WO2016202880A1 - Procédé de fermentation - Google Patents

Procédé de fermentation Download PDF

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Publication number
WO2016202880A1
WO2016202880A1 PCT/EP2016/063792 EP2016063792W WO2016202880A1 WO 2016202880 A1 WO2016202880 A1 WO 2016202880A1 EP 2016063792 W EP2016063792 W EP 2016063792W WO 2016202880 A1 WO2016202880 A1 WO 2016202880A1
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Prior art keywords
cell
fermentation
interest
compound
enzyme
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PCT/EP2016/063792
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English (en)
Inventor
Mickel Leonardus August Jansen
Remko Tsjibbe Winter
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Dsm Ip Assets B.V.
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Publication of WO2016202880A1 publication Critical patent/WO2016202880A1/fr

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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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously

Definitions

  • the present invention relates to a process for the production of a compound of interest by fermentation.
  • the invention also relates to an end-of-fermentation broth, for example one obtainable by a process of the invention.
  • Dicarboxylic acids such as malic acid, fumaric acid and succinic acid
  • dicarboxylic acids To meet the increasing need for dicarboxylic acids, more efficient and cost effective production methods are being developed.
  • dicarboxylic acids are made by fermentation of bacteria at neutral pH, for example described in US 5,573,931 .
  • other microorganisms like yeasts have been employed for the production of dicarboxylic acids at low pH, which has the advantage of producing the acid directly (WO2010/003728).
  • fermentation-based succinic acid production makes use of industrial feed-stocks.
  • industrial feedstocks are used that contain high fractions of fermentable sugars, such as glucose, fructose, maltose or sucrose.
  • examples of such feedstocks are sucrose-based raw materials (e.g., cane or beet molasses) or high quality dextrose obtained from starch hydrolysates.
  • the fermentable sugars are readily converted into succinic acid via microbial fermentation and low amounts of residual sugars are present in the end-of-fermentation broth.
  • the present invention relates to a process for the production of a compound of interest.
  • the process is a fermentation process, for example a process for the fermentative production of a dicarboxylic acid such as succinic acid.
  • fermentation is carried out in the presence of an enzyme, so as to reduce the amount of residual sugar present at the end of fermentation.
  • an enzyme in the process enables non-fermentable disaccharides and/or oligosaccharides present in the feedstock, for example a DE-95 type of glucose syrup, to be hydrolyzed during fermentation towards fermentable sugars.
  • Such fermentable sugars may in turn be converted towards a desired end-product, for example a dicarboxylic acid.
  • a higher product yield may be achieved in fermentation.
  • a higher yield in down-stream processing and/or a higher product quality may be realized.
  • the invention relates to a process for the production of a compound of interest, which process comprises fermenting a first cell capable of producing the compound of interest in a suitable fermentation medium under conditions which allow for production of the compound of interest,
  • the fermentation medium comprises disaccharides and/or oligosaccharides which are not completely fermentable by the first cell
  • the fermentation is carried out in the presence of an enzyme which is capable of hydrolyzing, at least in part, the disaccharides and/or the oligosaccharides;
  • the invention also relates to: an end-of-fermentation broth comprising a compound of interest obtainable by a process according to such a method;
  • an end-of-fermentation broth comprising a compound of interest and a lower amount of non-fermentable disaccharides and/or oligosaccharides than present at the beginning of fermentation.
  • Figure 1 sets out the plasmid map of pSUC223.
  • Figure 2 sets out a schematic depiction of the principle of PDC6 replacement by a FRD1 expression cassette by direct integration and in vivo recombination of the split Cre-recombinase construct. Expression of Cre-recombinase is regulated by the GAL1 promoter.
  • Figure 3 sets out a plasmid map of pSUC228.
  • Figure 4 shows the effect of incubation of fermentation supernatant without addition of Amigase Mega on individual sugar concentrations. All sugar concentrations are calculated back to glucose concentration.
  • Figure 5 shows the effect of addition of Amigase Mega to a fermentation supernatant on individual sugar concentrations. All sugar concentrations are calculated back to glucose concentration.
  • Figure 6 shows the effect of addition of Amigase Mega to a fermentation broth (including yeast cells) on individual sugar concentrations. All sugar concentrations are calculated back to glucose concentration.
  • Figures 7 shows the effect of incubating succinic fermentation supernatant with 5 g/l cellobiose and acid tolerant ⁇ -1 ,4-glucosidase.
  • Figure 8 shows the effect of incubating MilliQ water + 80 g/l succinic acid (pH 3) with 5.5 g/l cellobiose and acid tolerant ⁇ -1 ,4-glucosidase.
  • Figure 9 shows stability of sugars after 19 hrs incubation (no enzyme).
  • MQ EoF MilliQ water + succinic acid
  • SUC EoF succinic fermentation.
  • SEQ ID NO: 2 sets out the complete nucleic acid sequence of plasmid PSUC223.
  • SEQ ID NO: 3 sets out the amino acid sequence of FRD1 (encoded by YEL047c) lacking the first 19 amino acids.
  • SEQ ID NO: 4 sets out the nucleic acid sequence of the synthetic TDH3p-FRD1 - TDH3t gene.
  • SEQ ID NO: 5 sets out the nucleic acid sequence of primer 1 (TDH3p FW with PDC6 5' overhang).
  • SEQ ID NO: 6 sets out the nucleic acid sequence of primer 2 (TDH3t REV with overhang to pSUC228 Cre-1 ).
  • SEQ ID NO: 7 sets out the nucleic acid sequence of primer 3 (pSUC228 Cre-1 FW with overhang TDH3t).
  • SEQ ID NO: 8 sets out the nucleic acid sequence of primer 4 (DBC-03373, PSUC228 Cre-1 REV).
  • SEQ ID NO: 9 sets out the complete nucleic acid sequence of plasmid PSUC228.
  • SEQ ID NO: 10 sets out the nucleic acid sequence of primer 5 (DBC-03374, PSUC225 Cre-2 FW).
