Method for producing fructose-6-phosphate from dihydroxyacetone phosphate and glyceraldehyde-3-phosphate

Abstract

A method for producing fructose 6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) is described, the method comprising the steps of: (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA); and (b) enzymatically converting the dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P) so produced into fructose 6-phosphate (F6P); or comprising the steps of: (a') enzymatically converting glyceraldehyde 3-phosphate (G3P) into glyceraldehyde; and (b') enzymatically converting the glyceraldehyde so produced together with dihydroxyacetone phosphate (DHAP) into fructose 1-phosphate (F1P); and (c') enzymatically converting the fructose 1-phosphate (F1P) so produced into fructose 6-phosphate (F6P).

Description

[0001] The present invention relates to a method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), the method comprising the steps of:

[0002] (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA); and

[0003] (b) enzymatically converting the thus produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P); or

[0004] The method comprises the steps of:

[0005] (a') enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde; and

[0006] (b') enzymatically converting the glyceraldehyde thus produced together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P); and

[0007] (c') enzymatically converting the fructose-1-phosphate (F1P) thus produced into fructose-6-phosphate (F6P).

[0008] For the past several decades, metabolic engineering practitioners have been committed to providing biological solutions for producing chemicals, therefore, providing alternatives to more traditional chemical methods. Generally speaking, biological solutions allow the use of renewable raw materials (such as sugar) and compete with methods based on existing petrochemicals. A multi-step biological solution for the production of chemicals generally includes microorganisms as catalysts that convert raw materials into target molecules. The full set of enzyme reactions for producing specific target molecules can be divided into those enzyme reactions belonging to central carbon pathways and those enzyme reactions belonging to product-specific pathways. The reactions belonging to central carbon pathways and product-specific pathways are associated, in that the redox constraints (generally NAD (P) H) and energetic constraints (generally ATP) of each and every enzyme reaction must be considered under the overall balance of competitiveness that contributes to the method. Historically, the central carbon pathways of heterotrophs that depend on sugars for growth have been described as the Embden-Meyerhoff-Parnas pathway (EMPP), the pentose phosphate pathway (PPP), the Entner-Doudoroff pathway (EDP), and the phosphoketolase pathway (PKP) (see Gottschalk (1986), Bacterial Metabolism, 2nd edition Springer-Verlag, New York). Each central pathway or combination of central pathways provides advantages and disadvantages relative to a specific target molecule. In order to provide competitive bioprocessing, recombinant microorganisms with modifications involving the EMPP, PPP, and EDP have been described (M. Emmerling et al., Metab. Eng. 1: 117 (1999); L. O. Ingram et al., Appl. Environ. Microbiol. 53: 2420 (1987); CT Trinh et al., Appl. Environ. Microbiol. 74: 3634 (2008)). Recently, recombinant microorganisms with modifications involving PKP have been described (see Sonderegger et al., Appl. Environ. Microbiol. 70 (2004), 2892-2897, U.S. Patent 7,253,001, Chinen et al., J. Biosci. Bioeng. 103 (2007), 262-269, U.S. Patent 7,785,858; Fleige et al., Appl. Microbiol. Cell Physiol. 91 (2011), 769-776).

[0009] EMPP converts 1 mol of glucose into 2 mol of pyruvate (PYR). When acetyl-CoA is needed, 1 mol of PYR can be converted into 1 mol of acetyl-CoA, while generating 1 mol of CO2 and 1 mol of NADH. The sum of the reactions is given in Equation 1.

[0010] Glucose + 2ADP + 2H3PO4 + 2CoA + 4NAD + →2Acetyl-CoA+2CO2+2ATP+2H2O+4NADH+4H + (Equation 1)

[0011] PPP provides the means for converting 1 mol of glucose into 1 mol of CO2 and 2 mol of NADPH, while producing 0.67 mol of fructose-6-phosphate (F6P) and 0.33 mol of glyceraldehyde-3-phosphate (GAP). The F6P and GAP formed in this way must be metabolized by other reaction pathways, such as by EMPP metabolism. EDP converts 1 mol of glucose into 1 mol of GAP and 1 mol of PYR, while producing 1 mol of NADPH. Same as PPP, the GAP formed in this way must be metabolized by other reaction pathways. PKP provides the means for converting 1 mol of glucose into 1 mol of GAP and 1.5 mol of acetyl phosphate (AcP). When acetyl-CoA is needed, 1 equivalent of AcP plus 1 equivalent of coenzyme A (CoA) can be converted into 1 equivalent of acetyl-CoA and 1 equivalent of inorganic phosphate (Pi) by the action of phosphotransacetylase.

[0012] For specific target molecules derived from the AcCoA moiety produced near redox neutrality with respect to the AcCoA moiety by means of PKP, there is a lack of overall energy balance. PKP (and, similarly, PPP and EDP) do not generate ATP for the conversion of glucose to glucose-6-phosphate. In the case of phosphoenolpyruvate (PEP)-dependent glucose uptake, PEP must be generated by other means (e.g., by means of EMPP). Recycling GAP by means of PKP exacerbates this situation, especially when product-specific pathways provide small amounts of ATP.

[0013] Sonderegger (see above citation) and U.S. Pat. No. 7,253,001 disclose recombinant Saccharomyces cerevisiae strains comprising native or overexpressed phosphoketolase activity, together with overexpressed phosphotransacetylase to increase the yield of glucose / xylose mixtures into ethanol. These strains are characterized by PEP-independent glucose uptake in which both EMPP and PPP can operate.

