modification of the IdhA-gene, the pflD-gene and the wcaJ-gene, in particular by a modification of the Idh
  • the coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase, the pyruvate formate lyase and the enzyme encoded by the pykA-gene is reduced, preferably by a modification of the IdhA-gene, the pflA-gene and the pykA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12 and by a modification of the pykA-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 19, or a modification of the IdhA-gene, the pflD-gene and the pykA-gene, in particular by a modification
  • a contribution to solving the problems mentioned at the outset is furthermore provided by a method of producing an organic compound comprising:
  • the modified microorganism according to the present invention is cultured in a culture medium comprising at least one assimilable carbon source to allow the modified microorganism to produce the organic compound, thereby obtaining a fermentation broth comprising the organic compound.
  • Preferred organic compounds that can be produced by the process according to the present invention comprise carboxylic acids such as formic acid, lactic acid, propionic acid, 2-hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, acrylic acid, pyruvic acid or salts of these carboxylic acids, dicarboxylic acids such as malonic acid, succinic acid, malic acid, tartaric acid, glutaric acid, itaconic acid, adipic acid or salts thereof, tricarboxylic acids such as citric acid or salts thereof, alcohols such as methanol or ethanol, amino acids such as L-asparagine, L-aspartic acid, L-arginine, L-isoleucine, L-glycine, L- glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, L-tryptophan, L-threonine, L-valine, L-histidine, L-proline, L-me
  • the organic compound is succinic acid.
  • succinic acid' as used in the context of the present invention, has to be understood in its broadest sense and also encompasses salts thereof (i. e. succinate), as for example alkali metal salts, like Na + and K + -salts, or earth alkali salts, like Mg 2+ and Ca 2+ -salts, or ammonium salts or anhydrides of succinic acid.
  • the modified microorganism according to the present invention is preferably incubated in the culture medium at a temperature in the range of about 10 to 60°C or 20 to 50°C or 30 to 45°C at a pH of 5.0 to 9.0 or 5.5 to 8.0 or 6.0 to 7.0.
  • the organic compound, especially succinic acid is produced under anaerobic conditions.
  • Anaerobic conditions may be established by means of conventional techniques, as for example by degassing the constituents of the reaction medium and maintaining anaerobic conditions by introducing carbon dioxide or nitrogen or mixtures thereof and optionally hydrogen at a flow rate of, for example, 0.1 to 1 or 0.2 to 0.5 vvm.
  • Aerobic conditions may be established by means of conventional techniques, as for example by introducing air or oxygen at a flow rate of, for example, 0.1 to 1 or 0.2 to 0.5 vvm. If appropriate, a slight over pressure of 0.1 to 1.5 bar may be applied in the process.
  • the assimilable carbon source is preferably selected from sucrose, maltose, maltotriose, malto- tetraose, maltopentaose, maltohexaose, maltoheptaose, D-fructose, D-glucose, D-xylose, L- arabinose, D-galactose, D-mannose, glycerol and mixtures thereof or compositions containing at least one of said compounds, or is selected from decomposition products of starch, cellulose, hemicellulose and/or lignocellulose.
  • a preferred assimiable carbon source is sucrose.
  • sucrose and at least one further assimilable carbon source such as a mixture of sucrose and maltose, sucrose and D-fructose, sucrose and D-glucose, sucrose and D-xylose, sucrose and L-arabinose, sucrose and D-galactose, sucrose and D- mannose.
  • the initial concentration of the assimilable carbon source is preferably adjusted to a value in a range of 5 to 100 g/l, preferably 5 to 75 g/l and more preferably 5 to 50 g/l and may be maintained in said range during cultivation.
  • the pH of the reaction medium may be controlled by addition of suitable bases as for example, gaseous ammonia, NH4HCO3, (NhU ⁇ COs, NaOH, Na 2 C0 3 , NaHCOs, KOH, K2CO3, KHCOs, Mg(OH) 2 , MgCOs, Mg(HC0 3 ) 2 , Ca(OH) 2 , CaCOs, Ca(HC03) 2 , CaO, CH6N 2 0 2 , C 2 H 7 N and/or mixtures thereof.