  • SEQ I D NO: 1 1 sets out the nucleic acid sequence of primer 6 (pSUC225 Cre-2 REV with PDC6 3' overhang).
  • SEQ ID NO: 12 sets out the glycosomal Trypanosoma brucei fumarate reductase (FRDg) amino acid sequence lacking a 3 amino acid C-terminal targeting signal.
  • SEQ ID NO: 13 sets out the Rhizopus oryzae fumarase amino acid sequence, lacking the first 23 N-terminal amino acids.
  • SEQ ID NO: 14 sets out the Actinobacillus succinogenes phosphoenolpyruvate carboxykinase amino acid sequence, with EGY to DAF modification at position 120 - 122.
  • SEQ ID NO: 15 sets out the Saccharomyces cerevisiae peroxisomal malate dehydrogenase (Mdh3) amino acid sequence, lacking the C-terminal peroxisomal targeting sequence (amino acids SKL).
  • SEQ ID NO: 16 sets out the Saccharomyces cerevisiae pyruvate carboxylase amino acid sequence.
  • SEQ ID NO: 17 sets out the Kluyveromyces lactis isocitrate lyase amino acid sequence.
  • SEQ ID NO: 18 sets out Saccharomyces cerevisiae peroxisomal malate synthase (Mls1 ) amino acid sequence, lacking the 3 C-terminal peroxisomal targeting sequence.
  • SEQ ID NO: 19 sets out the amino acid sequence of a dicarboxylic acid transporter from Aspergillus niger.
  • the invention relates to a process for the production of a compound of interest, which process comprises fermenting a first cell capable of producing the compound of interest in a suitable fermentation medium under conditions which allow for production of the compound of interest,
  • the fermentation medium comprises disaccharides and/or oligosaccharides which are not completely fermentable by the first cell
  • the fermentation is carried out, at least in part, in the presence of an enzyme which is capable of hydrolyzing, at least in part, the disaccharides and/or the oligosaccharides;
  • the invention relates to a process for the production of any compound of interest.
  • the process comprises fermenting a first cell capable of producing the compound of interest in a suitable fermentation medium under conditions which allow for production of the compound of interest.
  • the fermentation medium is one which comprises disaccharides and/or oligosaccharides which are not completely fermentable by the first cell. That is to say, the first cell is a cell capable of producing the compound of interest, but typically not capable of fermentation of all of the disaccharides and/or the oligosaccharides in the fermentation medium. However, the first cell may be modified according to the invention as described herein, so that it is capable of fermentation of a greater fraction of the disaccharides and/or the oligosaccharides that it otherwise would be.
  • a typical fermentation process in which a feedstock is used which comprises non-fermentable disaccharides and/or oligosaccharides will result in an amount of residual sugar being present in the fermentation broth at the end of fermentation. That is to say, residual sugar is typically present in the form of non-fermentable disaccharides and/or oligosaccharides.
  • an oligosaccharide is a saccharide polymer comprising a small number, typically from three to about 10 or more of simply sugars (monosaccharides).
  • a disaccharide is composed of two monosaccharides.
  • Disaccharides and/or oligosaccharides are non-fermentable in the sense intended in the invention if (in the absence of enzyme according to the invention) they are not available to the first cell in a form in which that cell can convert the sugars to the compound of interest.
  • Example of non-fermentable disaccharides may include isomaltose and cellobiose.
  • the process is carried out in the presence of an enzyme which is capable of hydrolyzing, at least in part, the non-fermentable disaccharides and/or oligosaccharides.
  • the fermentation broth at the end of fermentation will comprise less residual sugar (than would be present for an equivalent process carried out in the absence of the enzyme).
  • the enzyme may be introduced into the fermentation in any convenient fashion.
  • enzyme per se is added to the fermentation medium. That is to say, the enzyme, for example in the form of a suitably formulated composition may be added directly to the fermentation medium.
  • the fermentation process of the invention may be carried out in the presence of a second cell, different to the first cell, which is capable of expressing the enzyme.
  • the fermentation process of the invention may be carried out wherein the first cell is modified so that it is capable of expressing the enzyme to a greater degree that an unmodified form of the cell.
  • Expression of the enzyme by a second cell or by a modified first cell may be inducible.
  • the enzyme may be present for only a portion of the fermentation period or for the entire course of fermentation.
  • the production phase may preferably be preceded by a biomass formation phase for optimal biomass production.
  • the enzyme may be present for the entire or part of the production phase.
  • Enzyme may also be present in the biomass formation phase.
  • the enzyme is preferably present for up to about the final 20% of the fermentation period, for example up to about the final 15% of the fermentation period, such as up to about the final 10% of the fermentation period (the percentage expressed being in terms of the total fermentation period, for example the total production period).
  • the enzyme may be any enzyme capable of hydrolysing a disaccharide and/or an oligosaccharide.
  • a suitable enzyme may an amylase such as an alpha-amylase, a beta-amlyase or a gluco-amylase, a pullulanase, an alpha-glucosidase, a beta-glucosidase or a trehalase.
  • Amylase is any glycoside hydrolase which is capable of acting on a-1 ,4- glycosidic bonds.
  • the amylase may be an a-amylase (EC 3.2.1 .1 ), a ⁇ -amylase (EC 3.2.1 .2) or a glucoamylase (EC 3.2.1 .3).
  • a suitable pullulanase (EC 3.1 .41 ) may be a type I pullulanase, which specifically attacks a-1 ,6 linkages, or a type II pullulase, which is also able to hydrolyze a-1 ,4 linkages.
  • a suitable enzyme may be added in any suitable amount.
  • the skilled person will readily be able to determine an appropriate dosage of enzyme in order to reduce the amount of residual sugar, for example dependent on the specific enzyme being used.
  • the invention also relates to an end-of-fermentation broth comprising a compound of interest obtainable by a process of the invention.
  • the invention also relates to an end-of-fermentation broth comprising a compound of interest and a lower amount of non-fermentable disaccharides and/or oligosaccharides than present at the beginning of fermentation, for example as obtained by a process of the invention.