[0014] Chinen (see above citation) and U.S. Pat. No. 7,785,858 disclose recombinant bacteria selected from Enterobacteriaceae, Coryneform bacteria and Bacillus bacteria, the recombinant bacteria comprising increased phosphoketolase activity for converting glucose into target molecules produced by the intermediate acetyl-CoA, the target molecules comprising a group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, L-leucine, L-cysteine, succinate and polyhydroxybutyrate. These strains are characterized by EMPP-operable PEP-dependent glucose uptake. Notably, the activity of phosphofructokinase in the bacteria of U.S. Pat. No. 7,785,858 is reduced compared to wild-type or unmodified strains (see page 33).

[0015] WO 2013 / 007786 describes a recombinant microorganism with phosphoketolase activity and wherein inactivates or weakens EMPP and wherein inactivates or weakens the oxidation branch of PPP by eliminating or reducing phosphofructokinase. These measures result in an increase in the amount of fructose-6-phosphate (F6P) converted by phosphoketolase and transported to the non-oxidative branch of PPP. In this case, the glyceraldehyde-3-phosphate (G3P) produced from the non-oxidative branch of PPP is recycled to fructose-1,6-bisphosphate (FBP) by condensation with dihydroxyacetone phosphate (DHAP). The reaction is catalyzed by fructose-bisphosphate aldolase (EC 4.1.2.13). Subsequently, fructose-1,6-bisphosphate (FBP) is converted into fructose-6-phosphate (F6P) by the action of fructose bisphosphatase (EC3.1.3.11).

[0016] Fructose bisphosphatase (EC 3.1.3.11) is highly regulated (see, e.g., G.A. Tejwani, Advances in Enzymology and Related Areas of Molecular Biology 54: 121-194 (1983)). To avoid ineffective cycling during glycolysis, fructose bisphosphatase activity is downregulated in the presence of glucose. Allosteric inhibition of E. coli type I fructose bisphosphatase, required for growth on neoglucogenic substrates, is mediated by glucose-6-phosphate, the first metabolite generated when glucose is transported into the cell (and is also part of the sucrose import and metabolic pathway in E. coli), and AMP (J. Hines et al., J. Biol. Chem. 282: 24697-24706 (2007)). This enzyme is essentially inactive if glucose or sucrose is present in the culture medium.

[0017] Since glucose or sucrose is often used as a carbon source and also used at a relatively high concentration in a fermentation process using a microorganism, such fermentation conditions may hinder the efficiency of conversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) to fructose-6-phosphate (F6P). Therefore, it is necessary to develop a pathway that allows conversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) to fructose-6-phosphate (F6P) even under conditions of high glucose concentration or sucrose concentration in a culture medium or a reaction vessel.

[0018] The present invention meets this need by providing a method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), the method comprising the steps of:

[0019] (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA); and

[0020] (b) enzymatically converting the dihydroxyacetone (DHA) thus produced together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P);

[0021] Or include the steps:

[0022] (a') enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde; and

[0023] (b') enzymatically converting the glyceraldehyde thus produced together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P); and

[0024] (c') and enzymatically converting the fructose-1-phosphate (F1P) thus produced into fructose-6-phosphate (F6P).

[0025] Dihydroxyacetone phosphate (DHAP) is first converted into dihydroxyacetone (DHA) and its subsequent condensation with GAP to produce F6P, which provides an alternative route for the condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) to fructose-1,6-bisphosphate (FBP) and its subsequent conversion to fructose-6-phosphate (F6P), with the advantage that it can be achieved by utilizing enzymes that are not regulated (especially inhibited) by glucose or sucrose. Therefore, this conversion can be performed even at high concentrations of glucose or sucrose. For the conversion of DHAP and G3P as described in steps (a') to (c'), this also applies.

[0026] Of course, the two alternative methods for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) as described above can also be applied in combination (in vitro or in vivo).

[0027] Thus, in a first aspect, the present invention relates to a method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), the method comprising the steps of:

[0028] (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA); and

[0029] (b) enzymatically converting the thus produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P).

[0030] The enzymatic conversion of dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) according to step (a) of the method of the invention can be achieved, for example, by using enzymes classified as EC 3.1.3.-. These enzymes are also called phosphorus monoester hydrolases (or phosphorus monoesterases). Phosphorus monoester hydrolases are enzymes that catalyze the hydrolysis of OP bonds by nucleophilic attack on phosphorus by cysteine ​​residues or coordinated metal ions.

[0031] In a preferred embodiment, the enzyme classified as EC 3.1.3.- is selected from:

[0032] - Sugar phosphatase (EC 3.1.3.23);

[0033] -6-phosphogluconate phosphatase (EC 3.1.3.-);

[0034] - pyridoxal phosphate phosphatase (EC 3.1.3.74);

[0035] - fructose-1-phosphate phosphatase (EC 3.1.3.-);

[0036] - dihydroxyacetone phosphatase (EC 3.1.3.-);

[0037] - Hexitol phosphatase (EC 3.1.3.-)

[0038] - Acid phosphatase (EC 3.1.3.2);

[0039] - alkaline phosphatase (EC 3.1.3.1);

[0040] - glycerol-1-phosphate phosphatase (EC 3.1.3.21); and

[0041] -3-Phosphoglycerate phosphatase (EC 3.1.3.38).