  • suitable bases as for example, gaseous ammonia, NH4HCO3, (NhU ⁇ COs, NaOH, Na 2 C0 3 , NaHCOs, KOH, K2CO3, KHCOs,
  • alkaline neutralization agents are especially required if the organic compounds that are formed in the course of the fermentation process are carboxylic acids or dicarboxylic acids.
  • succinic acid as the organic compound, Mg(OH)2 and MgC03 are particular preferred bases.
  • the fermentation step I) according to the present invention can, for example, be performed in stirred fermenters, bubble columns and loop reactors.
  • a comprehensive overview of the possible method types including stirrer types and geometric designs can be found in Chmiel: "Bio- reatechnik: Einbowung in die Biovonstechnik' , Volume 1.
  • typical variants available are the following variants known to those skilled in the art or explained, for example, in Chmiel, Hammes and Bailey: "Biochemical Engineering”: such as batch, fed-batch, repeated fed-batch or else continuous fermentation with and without recycling of the biomass.
  • sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or appropriate gas mixtures may be effected in order to achieve good yield (YP/S).
  • Particularly preferred conditions for producing the organic acid, especially succinic acid, in process step I) are:
  • the assimilable carbon source is converted to the organic compound, preferably to succinic acid, with a carbon yield YP/S of at least 0.5 g/g up to about 1 .28 g/g; as for example a carbon yield YP/S of at least 0,6 g/g, of at least 0.7 g/g, of at least 0.75 g/g, of at least 0.8 g/g, of at least 0.85 g/g, of at least 0.9 g/g, of at least 0.95 g/g, of at least 1.0 g/g, of at least 1 .05 g/g, of at least 1.1 g/g, of at least 1 .15 g/g, of at least 1 .20 g/g, of at least 1 .22 g/g, or of at least 1 .24 g/g (organic compound/carbon, preferably succinic acid/carbon).
  • organic compound/carbon preferably succinic acid/carbon
  • the assimilable carbon source is converted to the organic compound, preferably to succinic acid, with a specific productivity yield of at least 0.6 g g DCW- 1 IT 1 organic compound, preferably succinic acid, or of at least of at least
  • the assimilable carbon source is converted to the organic compound, preferably to succinic acid, with a space time yield for the organic compound, preferably for succinic acid, of at least 2.2 g/(l_xh) or of at least 2.5 g/(Lxh) , at least 2.75 g/(Lxh), at least 3 g/(Lxh), at least 3.25 g/(Lxh), at least 3.5 g/(Lxh), at least 3.7 g/(Lxh), at least 4.0 g/(Lxh) at least 4.5 g/(Lxh) or at least 5.0 g/(Lxh) of the organic compound, preferably succinic acid.
  • a space time yield for the organic compound, preferably for succinic acid of at least 2.2 g/(l_xh) or of at least 2.5 g/(Lxh) , at least 2.75 g/(Lxh), at least 3 g/(Lxh), at least 3.25
  • the modified microorganism is converting at least 20 g/L, more preferably at least 25 g/l and even more preferably at least 30 g/l of the assimilable carbon source to at least 20 g/l, more preferably to at least 25 g/l and even more preferably at least 30 g/l of the organic compound, preferably succinic acid.
  • the different yield parameters as described herein are well known in the art and are determined as described for example by Song and Lee, 2006.
  • Carbon yield' and “YP/S” (each expressed in mass of organic compound produced/mass of assimilable carbon source consumed) are herein used as synonyms.
  • the specific productivity yield describes the amount of a product, like succinic acid, that is produced per h and L fermentation broth per g of dry biomass.
  • the amount of dry cell weight stated as "DCW describes the quantity of biologically active microorganism in a biochemical reaction. The value is given as g product per g DCW per h (i.e.
  • the space-time-yield is defined as the ratio of the total amount of organic compound formed in the fermentation process to the volume of the culture, regarded over the entire time of cultivation.
  • the space-time yield is also known as the "volumetric productivity.
  • process step II the organic compound, preferably succinic acid, is recovered from the fermentation broth obtained in process step I).
  • the recovery process comprises the step of separating the recombinant microorganism from the fermentation broth as the so called "biomass".