  • Such an end-of-fermentation broth may comprise an amount of non-fermentable disaccharides and/or oligosaccharides of less than 5g/L disaccharides and/or oligosaccharides, such as less than 4g/L disaccharides and/or oligosaccharides, for example less than 3g/L disaccharides and/or oligosaccharides, less than 2g/L disaccharides and/or oligosaccharides or less.
  • the compound of interest in the method according to the invention can be any biological compound.
  • the compound of interest may be a dicarboxylic acid, for example succinic acid.
  • the compound of interest may be the cells themselves, i.e. biomass, in which case cells may be recovered from the medium following cultivation according to the method of the invention.
  • the biological compound may be biomass (i.e. the cells in the fermentation themselves) or any biopolymer or metabolite.
  • the biological compound may be encoded by a single polynucleotide or a series of polynucleotides composing a biosynthetic or metabolic pathway or may be the direct result of the product of a single polynucleotide or products of a series of polynucleotides.
  • the biological compound may be native to the host cell or heterologous.
  • heterologous biological compound is defined herein as a biological compound which is not native to the cell; or a native biological compound in which structural modifications have been made to alter the native biological compound.
  • biopolymer is defined herein as a chain (or polymer) of identical, similar, or dissimilar subunits (monomers).
  • the biopolymer may be any biopolymer.
  • the biopolymer may for example be, but is not limited to, a nucleic acid, polyamine, polyol, polypeptide (or polyamide), or polysaccharide.
  • the biopolymer may be a polypeptide.
  • the polypeptide may be any polypeptide having a biological activity of interest.
  • the term "polypeptide" is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins.
  • the polypeptide may be a pharmaceutical polypeptide and/or a bioactive. Polypeptides further include naturally occurring allelic and engineered variations of the above- mentioned polypeptides and hybrid polypeptides.
  • the polypeptide may be native or may be heterologous to the host cell.
  • the polypeptide may be a collagen or gelatin, or a variant or hybrid thereof.
  • the polypeptide may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide or intracellular protein.
  • the intracellular protein may be an enzyme such as, a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase.
  • the polypeptide may be an enzyme secreted extracellularly.
  • Such enzymes may belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase.
  • the enzyme may be a carbohydrase, e.g.
  • cellulases such as endoglucanases, ⁇ -glucanases, cellobiohydrolases or ⁇ -glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases
  • the enzyme may be a phytase.
  • the enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic amylase, carbohydrase, carboxypeptidase, endo- protease, metallo-protease, serine-protease, catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase,
  • the enzyme may be involved in the synthesis of a primary or secondary metabolite
  • a polypeptide or enzyme also can be a product as described in WO2010/102982.
  • a polypeptide can also be a fused or hybrid polypeptide to which another polypeptide is fused at the N- terminus or the C-terminus of the polypeptide or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.
  • fusion polypeptides include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter(s) and terminator.
  • the hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the host cell.
  • Example of fusion polypeptides and signal sequence fusions are for example as described in WO2010/121933.
  • the biopolymer may be a polysaccharide.
  • the polysaccharide may be any polysaccharide, including, but not limited to, a mucopolysaccharide ⁇ , g., heparin and hyaluronic acid) and nitrogen-containing polysaccharide (e.g., chitin).
  • the polysaccharide is hyaluronic acid.
  • the polynucleotide of interest according to the invention may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, carotenoids, (beta-lactam) antibiotics, and vitamins.
  • a primary or secondary metabolite such as organic acids, carotenoids, (beta-lactam) antibiotics, and vitamins.
  • Such metabolite may be considered as a biological compound according to the present invention.
  • metabolite encompasses both primary and secondary metabolites; the metabolite may be any metabolite, such as organic acids, carotenoids, (beta-lactam) antibiotics, and vitamins. Such metabolites may be considered as a biological compound according to the present invention. Metabolites include citric acid, gluconic acid, itaconic acid, a dicarboxylic acid such as fumaric acid, succinic acid, malic acid, or adipic acid, lactic acid or a keto acid such as levulinic acid.
  • the metabolite may be encoded by one or more genes, such as in a biosynthetic or metabolic pathway.
  • Primary metabolites are products of primary or general metabolism of a cell, which are concerned with energy metabolism, growth, and structure.
  • Secondary metabolites are products of secondary metabolism (see, for example, R. B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981 ).
  • Primary metabolites are for example intermediates of the main metabolic pathways such as the glycolytic pathway or the TCA cycle.
  • the primary metabolite may be, but is not limited to, an amino acid, fatty acid, such as a polyunsaturated fatty acid, nucleoside, nucleotide, sugar, triglyceride, or vitamin.
  • the secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, or terpene.
  • the secondary metabolite may be an antibiotic, antifeedant, attractant, bacteriocide, fungicide, hormone, insecticide, or rodenticide.
  • Antibiotics include cephalosporins, beta-lactams and macrolides, such as erythromycin.
  • ⁇ -lactams examples include clavulanic acid, penicillin (e.g. penicillin G, penicillin V or 6-aminopenicillinic acid) and semi synthetic penicillins such as amoxicillin and cephalosporins such as cephalosporin C.
  • penicillin e.g. penicillin G, penicillin V or 6-aminopenicillinic acid
  • semi synthetic penicillins such as amoxicillin and cephalosporins such as cephalosporin C.
  • the ⁇ -lactam may be an N-acylated derivative of ⁇ -lactam intermediates such as 7-amino-3-carbamoyloxymethyl-3-cephem- 4-carboxylic acid (7ACCCA), 7-aminocephalosporanic acid (7-ACA), 7-amino-3-chloro- 3-cephem-4-carboxylic acid (7-ACCA), 7-aminodesacetoxycephalosporanic acid (7-ADCA), 7-aminodesacetylcephalosporanic acid (7-ADAC), 7-amino-3-[(Z/£)-1 - propen-1 -yl]-3-cephem-4-carboxylic acid (7-PACA) and the like.