[0042] Thus, in one embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing sugar phosphatases (EC 3.1.3.23). Sugar phosphatases (EC 3.1.3.23) are enzymes that catalyze the following reaction:

[0043] Sugar phosphate + H2O → sugar + phosphate

[0044] This enzyme occurs in a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, protozoa, and bacteria. The enzyme has been described, for example, in Arabidopsis thaliana (UniProt Accession No. Q9ZVJ5), Plasmodium falciparum (UniProt Accession No. Q8IJ74), Streptococcus equinus, Streptococcus pyogenes, Saccharomyces cerevisia, Neisseria meningitidis, Lactococcus lactis, Klebsiella aerogenes, Escherichia coli (UniProt Accession No. P75792), Escherichia acidilactici, Enterococcus faecalis and Bacillus subtilis.

[0045] In principle, any sugar phosphatase of EC 3.1.3.23 can be used in the method of the present invention, as long as it has the ability to convert dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA). In a preferred embodiment, an enzyme from a bacterium of the genus Escherichia is used, more preferably an enzyme from an Escherichia coli species. Even more preferably, the YbiV protein or the YidA protein (UniProt accession number P75792 (SEQ ID NO: 1; encoded by the nucleotide sequence of SEQ ID NO: 39) and P0A8Y5 (SEQ ID NO: 2; encoded by the nucleotide sequence of SEQ ID NO: 41) from Escherichia coli are used.

[0046] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 1 or 2, or an amino acid sequence showing at least x% homology to SEQ ID NO: 1 or 2 and has sugar phosphatase (EC 3.1.3.23) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0047] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO: 1 or 2.

[0048] For determining sequence identity as described in this application, the following should generally apply: when the compared sequences do not have the same length, the degree of identity refers to the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence that are identical to the amino acid residues in the shorter sequence. Preferably, it refers to the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence.

[0049] The degree of sequence identity can be determined by performing pairwise alignments, preferably using algorithms and software well known in the art, such as the Needleman-Wunsch algorithm in conjunction with EMBOSSNEEDLE software.

[0050] When using this method to determine whether a particular sequence is, for example, at least 60% identical to a reference sequence, the default settings of the EMBOSS NEEDLE software can be used, which are defined as follows:

[0051] -Matrix: BLOSUM62

[0052] - Opening places: 10

[0053] - Vacancy extension: 0.5

[0054] -No end-gap penalty.

[0055] Preferably, the degree of identity is calculated over the entire length of the aligned sequences.

[0056] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing 6-phosphogluconate phosphatase (EC 3.1.3.-). 6-Phosphogluconate phosphatase is an enzyme that catalyzes the dephosphorylation of 6-phosphogluconate.

[0057] Such enzymes have been described, for example, for E. coli. Therefore, in a preferred embodiment, the corresponding enzyme from E. coli is used in the method according to the invention, more preferably the YieH protein (UniProt accession number P31467 (SEQ ID NO: 3; encoded by the nucleotide sequence of SEQ ID NO: 40)).

[0058] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence showing at least x% homology to SEQ ID NO:3 and has 6-phosphogluconate phosphatase activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0059] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:3.

[0060] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing pyridoxal phosphate phosphatase (EC 3.1.3.74). Pyridoxal phosphate phosphatase is an enzyme that catalyzes the following reaction:

[0061] Pyridoxal 5′-phosphate + H2O → pyridoxal + phosphate

[0062] This enzyme occurs in a variety of organisms, including eukaryotic and prokaryotic organisms, such as animals and bacteria. For example, the enzyme has been described in the following organisms: Homo sapiens, Rattus norvegicus, Brachylagus idahoensis, Bos taurus, Canis lupus, Feliscatus, Gallus gallus, Meriones unguiculatus, Mus musculus, Paenibacillus thiaminolyticus, Sinorhizobium meliloti, Sus scorfa, and Escherichia coli (UniProt Accession No. P27848). In principle, any pyridoxal phosphate phosphatase of EC 3.1.3.74 can be used in the method of the present invention, as long as it has the ability to convert dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA). In a preferred embodiment, an enzyme from a bacterium of the genus Escherichia is used, more preferably an enzyme from an Escherichia coli species. Even more preferably, the YigL protein from Escherichia coli (UniProt accession number P27848 (SEQ ID NO: 4; encoded by the nucleotide sequence of SEQ ID NO: 42)) is used.

[0063] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence showing at least x% homology to SEQ ID NO:4 and has pyridoxal phosphate phosphatase (EC 3.1.3.74) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0064] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:4.

[0065] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing fructose-1-phosphate phosphatase (EC 3.1.3.-). Fructose-1-phosphate phosphatase is an enzyme that catalyzes the dephosphorylation of fructose-1-phosphate.

[0066] Such enzymes have been described, for example, for E. coli. Therefore, in a preferred embodiment, the corresponding enzyme from E. coli is used in the process according to the invention, more preferably the YqaB protein (UniProt accession number P77475 (SEQ ID NO: 5; encoded by the nucleotide sequence of SEQ ID NO: 43)).

[0067] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence showing at least x% homology to SEQ ID NO:5 and has fructose-1-phosphate phosphatase (EC 3.1.3.-) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0068] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:5.

[0069] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing dihydroxyacetone phosphatase (EC 3.1.3.-).Dihydroxyacetone phosphatase is an enzyme that catalyzes the dephosphorylation of dihydroxyacetone phosphate (DHAP) to produce DHA.