  • biomass Processes for removing the biomass are known to those skilled in the art, and comprise filtration, sedimentation, flotation or combinations thereof. Consequently, the biomass can be removed, for example, with centrifuges, separators, decanters, filters or in a flotation apparatus. For maximum recovery of the product of value, washing of the biomass is often advisable, for example in the form of a diafiltration.
  • the selection of the method is dependent upon the biomass content in the fermentation broth and the properties of the biomass, and also the interaction of the biomass with the organic compound (e. the product of value).
  • the fermentation broth can be sterilized or pas- teurized.
  • the fermentation broth is concentrated. Depending on the requirement, this concentration can be done batch wise or continuously.
  • the pressure and temperature range should be selected such that firstly no product damage occurs, and secondly minimal use of apparatus and energy is necessary. The skillful selection of pressure and temperature levels for a multistage evaporation in particular enables saving of energy.
  • the recovery process may further comprise additional purification steps in which the organic compound, preferably succinic acid, is further purified. If, however, the organic compound is converted into a secondary organic product by chemical reactions as described below, a further purification of the organic compound is, depending on the kind of reaction and the reaction con- ditions, not necessarily required.
  • the purification of the organic compound obtained in process step II preferably for the purification of succinic acid, methods known to the person skilled in the art can be used, as for example crystallization, filtration, electrodialysis and chromatog- raphy.
  • succinic acid as the organic compound, for example, succinic acid may be isolated by precipitating it as a calcium succinate product by using calcium hydroxide, -oxide, - carbonate or hydrogen carbonate for neutralization and filtration of the precipitate.
  • the succinic acid is recovered from the precipitated calcium succinate by acidification with sulfuric acid fol- lowed by filtration to remove the calcium sulfate (gypsum) which precipitates.
  • the resulting solution may be further purified by means of ion exchange chromatography in order to remove un- desired residual ions.
  • the fermentation broth obtained in process step l) may be acidified to transform the magnesium succinate contained in the medium into the acid form (i. e. succinic acid), which subsequently can be crystallized by cooling down the acidified medium.
  • the process further comprises the process step:
  • succinic acid as the organic compound preferred secondary organic products are selected from the group consisting of succinic acid esters and polymers thereof, tetrahydrofuran (THF), 1 ,4-butanediol (BDO), gamma-butyrolactone (GBL), pyrrolidones, polyols and polyure- thanes.
  • THF tetrahydrofuran
  • BDO 1 ,4-butanediol
  • GBL gamma-butyrolactone
  • pyrrolidones polyols and polyure- thanes.
  • this process comprises: b1 ) either the direct catalytic hydrogenation of the succinic acid obtained in process steps I) or II) to THF and/or BDO and/or GBL or b2) the chemical esterification of succinic acid and/or succinic acid salts obtained in process steps I) or II) into its corresponding di-lower alkyl ester and subsequent catalytic hydrogenation of said ester to THF and/or BDO and/or GBL.
  • this process comprises: b) the chemical conversion of succinic acid ammonium salts obtained in process steps I) or II) to pyrrolidones in a manner known per se.
  • succinic acid ammonium salts obtained in process steps I) or II) to pyrrolidones in a manner known per se.
  • a contribution to solving the problems mentioned at the outset is furthermore provided by the use of the modified microorganism according to the present invention for the fermentative production of organic compounds.
  • Preferred organic compounds are those compounds that have already been mentioned in connection with the process according to the present invention, succinic acid being the most preferred organic compound.
  • preferred conditions for the fermentative production of organic compounds, preferably of succinic acid are those conditions that have already been described in connection with process step I) of the process according to the present invention.
  • Figure 1 shows a schematic map of plasmid pSacB (SEQ ID NO: 22).
  • Figure 2 shows a schematic map of plasmid pSacB AldhA (SEQ ID NO: 23).
  • Figure 3 shows a schematic map of plasmid pSacB ApfIA (SEQ ID NO: 24).
  • Figure 4 shows a schematic map of plasmid pSacB wcaJ * (SEQ ID NO: 25).
  • Figure 5 shows a schematic map of plasmid pSacB AptsG cscA (SEQ ID NO: 26).
  • Figure 6 shows a schematic map of plasmid pSacB AptsG argD::cscA-fusion (SEQ ID NO: 27).
  • Figure 7 shows a schematic map of plasmid pSacB pykA1 (SEQ ID NO: 29).