  • 7ACCCA 7-amino-3-carbamoyloxymethyl-3-cephem- 4-carboxylic acid
  • 7-ACA 7-aminocephalosporanic acid
  • 7-ACCA 7-amino-3-chloro- 3-
  • the acyl group at the 7-amino position is preferably adipic acid yielding the corresponding adipoyl derivate as disclosed in WO 93/05158, WO 93/08287 or WO 2004/106347.
  • Alternative suitable side chains have been disclosed in WO 95/04148, WO 95/04149, WO 96/38580, WO 98/48034 and WO 98/48035.
  • adipoyl-7-ADCA is adipoyl-7-ADCA
  • adipoyl-7-ACA is adipoyl-7-ADAC
  • adipoyl-7-ACCA is adipoyl-7-PACA or adipoyl-7-ACCCA
  • most preferred is adipoyl-7-ADCA.
  • the biological compound may also be the product of a selectable marker.
  • a selectable marker is a product of a polynucleotide of interest which product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'- phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), hyg (hygromycin), NAT or NTC (Nourseothricin) as well as equivalents thereof.
  • amdS acetamidase
  • argB ornithinecarbamoyltransferase
  • bar phosphinothricinacetyltransferase
  • hygB hygromycin
  • a suitable first cell for use in the method of the invention may be any wild type strain producing a compound of interest.
  • a suitable first cell may be a strain which has been obtained and/or improved by subjecting a parent or wild type strain of interest to a classical mutagenic treatment or to recombinant nucleic acid transformation.
  • a cell suitable for use in the method of the invention may already be capable of producing the compound of interest.
  • the cell may also be provided with a homologous or heterologous expression construct that encodes a polypeptide involved in the production of the compound of interest.
  • a genetically modified cell such as a genetically modified yeast
  • a genetically modified yeast is defined as a cell which contains, or is transformed or genetically modified with or a nucleotide sequence or polypeptide that does not naturally occur in the cell, or it contains additional copy or copies of an endogenous nucleic acid sequence, or it contains a deletion or disruption of an endogenous or homologous nucleotide sequence.
  • a wild-type eukaryotic cell is herein defined as the parental cell of the recombinant cell.
  • nucleic acid or polypeptide molecule when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably of the same variety or strain.
  • heterologous when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature.
  • Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced.
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include BLASTP and BLASTN, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 1 1 .0, gap extend 1 , Blosum 62 matrix.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single- stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
  • nucleotide sequences encoding enzymes in a yeast there are various means available in the art for overexpression of nucleotide sequences encoding enzymes in a yeast in the process of the invention.
  • a nucleotide sequence encoding an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the cell, e.g. by integrating additional copies of the gene in the cell's genome, by expressing the gene from a centromeric vector, from an episomal multicopy expression vector or by introducing an (episomal) expression vector that comprises multiple copies of the gene.
  • overexpression of the enzyme according to the invention is achieved with a (strong) constitutive promoter.
  • Suitable promoters in fungal cells are known to the skilled man in the art.
  • Suitable promotors may be, but are not limited to, TDH 1 , TDH3, GAL7, GAL10, GAL1 , CYC1 , HIS3, ADH1 , PH05, ADC1 , ACT1 , TRP1 , URA3, LEU2, EN01 , TPI 1 , AOX1 , PGL, GPDA and GAPDH.
  • Other suitable promoters include PDC1 , GPD1 , PGK1 , and TEF1 .
  • a gene encoding an enzyme may be ligated into a nucleic acid construct, for instance a plasmid, such as a low copy plasmid or a high copy plasmid.
  • the fungal cell according to the present invention may comprise a single copy, but preferably comprises multiple copies of a gene, for instance by multiple copies of a nucleotide construct.
  • a nucleic acid construct may be maintained episomally and thus comprises a sequence for autonomous replication, such as an autonomously replicating sequence and a centromere (Sikorski and Hieter ,1989, Genetics 122, 19-27).
  • a suitable episomal nucleic acid construct may e.g. be based on the yeast 2 ⁇ or pKD1 plasmids (Gleer et a/., 1991 , Biotechnology 9: 968-975), or the AMA plasmids (Fierro et a/., 1995, Curr. Genet. 29:482-489).
  • each nucleic acid construct may be integrated in one or more copies into the genome of the fungal cell. Integration into the cell's genome may occur at random by non-homologous recombination but preferably, the nucleic acid construct may be integrated into the cell's genome by homologous recombination as is well known in the art.
  • an expression construct or nucleic acid construct
  • Such an expression cassette/construct typically may contain all the control sequences required for expression of a coding sequence, wherein said control sequences are operably linked to said coding sequence.
  • operably linked is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide.
  • control sequences is defined herein to include all components, which are necessary or advantageous for the expression of mRNA and / or a polypeptide, either in vitro or in a host cell. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, Shine-Delgarno sequence, optimal translation initiation sequences (as described in Kozak, 1991 , J. Biol. Chem. 266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be optimized to their specific purpose.
  • a first cell suitable for use in the method according to the invention may be a prokaryotic cell.
  • the prokaryotic cell is a bacterial cell.
  • the term "bacterial cell” includes both Gram-negative and Gram-positive microorganisms.
  • the bacterium may be an Actinomycete.
  • Suitable bacteria may be selected from e.g. Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Acintomycetes such as Streptomyces and Actinoplanes species.
  • the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B.
  • the first cell suitable for use in the method according to the invention may be a eukaryotic host cell.
  • the eukaryotic cell is a fungal or an algal cell.
  • the eukaryotic cell may be a fungal cell, for example a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Issatchenkia orientalis (also known as Candida krusei) and Pichia pastoris.
  • the eukaryotic cell may be a filamentous fungal cell.
  • Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Pleurotus, Schizophyllum, Talaromyces/Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
  • Preferred filamentous fungal cells belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces/Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Acremonium chrysogenum, Acremonium alabamense, Aspergillus awamori, Aspergillus sojae, Aspergillus fumigatus, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris, Penicillium chrysogenum or Penicillium citrium.
  • a more preferred host cell belongs to the genus Aspergillus, more preferably the host cell belongs to the species Aspergillus niger.