[0070] For example, this enzyme has been described for Corynebacterium glutamicum. Therefore, in a preferred embodiment, the method of the present invention uses the corresponding enzyme from Corynebacterium glutamicum, more preferably the HdpA protein (UniProt accession number A4QFW4 (SEQ ID NO:6; encoded by the nucleotide sequence of SEQ ID NO:48)).

[0071] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence showing at least x% homology to SEQ ID NO: 6 and has dihydroxyacetone phosphatase (EC 3.1.3.-) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0072] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:6.

[0073] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing hexitol phosphatases (EC 3.1.3.-). Hexitol phosphatases are enzymes that catalyze the dephosphorylation of D-mannitol 1-phosphate and D-sorbitol 6-phosphate.

[0074] Such an enzyme has been described, for example, for E. coli. Therefore, in a preferred embodiment, the corresponding enzyme from E. coli is used in the method according to the invention, more preferably the HxpA protein (UniProt accession number P77625 (SEQ ID NO: 7; encoded by the nucleotide sequence of SEQ ID NO: 44)).

[0075] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence showing at least x% homology to SEQ ID NO:7 and has hexitol phosphatase (EC 3.1.3.-) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0076] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:7.

[0077] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing acid phosphatase (EC 3.1.3.2). Acid phosphatase is an enzyme that catalyzes the following reaction:

[0078] Phosphate monoester + H2O → alcohol + phosphate

[0079] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any acid phosphatase (EC 3.1.3.2) can be used in the method of the invention, as long as it can convert dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA).

[0080] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing alkaline phosphatase (EC 3.1.3.1). Similar to acid phosphatase, alkaline phosphatase is an enzyme that catalyzes the following reaction:

[0081] Phosphate monoester + H2O → alcohol + phosphate

[0082] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any alkaline phosphatase (EC 3.1.3.1) can be used in the method of the present invention, as long as it can convert dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA).

[0083] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing glycerol-1-phosphate phosphatase (EC 3.1.3.21). Glycerol-1-phosphate phosphatase naturally catalyzes the following reaction:

[0084] Glycerol 1-phosphate + H2O → Glycerol + phosphate

[0085] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any glycerol-1-phosphate phosphatase (EC 3.1.3.21) can be used in the method of the present invention, as long as it can convert dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA).

[0086] In another embodiment, the conversion of dihydroxyacetone phosphate (DHAP) to dihydroxyacetone (DHA) is achieved by utilizing 3-phosphoglycerate phosphatase (EC 3.1.3.38). 3-phosphoglycerate phosphatase naturally catalyzes the following reaction:

[0087] D-glycerate 3-phosphate + H2O → D-glycerate + phosphate

[0088] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as plants, fungi and bacteria. In principle, 3-phosphoglycerate phosphatase (EC 3.1.3.38) can be used in the method of the present invention, as long as it can convert dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA).

[0089] As mentioned above, the dihydroxyacetone (DHA) obtained in step (a) of the method of the present invention can then be further converted into fructose-6-phosphate (F6P) together with glyceraldehyde-3-phosphate (G3P) as described in step (b) of the method. In the case of an in vitro reaction, glyceraldehyde-3-phosphate (G3P) can be simply added to the reaction. In the case of an in vivo reaction, glyceraldehyde-3-phosphate (G3P) is provided by other metabolic pathways. Since glyceraldehyde-3-phosphate (G3P) is an intermediate in glycolysis and the Entner-Douderoff pathway, it occurs in almost all organisms. In addition, it is an isomer of dihydroxyacetone phosphate (DHAP) and can be produced from dihydroxyacetone phosphate (DHAP) by the action of triose phosphate isomerase.

[0090] In one embodiment, the conversion of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) to fructose-6-phosphate (F6P) according to step (b) of the method of the present invention can be achieved using enzymes called aldehyde lyases (sometimes also called carbon-carbon lyases). These enzymes are classified in EC 4.1.2.-. This type of enzyme catalyzes the cleavage of CC bonds in molecules with carbonyl and hydroxyl groups to form two molecules, an aldehyde and a ketone, respectively. However, it has been found that these enzymes can also catalyze the condensation of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone (DHA) to fructose-6-phosphate (F6P).

[0091] In a preferred embodiment, the aldehyde lyase used in the process of the invention to convert dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) is a fructose-6-phosphate aldolase, for example, fructose-6-phosphate aldolase 1. An example and preferred embodiment is fructose-6-phosphate aldolase 1 from Escherichia coli encoded by the gene fsaA. The amino acid sequence of this protein is available, for example, according to UniProt accession number P78055 (SEQ ID NO: 8; encoded by the nucleotide sequence of SEQ ID NO: 34).

[0092] In another preferred embodiment, the aldehyde lyase used in the process of the present invention to convert dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) is fructose-6-phosphate aldolase 2. Fructose-6-phosphate aldolase 2 from Escherichia coli encoded by the gene fsaB is an example and a preferred embodiment. The amino acid sequence of this protein is available, for example, according to UniProt accession number P32669 (SEQ ID NO: 16; encoded by the nucleotide sequence of SEQ ID NO: 36).

[0093] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 8, 9 or 16, or an amino acid sequence showing at least x% homology to SEQ ID NO: 8, 9 or 16 and has fructose-6-phosphate aldolase activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) as described above.

[0094] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO: 8, 9 or 16. The amino acid sequence of SEQ ID NO: 9 (encoded by the nucleotide sequence of SEQ ID NO: 35) is a mutant form of the sequence of SEQ ID NO: 8 having higher enzyme activity.