  • DD1 For preparing a pre-culture DD1 was inoculated from frozen stock into 40 ml BHI (brain heart infusion; Becton, Dickinson and Company) in 100 ml shake flask. Incubation was performed over night at 37°C; 200 rpm. For preparing the main-culture 100 ml BHI were placed in a 250 ml shake flask and inoculated to a final OD (600 nm) of 0.2 with the pre-culture. Incubation was performed at 37°C, 200 rpm. The cells were harvested at an OD of approximately 0.5, 0.6 and 0.7, pellet was washed once with 10% cold glycerol at 4°C and re-suspended in 2 ml 10% glyc- erol (4°C).
  • Plasmid-DNA 100 ⁇ of competent cells were the mixed with 2-8 ⁇ g Plasmid-DNA and kept on ice for 2 min in an electroporation cuvette with a width of 0.2 cm. Electroporation under the following conditions: 400 ⁇ ; 25 ⁇ F; 2.5 kV (Gene Pulser, Bio-Rad). 1 ml of chilled BHI was added immediately after electroporation and incubation was performed for approximately 2 h at 37°C.
  • Mutation/deletion plasmids were constructed based on the vector pSacB (SEQ ID NO: 22).
  • Figure 1 shows a schematic map of plasmid pSacB. 5'- and 3'- flanking regions (approx. 1500 bp each) of the chromosomal fragment, which should be deleted were amplified by PCR from chromosomal DNA of Basfia succiniciproducens and introduced into said vector using standard techniques. Normally, at least 80 % of the ORF were targeted for a deletion. In such a way, the mutation/deletion plasmids for the lactate dehydrogenase IdhA, pSacB_delta_/c/M
  • Figures 1 , 2, 3, 4, 5 and 6 show schematic maps of plasmid pSacB, pSacB_delta_/c/M, pSacB_delta_p/7A pSacB_wcaJ ⁇ and pSacB_delta_pfsG_cscA
  • Basfia succiniciproducens with pSacB_ivcaJ * leads to the expression of a truncated enzyme encoded by the wcaJ-gene
  • a transformation with pSacB pykA1 leads to the expression of a pyruvate kinase in which at amino acid position 167 glycine is substituted by cysteine.
  • Basfia succiniciproducens with pSacB_delta_pfsG_cscA leads to a deletion of the pfsG-gene and an introduction of the cscA-gene from E. coli ⁇ N (ATCC 9637) into the cell
  • a transformation with pSacB_delta_pfsG_ leads to a deletion of the pfsG-gene and an introduction of a fusion gene comprising nuelceotides 1 to 869 of the argD- gene and nucleotides 524 to 1434 of the cscA-gene from E. co// ' W (ATCC 9637) into the cell.
  • the sacS-gene is contained from bases 2380-3801 .
  • the sacS-promotor is contained from bases 3802-4264.
  • the chloramphenicol gene is contained from base 526-984.
  • the origin of replication for E. coli (ori EC) is contained from base 1477-2337 (see fig. 1 ).
  • the 5' flanking region of the IdhA-gene which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1519-2850, while the 3' flanking region of the IdhA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 62-1518.
  • the sacS-gene is contained from bases 5169-6590.
  • the sacS-promoter is contained from bases 6591 -7053.
  • the chloramphenicol gene is contained from base 3315-3773.
  • the origin of replication for E. coli (ori EC) is contained from base 4266-5126 (see fig. 2).
  • the 5' flanking region of the pflA- gene which is homologous to the genome of Basfia succiniciproducens, is contained from ba- ses 1506-3005, while the 3' flanking region of the pflA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1505.
  • the sacS-gene is contained from bases 5278-6699.
  • the sacS-promoter is contained from bases 6700-7162.
  • the chloramphenicol gene is contained from base 3424-3882.
  • the origin of replication for E. coli (ori EC) is contained from base 4375-5235 (see fig. 3).
  • the 5' flanking region of the wcaJ- gene which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1838-3379, while the 3' flanking region of the wcaJ-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1236.
  • the sacS-gene is con- tained from bases 5652-7073.
  • the sacS-promoter is contained from bases 7074-7536.
  • the chloramphenicol gene is contained from base 3798-4256. The origin of replication for E.