  • the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.
  • Useful strains in the context of the present invention may be Aspergillus niger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 101 1 , CBS205.89, ATCC 9576, ATCC14488-14491 , ATCC 1 1601 , ATCC12892, P. chrysogenum CBS 455.95, P.
  • a fungal cell in a process as disclosed herein may be any suitable wild type or recombinant or genetically modified fungal cell, in particular a recombinant or genetically modified yeast cell.
  • a genetically modified fungal cell may comprise a genetic modification of a gene selected from the group consisting of a gene encoding a pyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a malate dehydrogenase, a fumarase, a fumarate reductase, an isocitrate lyase, a malate synthase and a dicarboxylic acid transporter.
  • a genetically modified yeast cell suitable for use in the process of the invention may comprise any suitable genetic modifications, such as deletions or disruptions, and insertions of homologous or heterologous nucleotides sequences.
  • a yeast cell suitable for use in the process of the invention may be genetically modified or transformed with nucleotide sequences that encode homologous and/or heterologous enzymes that catalyse reactions in the cell resulting in an increased flux towards a dicarboxylic acid such malic acid, fumaric acid and/ or succinic acid.
  • nucleotide sequences encoding i) a malate dehydrogenase which catalyses the conversion from OAA to malic acid; ii) a fumarase, which catalyses the conversion of malic acid to fumaric acid; or iii) a fumarate reductase that catalyses the conversion of fumaric acid to succinic acid, depending on the dicarboxylic acid to be produced.
  • a genetically modified yeast cell may be used.
  • a yeast cell used in the process according to the present invention comprises genetic modifications according to the preferred embodiments as described herein below.
  • a recombinant fungal cell may comprise a genetic modification with a pyruvate carboxylase (PYC), that catalyses the reaction from pyruvate to oxaloacetate (EC 6.4.1 .1 ).
  • the pyruvate carboxylase may for instance be active in the cytosol upon expression of the gene.
  • the fungal cell overexpresses a pyruvate carboxylase, for instance an endogenous or homologous pyruvate carboxylase is overexpressed.
  • a host cell according to the present invention may be genetically modified with a pyruvate carboxylase which has at least 80, 85, 90, 95, 99 or 100% sequence identity with amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 16.
  • the genetically modified yeast cell expresses a nucleotide sequence encoding a phosphoenolpyruvate (PEP) carboxykinase in the cytosol.
  • a nucleotide sequence encoding a phosphoenolpyruvate (PEP) carboxykinase is overexpressed.
  • the PEP carboxykinase (EC 4.1 .1 .49) preferably is a heterologous enzyme, preferably derived from bacteria, more preferably the enzyme having PEP carboxykinase activity is derived from Escherichia coii, Mannheimia sp., Actinobacillus sp., or Anaerobic-spirillum sp., more preferably Mannheimia succiniciproducens.
  • a gene encoding a PEP carboxykinase may be overexpressed and may be expressed and active in the cytosol of a fungal cell.
  • a yeast cell according to the present invention is genetically modified with a PEP carboxykinase which has at least 80, 85, 90, 95, 99 or 100% sequence identity with amino acid sequence of SEQ ID NO: 14.
  • a fungal cell is further genetically modified with a gene encoding a malate dehydrogenase (MDH) active in the cytosol upon expression of the gene.
  • MDH malate dehydrogenase
  • Cytosolic expression may be obtained by deletion of a peroxisomal targeting signal.
  • the malate dehydrogenase may be overexpressed.
  • a cytosolic MDH may be any suitable homologous or heterologous malate dehydrogenase, catalyzing the reaction from oxaloacetate to malate (EC 1 .1 .1 .37), for instance derived from S. cerevisiae.
  • the MDH is S. cerevisiae MDH3, more preferably one which has a C- terminal SKL deletion such that it is active in the cytosol.
  • a yeast cell according to the present invention comprises a nucleotide sequence encoding a malate dehydrogenase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15.
  • a fungal cell of the present disclosure is further genetically modified with a gene encoding a fumarase, that catalyses the reaction from malic acid to fumaric acid (EC 4.2.1 .2).
  • a gene encoding fumarase may be derived from any suitable origin, preferably from microbial origin, for instance a yeast such as Saccharomyces or a filamentous fungus, such Rhizopus oryzae, or a bacterium such a Escherichia coli.
  • a fungal cell of the present disclosure may overexpress a nucleotide sequence encoding a fumarase.
  • the fumarase may be active in the cytosol upon expression of the nucleotide sequence, for instance by deleting a peroxisomal targeting signal. It was found that cytosolic activity of a fumarase resulted in a high productivity of a dicarboxylic acid by the fungal cell.
  • a yeast in the process according to the present invention overexpresses a nucleotide sequence encoding a fumarase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 13.
  • the fungal cell is genetically modified with any suitable heterologous or homologous gene encoding a NAD(H)-dependent fumarate reductase, catalyzing the reaction from fumarate to succinate (EC 1 .3.1 .6).
  • the NADH-dependent fumarate reductase may be a heterologous enzyme, which may be derived from any suitable origin, for instance bacteria, fungi, protozoa or plants.
  • a fungal cell of the present disclosure comprises a heterologous NAD(H)-dependent fumarate reductase, preferably derived from a Trypanosoma sp, for instance a Trypanosoma brucei.
  • the NAD(H)-dependent fumarate reductase is expressed and active in the cytosol, for instance by deleting a peroxisomal targeting signal.
  • the fungal cell may overexpress a gene encoding a NAD(H)-dependent fumarate reductase.
  • a yeast cell in the process according to the present invention is genetically modified with a NAD(H)-dependent fumarate reductase, which has at least 80, 85, 90, 95, 99 or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12.
  • a genetically modified yeast in the process according to the invention expresses a nucleotide sequence encoding a dicarboxylic acid transporter protein.
  • the dicarboxylic acid transporter protein is overexpressed.
  • a dicarboxylic acid transporter protein may be any suitable homologous or heterologous protein.
  • the dicarboxylic acid transporter protein is a heterologous protein.