[0095] In another embodiment, the conversion of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) to fructose-6-phosphate (F6P) according to step (b) of the method of the present invention can be achieved using enzymes called transaldolases. These enzymes are classified in EC 2.2.1.2. This class of enzymes catalyzes the following reaction:

[0096]

[0097] This enzyme occurs in a wide variety of organisms, both eukaryotic and prokaryotic, such as plants, algae, animals, fungi, and bacteria. The enzyme has been described, for example, in the following organisms: Acidithiobacillus ferrooxidans, Arthrobacter sp., Bifidobacterium bifidum, Blastobotrys adeninivorans, Bostaurus, Carcinus maenas, Chlorella sp., Chlorobium vibriforme f. thiosulfatophilum, Chromatium sp., Clostridium acetobutylicum, Cryptococcus neoformans, Cyberlindnera jadinii, Escherichia coli, Euglena sp., Francisella tularensis, tularensis) (UniProt accession number Q5NFX0), Fusarium oxysporum, Gluconobacter oxydans (UniProt accession number Q76EM7), Homo sapiens, Methanocaldococcus jannaschii, Moniiella megachiliensis, Mus musculus, Musca domestica, Oryctolagus cuniculus, Rattus norvegicus, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Scheffersomyces stipitis, Solanum lycopersicum, Spinacia ileracea, Tetranychus telarius, Thermoplasma acidophilus acidophilum), Thermotogamaritima (UniProt Accession No. Q9WYD1), Streptococcus pyogenes (UniProt Accession No. Q99XT4; SEQ ID NO: 12 (encoded by the nucleotide sequence of SEQ ID NO: 50)), Clostridium beijerinckii (UniProt Accession No. A0A0B5QQ90; SEQ ID NO: 13 (encoded by the nucleotide sequence of SEQ ID NO: 53)), Caulobacter vibrioides (UniProt Accession No. Q9A2F1; SEQ ID NO: 14 (encoded by the nucleotide sequence of SEQ ID NO: 54)), Streptococcus mutans (UniProt Accession No. Q8DVJ4; SEQ ID NO: 15 (encoded by the nucleotide sequence of SEQ ID NO: 56)), Escherichia coli (UniProt Accession No. P0A870; SEQ ID NO: 17 (encoded by the nucleotide sequence of SEQ ID NO: NO:37), Enterococcus faecalis (UniProt Accession No. A0A0M2AGL1; SEQ ID NO:18 (encoded by the nucleotide sequence of SEQ ID NO:52)), Streptococcus suis (UniProt Accession No. A0A0E4C393; SEQ ID NO:19 (encoded by the nucleotide sequence of SEQ ID NO:55)), Streptococcus pneumoniae (UniProt Accession No. A0A0D6J3Z8; SEQ ID NO:20 (encoded by the nucleotide sequence of SEQ ID NO:58)), Streptococcus gordonii (UniProt Accession No. A8AZ46; SEQ ID NO:10 (encoded by the nucleotide sequence of SEQ ID NO:51)), Streptococcus agalactiae (UniProt Accession No. Q8E738; SEQ ID NO:31 (encoded by the nucleotide sequence of SEQ ID NO:57)) and Listeria monocytogenes (UniProt Accession No. A0A0H3GHX1; SEQ ID NO:11 (encoded by the nucleotide sequence of SEQ ID NO:49)).

[0098] In principle, any transaldolase of EC 2.2.1.2 can be used in the method of the present invention, as long as it has the ability to convert dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P). In a preferred embodiment, an enzyme from a bacterium of the genus Streptococcus or Listeria is used, more preferably an enzyme of a species of Streptococcus gordonii or a species of Listeria monocytogenes is used.

[0099] In a preferred embodiment, the protein from Streptococcus gordonii encoded by the S. gordonii SGO_1787 gene (UniProt accession number A8AZ46) is used.

[0100] In a preferred embodiment, the enzyme has an amino acid sequence as shown in any one of SEQ ID NOs: 10 to 15, SEQ ID NOs: 17 to 20, SEQ ID NO: 64 and SEQ ID NO: 31, or an amino acid sequence showing at least x% homology to any one of SEQ ID NOs: 10 to 20, SEQ ID NO: 64 and SEQ ID NO: 31 and has transaldolase (EC 2.2.1.2) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) as described above. Particularly preferred is the enzyme from Streptococcus suis (UniProt Accession No. A0A0E4C393; SEQ ID NO: 19 (encoded by the nucleotide sequence of SEQ ID NO: 55)).

[0101] The amino acid sequence of SEQ ID NO: 64 (encoded by the nucleotide sequence of SEQ ID NO: 38) is a mutant form (talB F178Y) of the sequence of SEQ ID NO: 17 having higher enzyme activity.

[0102] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO: 10 or 11.

[0103] In another aspect, the present invention relates to a method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), the method comprising the steps of:

[0104] (a') enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde; and

[0105] (b') enzymatically converting the glyceraldehyde thus produced together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P); and

[0106] (c') enzymatically converting the fructose-1-phosphate (F1P) thus produced into fructose-6-phosphate (F6P).