  • coli ori EC
  • the wcaJ-gene is contained from bases 1237-1837 with an insertion of a nucleotide in the codon that encodes of lysine between thymine at position 81 and adenine at position 82 of at position of the wcaJ-gene (which corresponds to position 1756 of plasmid pSacB-ivcaJ * .
  • This insertion leads to a frame shift mutation, wherein by means of this frame shift mutation a stop codon is introduced, leading to the expression of a truncated enzyme.
  • the 5' flanking region of the pfsG-gene which is homologous to the genome of Basfia succiniciproducens, is contained from bases 4138-5698, while the 3' flanking region of the pfsG-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 7133-8692.
  • the sacS-gene is contained from bases 2272-3693.
  • the sacS-promoter is contained from bases 3694-4156.
  • the chloramphenicol gene is contained from base 418-876.
  • the origin of replication for E. coli (ori EC) is contained from base 1369-2229 and the cscA-gene from E. coli ⁇ N is contained from bases 5699-7132 (see fig. 5).
  • the 5' flanking region of the pfsG-gene which is homologous to the genome of Basfia succiniciproducens, is contained from bases 3384-4838, while the 3' flanking region of the pfsG-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 5738-7300.
  • the sacS-gene is contained from bases 1473-2894.
  • the sacS-promoter is contained from bases
  • the chloramphenicol gene is contained from base 7719-77.
  • the origin of replication for E. coli is contained from base 570-1430 and the fusion gene (SEQ ID NO: 28) argDv.cscA is contained from bases 3959-5737, consisting of a truncated version of the argD- gene (3959-4826) and a truncated version of the cscA-gene (4827-5737) (see fig. 6).
  • the part of the py/oA-gene which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1 185.
  • the sacS-gene is contained from bases 3458-4879.
  • the sacS-promoter is contained from bases 4880-5342.
  • the chloramphenicol gene is contained from bases 1604-2062.
  • the origin of repli- cation for E. coli (ori EC) is contained from bases 2555-3415 (see fig. 7).
  • Example 3 Generation of improved succinate producing strains a) Basfia succiniciproducens DD1 was transformed as described above with the
  • the "Campbell in” strains were incubated in 25-35 ml of non selective medium (BHI containing no antibiotic) at 37°C, 220 rpm over night. The overnight culture was then streaked onto freshly prepared BHI containing sucrose plates (10%, no antibiotics) and in- cubated overnight at 37°C ("first sucrose transfer”). Single colony obtained from first transfer were again streaked onto freshly prepared BHI containing sucrose plates (10%) and incubated overnight at 37°C ("second sucrose transfer”). This procedure was repeated until a minimal completion of five transfers ("third, forth, fifth sucrose transfer”) in sucrose.
  • first to fifth sucrose transfer refers to the transfer of a strain after chromosomal integration of a vector containing a sacB levan-sucrase gene onto sucrose and growth medium containing agar plates for the purpose of selecting for strains with the loss of the sacB gene and the surrounding plasmid sequences.
  • Single colony from the fifth transfer plates were inoculated onto 25-35 ml of non selective medium (BHI containing no antibiotic) and incubated at 37°C, 220 rpm over night. The overnight culture was serially diluted and plated onto BHI plates to obtain isolated single colonies.
  • the "Campbelled out" strains containing the mutation/deletion of the IdhA-ge e were confirmed by chloramphenicol sensitivity.
  • Basfia succiniciproducens DD1 AldhA ApfIA was transformed with pSacB_ivcaJ * as described above and "Campbelled in” to yield a "Campbell in” strain. Transformation and integration was confirmed by PCR. The "Campbell in” strain was then “Campbelled out” as described previously. The deletion mutants among these strains were identified and confirmed by
  • Basfia succiniciproducens DD1 AldhA ApfIA wcaJ * in which IdhA and pfIA are deleted and which expresses a truncated enzyme encoded by the wcaJ-gene.
  • Basfia succiniciproducens DD1 AldhA ApfIA wcaJ * was transformed with pSacB_pykA1 as described above and “Campbelled in” to yield a "Campbell in” strain. Transformation and integration was confirmed by PCR. The "Campbell in” strain was then “Campbelled out” as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis.