  • a dicarboxylic acid transporter protein may be derived from any suitable organism, preferably from yeast or fungi such as Schizosaccharomyces pombe or Aspergillus niger.
  • a dicarboxylic acid transporter protein is a dicarboxylic acid transporter/malic acid transporter protein, eg. from Aspergillus niger which has at least 80, 85, 90, 95 or 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 19.
  • a genetically modified fungal cell may further comprise a genetic modification with a gene encoding an isocitrate lyase (EC 4.1 .3.1 ), which may be any suitable heterologous or homologous enzyme.
  • the isocitrate lyase may for instance be obtained from Kluyveromyces lactis or Escherichia coli.
  • a first cell in the process according to the present invention is genetically modified with a isocitrate lyase which has at least 80, 85, 90, 95, 99 or 100% sequence identity with amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 17.
  • a genetically modified fungal cell may further comprise as genetic modification with a malate synthase (EC 2.3.3.9).
  • the malate synthase may be overexpressed and / or active in the cytosol, for instance by deletion of a peroxisomal targeting signal.
  • the malate synthase is a S. cerevisiae malate synthase, for instance the native malate synthase is altered by the deletion of the SKL carboxy-terminal sequence.
  • a first cell in the process according to the present invention is genetically modified with a malate synthase which has at least 80, 85, 90, 95, 99 or 100% sequence identity with amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 18.
  • a recombinant fungal cell in the process for producing a dicarboxylic acid disclosed herein comprises a disruption of a gene encoding an enzyme of the ethanol fermentation pathway.
  • a gene encoding an enzyme of an ethanol fermentation pathway may be pyruvate decarboxylase (EC 4.1 .1 .1 ), catalyzing the reaction from pyruvate to acetaldehyde, or alcohol dehydrogenase (EC 1 .1 .1 .1 ), catalyzing the reaction from acetaldehyde to ethanol.
  • a fungal cell in the process as disclosed herein comprises a disruption of one, two or more genes encoding an alcohol dehydrogenase.
  • the fungal cell is a yeast, e.g. S. cerevisiae
  • the yeast preferably comprises a disruption of an alcohol dehydrogenase gene adhl and / or adh2.
  • the yeast in the process of the invention comprises at least one gene encoding glycerol-3-phosphate dehydrogenase which is not functional.
  • a glycerol-3-phosphate dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced glycerol-3-phosphate dehydrogenase activity, for instance by mutation, disruption, or deletion of the gene encoding glycerol-3-phosphate dehydrogenase, resulting in a decreased formation of glycerol as compared to a wild-type cell.
  • a genetically modified yeast in the process according to the present invention overexpresses a nucleotide sequence encoding a PEP carboxykinase, a nucleotide sequence encoding a malate dehydrogenase, a nucleotide sequence encoding a fumarase, a nucleotide sequence encoding a NAD(H) dependent fumarate reductase, and/or a nucleotide sequence encoding a malic acid transporter protein, preferably wherein the enzymes are active in the cytosol.
  • Preferred embodiments of the enzymes are as described herein above.
  • Cytosolic expression of the enzymes described above may be obtained by deletion of a peroxisomal or mitochondrial targeting signal.
  • the presence of a peroxisomal or mitochondrial targeting signal may for instance be determined by the method disclosed by Schluter et al., Nucleid Acid Research 2007, 35, D815-D822.
  • the cells are cultivated in a reaction medium and under conditions suitable for production of the compound of interest.
  • the cells may be cultivated at a large-scale (including, for example, in the form of a continuous, batch or fed-batch cultivations) in a suitable reaction medium and under conditions allowing the compound of interest to be produced and/or isolated.
  • the amount of reaction medium may be maintained at an approximately constant volume in the method of the invention. That is to say, evaporated reaction medium may be replenished so that the volume of reaction medium remains approximately constant.
  • the cultivation takes place in a suitable reaction medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e. g., Bennett, J. W. and LaSure, L, eds., More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • suitable media are available from commercial suppliers or may be prepared using published compositions (e. g., in catalogues of the American Type Culture Collection).
  • a process for producing a dicarboxylic acid may be carried out at any suitable temperature.
  • a suitable temperature may for instance be between about 10 and about 40 degrees Celsius, for instance between about 15 and about 30 degrees Celsius, in particular at about 30°C.
  • the compound of interest can be isolated directly from the reaction medium. If the compound of interest is not secreted, it can be isolated from, for example, cell lysates.
  • the compound of interest may be isolated by methods known in the art.
  • the compound of interest may be isolated from the reaction medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.
  • the isolated compound of interest may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e. g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e. g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • the compound of interest may be used without substantial isolation from the culture broth; separation of the reaction medium from the biomass may be adequate.
  • the process for the production of a dicarboxylic acid further comprises recovering the dicarboxylic acid.
  • Recovery of the dicarboxylic acid may be carried out by any suitable method.
  • a dicarboxylic acid that is produced in a process as disclosed herein is recovered from the fermentation medium.
  • Recovery of a dicarboxylic acid may be carried out by any suitable method known in the art, for instance by crystallization, ammonium precipitation, ion exchange technology, centrifugation or filtration or any suitable combination of these methods.
  • the recovery of dicarboxylic acid comprises crystallizing the dicarboxylic acid and forming dicarboxylic acid crystals.
  • the crystallizing of dicarboxylic acid comprises removing part of the fermentation medium, preferably by evaporation, to obtain a concentrated medium.
  • the process according to the present invention comprises recovering a dicarboxylic acid which is succinic acid and wherein the recovering comprises crystallizing succinic acid from an aqueous solution having a pH of between 1 and 5 and comprising succinic acid, comprising evaporating part of the aqueous solution to obtain a concentrated solution, lowering the temperature of the concentrated solution to a value of between 5 and 35 degrees Celsius, wherein succinic acid crystals are formed.
  • the crystallizing comprises bringing the temperature of the concentrated medium to a temperature of between 10 and 30 degrees Celsius, preferably between 15 and 25 degrees Celsius.