[0107] According to step (a') of the method of the present invention, the enzymatic conversion of glyceraldehyde-3-phosphate (G3P) into glyceraldehyde is a dephosphorylation reaction according to the following scheme:

[0108] D-Glyceraldehyde-3-phosphate + H2O → D-Glyceraldehyde + H3PO4

[0109] This hydrolytic cleavage of the phosphate group is an irreversible reaction. This reaction can be achieved, for example, by using enzymes classified as EC 3.1.3.-. These enzymes are also referred to as phosphorus monoester hydrolases (or phosphorus monoesterases). Phosphorus monoester hydrolases are enzymes that catalyze the hydrolysis of OP bonds by nucleophilic attacks on phosphorus by cysteine ​​residues or coordinated metal ions.

[0110] In a preferred embodiment, the enzyme classified as EC 3.1.3.- is selected from:

[0111] - Glyceraldehyde 3-phosphate phosphatase (EC 3.1.3.-);

[0112] - alkaline phosphatase (EC 3.1.3.1);

[0113] - Acid phosphatase (EC 3.1.3.2);

[0114] - sugar phosphatase (EC 3.1.2.23); and

[0115] - Hexitol phosphatase (EC 3.1.3.-)

[0116] Thus, in one embodiment, the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde is achieved by utilizing glyceraldehyde 3-phosphate phosphatases (EC 3.1.3.-).These enzymes catalyze the dephosphorylation of glyceraldehyde-3-phosphate.

[0117] This activity has been described, for example, for the protein encoded by the gene of Pyrococcus horikoshii PH1655 or the protein encoded by the gene of Methanocaldococcus iannaschii MJ1437. Therefore, in a preferred embodiment, the corresponding protein from Pyrococcus horikoshii (UniProt accession number O59346 (SEQ ID NO: 21; encoded by the nucleotide sequence of SEQ ID NO: 59)) or from Methanocaldococcus jannaschii (UniProt accession number Q58832 (SEQ ID NO: 22; encoded by the nucleotide sequence of SEQ ID NO: 60)) is used.

[0118] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 21 or as shown in SEQ ID NO: 22, or an amino acid sequence showing at least x% homology to SEQ ID NO: 21 or SEQ ID NO: 22 and has glyceraldehyde 3-phosphate phosphatase (EC 3.1.3.-) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde as described above.

[0119] In another embodiment, the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde is achieved by utilizing alkaline phosphatase (EC 3.1.3.1). Alkaline phosphatase is an enzyme that catalyzes the following reaction:

[0120] Phosphate monoester + H2O → alcohol + phosphate

[0121] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any alkaline phosphatase (EC 3.1.3.1) can be used in the method of the invention, as long as it can convert glyceraldehyde-3-phosphate (G3P) into glyceraldehyde.

[0122] In another embodiment, the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde is achieved by utilizing acid phosphatase (EC 3.1.3.2). Similar to alkaline phosphatase, acid phosphatase is an enzyme that catalyzes the following reaction:

[0123] Phosphate monoester + H2O → alcohol + phosphate

[0124] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any acid phosphatase (EC 3.1.3.2) can be used in the method of the invention, as long as it can convert glyceraldehyde-3-phosphate (G3P) into glyceraldehyde.

[0125] In yet another preferred embodiment, the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde is achieved by utilizing sugar phosphatases (EC 3.1.3.23). Sugar phosphatases (EC 3.1.3.23) are enzymes that catalyze the following reaction:

[0126] Sugar phosphate + H2O → sugar + phosphate

[0127] This enzyme occurs in a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, protozoa, and bacteria. For example, the enzyme has been described in the following organisms: Arabidopsis thaliana (UniProt Accession No. Q9ZVJ5), Plasmodium falciparum (UniProt Accession No. Q8IJ74), Streptococcus equinus, Streptococcus pyogenes, Saccharomyces cerevisia, Neisseria meningitidis, Lactococcus lactis, Klebsiella aerogenes, Escherichia coli (UniProt Accession No. P75792 (SEQ ID NO: 1; encoded by the nucleotide sequence of SEQ ID NO: 39)), Escherichia acidilactici, Enterococcus faecalis and Bacillus subtilis.

[0128] In principle, any sugar phosphatase of EC 3.1.3.23 can be used in the method of the present invention, as long as it has the ability to convert glyceraldehyde-3-phosphate (G3P) into glyceraldehyde. In a preferred embodiment, an enzyme from a bacterium of the genus Escherichia is used, more preferably an enzyme from an Escherichia coli species. Even more preferably, the YbiV protein from Escherichia coli (UniProt accession number P75792 (SEQ ID NO: 1; encoded by the nucleotide sequence of SEQ ID NO: 39)) is used.

[0129] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence showing at least x% homology to SEQ ID NO: 1 and has sugar phosphatase (EC 3.1.3.23) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) as described above.

[0130] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:1.

[0131] In another embodiment, the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde is achieved by utilizing hexitol phosphatases (EC 3.1.3.-). Hexitol phosphatases are enzymes that catalyze the dephosphorylation of D-mannitol 1-phosphate and D-sorbitol 6-phosphate.

[0132] Such enzymes have been described, for example, for E. coli. Thus, in a preferred embodiment, the corresponding enzyme from E. coli is used in the process according to the invention, more preferably the HxpB protein (UniProt accession number P77247 (SEQ ID NO: 23; encoded by the nucleotide sequence of SEQ ID NO: 45)). In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence showing at least x% homology to SEQ ID NO: 23 and has hexitol phosphatase (EC 3.1.3.-) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde as described above.

[0133] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:23.