  • Basfia succiniciproducens DD1 AldhA ApfIA wcaJ * was transformed with
  • Basfia succiniciproducens DD1 AldhA ApfIA wcaJ * pykA1 was transformed with pSacB_delta_pfsG argD::cscA- us ⁇ on as described above and "Campbelled in” to yield a "Campbell in” strain. Transformation and integration was confirmed by PCR. The "Campbell in” strain was then “Campbelled out” as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis.
  • Basfia succiniciproducens DD1 AldhA ApfIA wcaJ * pykA1 was transformed with pSacB_delta_pfsG cscA as described above and “Campbelled in” to yield a "Campbell in” strain. Transformation and integration was confirmed by PCR. The "Campbell in” strain was then “Campbelled out” as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis.
  • Basfia succiniciproducens DD1 was transformed with pSacB_delta_pfsG cscA to yield a "Campbell in” strain. Transformation and integration was confirmed by PCR. The "Campbell in” strain was then “Campbelled out” as described previously. The mutants among these strains were identified and confirmed by PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AptsG cscA.
  • the productivity of the DD1 AldhA ApfIA wcaJ * was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ * AptsG argD::cscA-fusion in the presence of sucrose as a carbon source. Furthermore, the productivity of the DD1 AldhA ApfIA wcaJ * pykA1 was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ * pykA1 AptsG argD::cscA-fusion in the presence of sucrose as a carbon source.
  • the productivity of the DD1 AldhA ApfIA wcaJ * pykA1 was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ * pykA1 AptsG cscA in the presence of sucrose as a carbon source. Furthermore, the productivity of the DD1 strain was compared with the productivity of the mutant strain DD1 ⁇ ptsG cscA in the presence of sucrose as a carbon source.
  • Nicotinic acid (B3) 1 .0 g/L
  • Pantothenic acid (B5) 1 .0 g/L
  • trace element solution 0.05 mL 21 g/L 0.02 g/L
  • Table 8 Cultivation of the DD1 AldhA ApfIA wcaJ * py/oAi-strain and the DD1 AldhA ApfIA wcaJ * pykA1 AptsG argD: :cscA-fusion-strain on sucrose (medium LSM_3_YE) cultivation time
  • Table 9 Cultivation of the DD1 AldhA ApfIA wcaJ * py/oAi-strain and the DD1 AldhA ApfIA wcaJ * pykA1 AptsG argD: :cscA-fusion-strain on sucrose (medium CGM) cultivation time
  • Table 10 Cultivation of the DD1 AldhA ApfIA wcaJ * py/oAi-strain and the DD1 AldhA ApfIA wcaJ * pykA1 AptsG cscA-strain on sucrose (medium LSM_3) cultivation time
  • Table 1 1 Cultivation of the DD1 AldhA ApfIA wcaJ * py/oAi-strain and the DD1 AldhA ApfIA wcaJ * pykA1 AptsG cscA-strain on sucrose (medium CGM) cultivation time
  • Table 12 Cultivation of the DD1 -strain and the DDIApfsG cscA-strain on sucrose (medium LSM_3) cultivation time
  • Table 13 HPLC method (ZX-THF50) for analysis of succinic acid, formic acid, lactic acid, acetic acid and pyruvic acid
  • SEQ ID NO: 1 nucleotide sequence of 16 S rDNA of strain DD1 .
  • SEQ ID NO: 2 nucleotide sequence of 23 S rDNA of strain DD1 .
  • SEQ ID NO: 3 nucleotide sequence of pfsG-gene from strain DD1 .
  • SEQ ID NO: 4 amino acid sequence of PtsG from strain DD1 .
  • SEQ ID NO: 6 amino acid sequence of CscA from E. coli W
  • SEQ ID NO: 7 (nucleotide sequence of argD-gene from strain DD1 )
  • SEQ ID NO: 8 amino acid sequence of ArgD from strain DD1 .
  • SEQ ID NO: 9 nucleotide sequence of IdhA-gene from strain DD1 .
  • SEQ ID NO: 10 amino acid sequence of LdhA from strain DD1 .
  • SEQ ID NO: 12 amino acid sequence of PflA from strain DD1 .