  • the fermentation medium has a pH of between 1.5 and 4.5, preferably between 2 and 4.
  • Another advantage of crystallizing succinic acid at a higher temperature is that it requires a lower amount of energy for cooling the aqueous solution as compared to a process wherein crystallizing succinic acid is carried out below 10 or 5 degrees Celsius, resulting in a more economical and sustainable process.
  • the crystallizing of succinic acid comprises a step of washing the succinic acid crystals.
  • Succinic acid may be crystallized directly from the fermentation medium having a pH of between 1 and 5 to a purity of at least 90% w/w, preferably at least 95, 96, 97, or at least 98%, or 99 to 100%w/w.
  • the recovery of the dicarboxylic acid, preferably succinic acid comprises removing the biomass from the fermentation medium and crystallizing the dicarboxylic acid, preferably crystallizing as described herein above.
  • the removing of biomass is carried out by filtration.
  • the process for the production of a dicarboxylic acid further comprises using the dicarboxylic acid in an industrial process.
  • An industrial process for a dicarboxylic acid may be the application as a cosmetic additive, deicing agent, food additive or as a building block for (bio)polymers.
  • the fermentation medium comprises an amount of succinic acid of between 1 and 150 g/l, preferably between 5 and 100 g/l, more preferably between 10 and 80 g/l or between 15 and 60 g/l of succinic acid.
  • the present invention relates to a process for crystallizing succinic acid from an aqueous solution having a pH of between 1 and 5 and comprising succinic acid, comprising removing part of the aqueous solution by evaporation to obtain a concentrated solution, and bringing the temperature of the concentrated solution to a value of between 10 and 30 degrees Celsius, wherein succinic acid cystals are formed.
  • the crystallizing comprises bringing the temperature of the concentrated solution between 15 and 25 degrees Celsius, preferably between 18 and 22 degrees Celsius.
  • the aqueous solution has a pH of between 1 .5 and 4.5, preferably between 2 and 4.
  • the aqueous solution may be any suitable solution comprising succinic acid.
  • the aqueous solution may comprise soluble constituents and insoluble constituents and, such as (fragments of) microbial cells, protein, plant biomass lignocellulose, cellulose and the like.
  • the aqueous solution is a fermentation medium, preferably a fermentation medium obtainable by a process for the production of a dicarboxylic acid as described herein.
  • Standard genetic techniques such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001 ) "Molecular Cloning: A Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g.
  • Saccharomyces cerevisiae strain SUC-632 was constructed as described in WO2013/004670. Saccharomyces cerevisiae strain SUC-632 was constructed as described in WO2013/004670. Strain SUC-632 was used as a starting point to construct strain SUC-947. Strain SUC-708 is a mutant of strain SUC-632 obtained by classical strain improvement.
  • a fumarase gene of E. coli was transformed to strain SUC-708 as described below.
  • SEQ ID NO: 1 describes the fumarase (fumB) protein sequence from Escherichia coli (E.C. 4.2.1 .2, UniProt accession number P14407).
  • the gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632.
  • the stop codon TAA was modified to TAAG.
  • Expression of the FUM_01 gene is controlled by the TDH1 promoter (600 bp directly before the start codon of the TDH1 gene) and the TDH1 terminator (300 bp directly after the stop codon of the TDH1 gene).
  • TDH1 promoter and TDH1 terminator sequences controlling expression of FUM_01 are native sequences derived from Saccharomyces cerevisiae S288C.
  • the synthetic promoter-gene sequence including appropriate restriction sites was synthesized by GenArt (Regensburg, Germany). This synthetic fragment is part of plasmid pSUC223 ( Figure 1 ), whose sequence is described in SEQ ID NO: 7. Plasmid pSUC223 contains a KanMX marker with allows for selection for growth in the presence of G418.
  • the KanMX marker flanked by lox66 and lox71 sites (Albert et al., Plant Journal, 7(4), 649-659), can be removed by the action of Cre- recombinase, as described by Gueldender et al., (Nucleic Acids Res. 1996 Jul 1 ;24(13):2519-24).
  • the TDH 1 p-fum B-TDH 11 and the lox66-KanMX-lox71 sequences were flanked by sequences that allow integration by double cross-over at the YPRCtau3 locus, which is located on chromosome XVI.
  • Plasmid pSUC223 was restricted using restriction enzymes Apa and Xho ⁇ .
  • a 5.408 bp fragment containing the 5' YPRCtau3 flank, the synthetic fumB construct, the KanMX selection marker flanked by lox66 and lox71 sites and the 3' YPRCtau3 flank was excised from an agarose gel and purified using the ZymocleanTM Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions.
  • the fragment was transformed to strain SUC-708.
  • Transformants were selected on Yeast Extract BactoPeptone (YEP) 2% galactose plates supplemented with 200 ⁇ g G418/milliliter for selection of transformants containing the KanMX marker, yielding multiple transformants. Presence of the introduced fumB gene was confirmed by PCR using primer sequences that can anneal to the coding sequences of the ORF encoded by SEQ ID NO: 2.
  • SUC-708 FUM_01 #3 was named SUC-813.
  • the S. cerevisiae Frd1 protein has multiple possible localizations.
  • the variant omitting the 19 aa signal peptide is described in SEQ ID NO: 3, which was subjected to the codon-pair method as disclosed in WO2008/000632 for expression in S. cerevisiae.
  • the stop codon TAA was modified to TAAG.
  • Expression of the FRD1 gene is controlled by the TDH3 promoter (600 bp directly before the start codon of the TDH3 gene) and the TDH3 terminator (300 bp directly after the stop codon of the TDH3 gene).
  • TDH3 promoter and TDH3 terminator sequences controlling expression of FRD1 are native sequences derived from Saccharomyces cerevisiae S288C.
  • the synthetic promoter-gene sequence including appropriate restriction sites was synthesized by DNA2.0 (Menlo Park, CA, USA) and is described in SEQ ID NO: 4.
  • the modified FRD1 gene was transformed into strain SUC-813 as depicted in Figure 2.