[0134] As mentioned above, the glyceraldehyde obtained in step (a') of the method of the present invention can then be further converted into fructose-6-phosphate (F1P) together with dihydroxyacetone phosphate (DHAP) as described in step (b') of the method. In the case of an in vitro reaction, dihydroxyacetone phosphate (DHAP) can be simply added to the reaction. In the case of an in vivo reaction, dihydroxyacetone phosphate (DHAP) is provided by other metabolic pathways. Since dihydroxyacetone phosphate (DHAP) is an intermediate in glycolysis and the Entner-Douderoff pathway, it occurs in almost all organisms. In addition, it is an isomer of glyceraldehyde-3-phosphate (G3P) and can be produced from glyceraldehyde-3-phosphate (G3P) by the action of triosephosphate isomerase.

[0135] The conversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde into fructose-1-phosphate (F1P) according to step (b') of the process of the present invention is an aldol condensation process and proceeds according to the following reaction:

[0136]

[0137] This conversion can be achieved, for example, by utilizing fructose bisphosphate aldolase (EC 4.1.2.13).

[0138] Fructose-bisphosphate aldolase (EC 4.1.2.13) is an enzyme that catalyzes the following reaction:

[0139]

[0140] The enzyme has been identified in a variety of organisms, and fructose-1,6-bisphosphate aldolase is divided into two classes that rely on different reaction mechanisms. Class I fructose-1,6-bisphosphate aldolase is mainly found in animals and higher plants, while class II fructose-1,6-bisphosphate aldolase is mainly found in algae, bacteria and yeast. Enzymes belonging to class II require divalent metal ions as cofactors.

[0141] Type I and type II fructose-1,6-bisphosphate aldolases have been isolated from different prokaryotic and eukaryotic sources and are therefore ubiquitous glycolytic enzymes that play key roles in glycolysis, gluconeogenesis, and fructose metabolism (Brovetto M. et al. Chem. Rev. 111 (2011), 4346-4403).

[0142] Thus, in a preferred embodiment, the fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is derived from a prokaryotic organism, preferably a bacterium. For example, the enzyme has been described in the following organisms: Peptoniphilus asaccharolyticus, Escherichia coli, Thermus aquaticus, Mycobacterium tuberculosis, Aspergillus oryzae, Bacillus cereus, Bacillus subtilis, Clostridium sp., Corynebacterium sp., Heliobacter pylori, Lactobacillus sp., Mycobacterium sp., Penicillinum sp., Pseudomonas sp., Plasmodium falciparum, Saccharomyces sp. sp.) and Methylococcus cuniculus.

[0143] In addition, in a preferred embodiment, the fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) is derived from a eukaryotic organism. For example, the enzyme has been described to occur in the following organisms: Homo sapiens, Drosophilamelanogaster, Oryctolagus cuniculus, Gallus gallus, Zea mays, Bos taurus, Mus musculus and Medicago sativa.

[0144] The study of Siebers et al. revealed for the first time that genes encoding classical class I and class II enzymes had not yet been identified in any sequenced archaeal genome (Siebers B. et al., J Bol. Chem. 276 (2001), 28710-28718). Later, biochemical and structural characterization of aldolases from two extremely thermophilic archaea, Thermoproteus tenax and Pyrococcus furiosus, showed that these enzymes utilize a Schiff base mechanism and therefore belong to class I aldolases (Siebers et al., loc. cit.; Lorentzen E. et al., Biochem. Soc. Trans. 32 (2004), 259-263).

[0145] Class I fructose-1,6-bisphosphate aldolases can be classified into three isozyme forms that can be distinguished based on immunoreactivity and turnover relative to fructose-1,6-bisphosphate substrates and fructose 1-phosphate substrates (Blonski et al., Biochem. J. 323 (1997), 71-77). Isozyme A from rabbit muscle has been the most well-studied class I fructose-1,6-bisphosphate aldolase (Gefflaut et al., Prog. Biophys. Mol. Biol. 63 (1995), 301-340). Dozens of different isozymes have been sequenced and the structures of several aldolase isozymes have been determined, including those from rabbit muscle (Sygusch et al., Proc. Natl. Acad. Sci. 84 (1987), 7846-7850), human muscle (Gamblin et al., FEBS Lett. 262 (1987), 282-286; Arakaki et al., Protein Sci. 13 (2004), 3077-3084) and Drosophila (Hester et al., FEBS Lett. 292 (1991), 237-242). The molecular architecture of these isozymes is highly conserved, except for the 20 amino acid residues occupying the C-terminal region. The polypeptide fold of each enzyme subunit of the homotetramer corresponds to that of a β-barrel, with the active site located in the center of the β-barrel (Sygusch et al., Proc. Natl. Acad. Sci. 84 (1987), 7846-7850). Unlike other β-barrel isozymes, the active site consists of a large number of charged amino acid residues, namely Asp-33, Lys-107, Lys-146, Glu-187 and Lys-229 (Blonski et al., Biochem. J. 323 (1997), 71-77).

[0146] Class II FBP-aldolases require divalent cations, usually Zn2+ , and are activated by monovalent cations (Horecker et al., In The Enzymes (Boyer, PD ed.), 1972, 3rd edition, Vol. 7, 213-258, Academic Press, New York). They share about 15% sequence identity with class I enzymes (Naismith et al., J. Mol. Biol. 225 (1992), 1137-1141). In a preferred embodiment, the fructose-1,6-bisphosphate aldolase used in the method of the present invention is activated by divalent cations, preferably Zn 2+ Provided when present and activated by monovalent cations.