  • SEQ ID NO: 13 (nucleotide sequence of pflD-gene from strain DD1 )
  • SEQ ID NO: 14 amino acid of PfID from strain DD1
  • SEQ ID NO: 15 (nucleotide sequence of wcaJ-gene from strain DD1 )
  • SEQ ID NO: 16 amino acid sequence of the enzyme encoded by the above wcaJ-gene
  • SEQ ID NO: 17 (nucleotide sequence of wcaJ-gene from strain DD1 with insertion of cytosine between nucleotides 81 and 82) atgataaaacgccttttcgatattgttgtcgcattgatagcattgattttgttttcgcccttatatttgttttgtggcttatcaaggtaaaacaaatt tgggatcaccggtgttatttaaacaaacccgccccggattgcatggtaaaccctttgagatgattaagttcagaacaatgaaagacgg cgcagatgaaaacggtaatattttgccggatgcggagcgcttaacacctttcggcaaaatgttggagttgcgctaccagtctggagagt
  • SEQ ID NO: 19 amino acid sequence of PykA from strain DD1 .
  • SEQ ID NO: 20 (nucleotide sequence of fruA-gene from strain DD1 )
  • SEQ ID NO: 21 (amino acid sequence of the enzyme encoded by the above fruA-gene) MKDKPMNIFLTQSPNLGRAKAFLLHQVLAAAVKQQNHQLVENAEQADLAIVFGKTLPNLTALLG KKVYLADEEQALNAPENTVAQALTEAVDYVQPAQQDVQPATASGMKNIVAVTACPTGVAHTF MSAEAITTYCQQQGWNVKVETRGQVGANNIISAEDVAAADLVFIATDINVDLSKFKGKPMYRTS TGLALKKTAQEFDKAFKEATIYQGEETTTTTETQTSGEKKGVYKHLMTGVSHMLPLVVAGGLLI AISFMFGIEAFKDENIAGGLPKALMDIGGGAAFHLMIAVFAGYVAFSIADRPGLAVGLIGGMLATS AGAGILGGIIAGFLAGYVVKFLNDAIQLPASLTSLKPILILPLLGSAIVGLAMIYLLNPPVAAAMNAL TE
  • SEQ ID NO: 22 complete nucleotide sequence of plasmid pSacB
  • SEQ ID NO: 23 complete nucleotide sequence of plasmid pSacB_delta_/c/M
  • SEQ ID NO: 25 complete nucleotide sequence of plasmid pSacB wcaJ *
  • SEQ ID NO: 26 (complete nucleotide sequence of plasmid pSacB_delta_pfsG_cscA) ctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcacaaatacaggg gtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtcagatggagatt gatttaatggcggatgtgctgagagcaccgcccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggccggaataat aaagccttaatagccttgattaagcccg
  • SEQ ID NO: 28 (nucleotide sequence of fusion-gene argD::cscA
  • SEQ ID NO: 29 complete nucleotide sequence of plasmid pSacB_py/oAi

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Abstract

La présente invention concerne un micro-organisme modifié ayant, par rapport à son type sauvage, une activité réduite de l'enzyme qui est codée par le gène pfsG, et le micro-organisme modifié exprimant une enzyme ayant l'activité de saccharose hydrolase et le type sauvage à partir duquel le micro-organisme modifié a été dérivé appartenant à la famille des Pasteurellaceae. La présente invention concerne également un procédé de production d'acide succinique, ainsi que l'utilisation de microorganismes modifiés.
PCT/EP2015/069445 2014-08-28 2015-08-25 Microorganisme modifié pour la production améliorée de produits chimiques fins sur du saccharose Ceased WO2016030373A1 (fr)

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US10597429B2 (en) 2015-12-02 2020-03-24 Basf Se Method of producing proteins in filamentous fungi with decreased CLR1 activity
US11299522B2 (en) 2015-12-02 2022-04-12 Basf Se Method of producing proteins in filamentous fungi with decreased CLR2 activity

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10597429B2 (en) 2015-12-02 2020-03-24 Basf Se Method of producing proteins in filamentous fungi with decreased CLR1 activity
US11299522B2 (en) 2015-12-02 2022-04-12 Basf Se Method of producing proteins in filamentous fungi with decreased CLR2 activity

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