  • the modified FRD1 gene replaced the PDC6 gene in strain SUC-813 using the "split Cre recombinase integration" or "direct Cre recombinase integration” (DCI) approach as described in PCT/EP2013/055047.
  • PCR fragments were generated using Phusion DNA polymerase (New England Biolabs, USA) according to manufacturer's instructions.
  • PCR fragment 1 was generated by using the primer sequences described in SEQ ID NO: 5 and SEQ ID NO: 6, using a cloning plasmid of DNA 2.0 containing SEQ ID NO: 4 as template.
  • SEQ ID NO: 5 contains a 66 bp overlap with the 5' region of the PDC6 gene, directly located before the start codon of the PDC6 gene.
  • PCR fragment 2 was generated by using the primer sequences described in SEQ ID NO: 7 and SEQ ID NO: 8 using plasmid pSUC228 ( Figure 3, SEQ ID NO: 9) as template.
  • Plasmid pSUC228 is a modified version of pSUC227, which is described in PCT/EP2013/055047.
  • Plasmid pSUC227 contains a KanMX marker, which is replaced by a nourseothricin (natMX4) marker (Goldstein and McCusker, Yeast.
  • PCR fragment 3 was generated by using the primer sequences described in SEQ ID NO: 10 and SEQ ID NO: 1 1 , using pSUC225 (described in PCT/EP2013/055047) as template.
  • SEQ ID NO: 1 1 contains a 64 bp overlap with the 3' region of the PDC6 gene, directly located after the stop codon of the PDC6 gene.
  • PCR fragments The size of the PCR fragments was checked with standard agarose electrophoresis techniques. PCR amplified DNA fragments were purified using the ZymocleanTM Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions.
  • Yeast transformation was done by a method known by persons skilled in the art.
  • S. cerevisiae strain SUC-813 was transformed with purified PCR fragments 1 , 2 and 3.
  • PCR fragment 1 contained an overlap with PCR fragment 2 at its 3' end.
  • PCR fragment 3 contained an overlap with PCR fragment 2 at its 5' end.
  • PCR fragment 2 contained an overlap at its 5' end with PCR fragment 1 and at its 3' end with PCR fragment 3, such that this allowed homologous recombination of all three PCR fragments ( Figure 2).
  • the 5' end of PCR fragment 1 and the 3' end of PCR fragment 3 were homologous to the PDC6 locus and enabled integration of all three PCR fragments in the PDC6 locus. This resulted in one linear fragment consisting of PCR fragments 1 to 3 integrated in the PDC6 locus ( Figure 2). This method of integration is described in patent application WO2013/076280.
  • Transformation mixtures were plated on YPD-agar (per liter: 10 grams of yeast extract, 20 grams per liter peptone, 20 grams per liter dextrose, 20 grams of agar) containing 100 ⁇ g nourseothricin (Jena Bioscience, Germany) per ml. After three to five days of growth at 30 ° C, individual transformants were re-streaked on fresh YPD-agar plates containing 100 ⁇ g nourseothricin per ml.
  • the marker cassette and Cre-recombinase and are effectively out-recombination by the method described in PCT/EP2013/055047, resulting in replacement of the PDC6 gene by SEQ ID NO: 4 and leaving a lox 72 site as a result of recombination between the lox66 and lox71 sites. Due to the activity of Cre- recombinase, the KanMX marker flanked by lox66 and lox71 sites, which was introduced into genomic DNA to create strain SUC-813 was also efficiently out- recombined. The resulting markerfree strain was named SUC-947.
  • Strain SUC-947 was able to grow on YPD-agar plates, but unable to grow on YPD-agar plates supplemented with either 200 ⁇ g G418/ml or 100 ⁇ g/ml nourseothricin or both, confirming out- recombination of both the KanMX and the natMX4 marker.
  • Example 2 Effect of addition of an amylo-glucosidase to a succinic acid fermentation on the sugar level end of fermentation
  • the medium was based on Verduyn et al. (Verduyn C, Postma E, Scheffers WA, Van Dijken JP. Yeast, 1992 Jul;8(7):501 -517), with modifications in the carbon and nitrogen sources, as described herein below.
  • Zincsulphate . 7H 2 0 ZnS0 4 .7H 2 0 4.50
  • Ironsulphate . 7H 2 0 FeS0 4 .7H 2 0 3.00
  • the pH was controlled at 5.0 by addition of ammonia (10 wt%). Temperature was controlled at 30°C. p0 2 was controlled at 25% (relative to air saturation) by adjusting the stirrer speed. Total airflow applied was 18 NL/h. Glucose concentration was kept limited by controlled feed to the fermenter (exponent of 0.15 was applied).
  • Succinic yeast strains were cultivated as described above in Example 2.
  • 5 g/l cellobiose Sigma Aldrich
  • 0.175 mg/ml of acid tolerant ⁇ -1 ,4-glucosidase from Talaromyces emersonii, heterologously expressed in Aspergillus niger EBA4 see patent: WO201 1/098577.
  • succinic acid (Sigma Aldrich) was dissolved in MilliQ water, adjusted to pH 3 with carbonate, 5.5 g/l cellobiose (Sigma Aldrich) was added along with 0.175 mg/ml of acid tolerant ⁇ -1 ,4-glucosidase described above. Both solutions were incubated at 30°C under gentle shaking.

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Abstract

La présente invention concerne un procédé de production d'un composé d'intérêt, lequel procédé comprend la fermentation d'une première cellule capable de produire le composé d'intérêt dans un milieu de fermentation approprié sous des conditions qui permettent la production du composé d'intérêt, le milieu de fermentation comprenant des disaccharides et/ou des oligosaccharides qui ne sont pas entièrement fermentescibles par la première cellule ; et la fermentation étant réalisée en présence d'une enzyme qui est capable d'hydrolyse, au moins en partie, des disaccharides et/ou des oligosaccharides ; et, éventuellement, la récupération du composé d'intérêt du milieu réactionnel. L'invention concerne également un bouillon de fermentation pouvant être obtenu grâce à un tel procédé.
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