[0147] Class II FBPases can be further divided into Class IIA and Class IIB families. Traditionally, Class IIA and Class IIB FBPases are classified according to sequence homology and their oligomeric states. Class IIA FBPases are considered dimers, while Class IIB FBPases can be dimers, tetramers, or octamers. (Izard and Sygush, J. Biol. Chem 279 (2004), 11825-11833; Galkin et al., Biochemistry 48 (2009), 3186-3196; Nakahara et al., Plant Cell Physiol. 44 (2003), 326-333). Comparison of the sequences of each FBP-protein showed that the members belonging to each family showed 40% sequence similarity, and the amino acid sequence identity between type A and type B class II FBP aldolases was of the order of 25-30% (Plaumann et al., Curr. Genet. 31 (1997), 430-438). Subsequent sequence comparisons of eight known class II FBP aldolases showed that Arg-331 was one of the highly conserved residues. Chemical modification and site-directed mutagenesis have confirmed the crucial role of this amino acid in the active site (Qamar et al., Protein Sci. 5 (1996), 154-161).

[0148] The crystal structures of several enzymes have been determined, namely those from Escherichia coli (Hall et al., J. Mol. Biol. 287 (1999), 383-394), Thermus aquaticus (Izard and Sygush; loc. cit.), Thermus caldophilus (Lee et al., Biochem. Biophys. Res. Commun. 347 (2006), 616-625), Giardia lamblia (Galkin et al.; loc. cit.), Mycobacterium tuberculosis (Pegan et al., J. Mol. Biol. 386 (2009), 1038-1053). The secondary structure of the M. tuberculosis FBP aldolase is similar to other bacterial class II aldolases (Pegan et al., loc. cit.). The enzyme has an eight-stranded β-sheet core, in which each β-strand (β1-β8) is typically followed by an α-helix (α1-α8a), resulting in an overall (β / α) 8-barrel fold, also known as the TIM barrel fold (reference number IPR013785 in the InterPro database).

[0149] In principle, any fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) can be used for converting D-erythrose into glycolaldehyde according to the method of the invention.

[0150] In a preferred embodiment, the fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) used in the method of the present invention is a fructose-1,6-bisphosphate aldolase (i.e., a class II fructose-bisphosphate aldolase) (Uniprot P0AB71) from Escherichia coli (strain K12) showing an amino acid sequence as set forth in SEQ ID NO: 24 (encoded by the nucleotide sequence of SEQ ID NO: 46 of the gene fbaA); or a fructose-1,6-bisphosphate aldolase (i.e., a class I fructose-bisphosphate aldolase) (Uniprot P0A991) from Escherichia coli (strain K12) showing an amino acid sequence as set forth in SEQ ID NO: 25 (encoded by the nucleotide sequence of SEQ ID NO: 47 of the gene fbaB); or a fructose-1,6-bisphosphate aldolase B (Uniprot P05062) from Homo sapiens showing an amino acid sequence as set forth in SEQ ID NO: 26. or fructose-1,6-bisphosphate aldolase A from Homo sapiens (Uniprot P04075), which shows the amino acid sequence set forth in SEQ ID NO:27, or fructose-1,6-bisphosphate aldolase C from Homo sapiens (Uniprot P09972), which shows the amino acid sequence set forth in SEQ ID NO:28.

[0151] Thus, in a preferred embodiment, the fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) used in the method of the invention has an amino acid sequence as shown in any one of SEQ ID NOs: 24 to 28 or an amino acid sequence showing at least x% homology to any one of SEQ ID NOs: 24 to 28 and has fructose-1,6-bisphosphate aldolase activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting D-erythrose into glycolaldehyde as described above. Preferably, the degree of identity is determined as described above.

[0152] The enzyme activity of fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) can be assessed by methods known to those skilled in the art. Such methods are described, for example, in Blonski K. et al., Biochem. J. 323 (1997), 71-77 and Szwergold et al., Arch. Biochem. Biophys. 317 (1995), 244-252.

[0153] As mentioned above, the fructose-1-phosphate (F1P) obtained in step (b') of the method of the present invention can then be converted into fructose-6-phosphate (F6P) in one step by enzyme promotion (see step (c')). This conversion is an isomerization process and proceeds according to the following reaction:

[0154]

[0155] The conversion can be achieved, for example, by using phosphoglucomutase (EC 5.4.2.2), phosphomannose mutase (EC 5.4.2.8) or β-phosphoglucomutase (EC 5.4.2.6).

[0156] Thus, in one embodiment, the conversion of fructose-1-phosphate (F1P) to fructose-6-phosphate (F6P) according to step (c') of the method of the invention is achieved by utilizing phosphoglucomutase (EC 5.4.2.2). Phosphoglucomutase is an enzyme that naturally catalyzes the following reaction:

[0157]

[0158] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any phosphoglucomutase (EC 5.4.2.2) can be used in the method of the present invention, as long as it can convert fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P). In a preferred embodiment, the enzyme is an enzyme encoded by the pgm gene of Aeromonas hydrophila, preferably Aeromonas hydrophila subsp. hydrophila, such as a protein having an amino acid sequence as shown in UniProt accession number A0KIH4 (SEQ ID NO: 29; encoded by the nucleotide sequence of SEQ ID NO: 62 of gene pgm).

[0159] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence showing at least x% homology to SEQ ID NO: 29 and has phosphoglucomutase (EC 5.4.2.2) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) as described above.

[0160] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:29.

[0161] In another embodiment, the conversion of fructose-1-phosphate (F1P) to fructose-6-phosphate (F6P) according to step (c') of the method of the present invention is achieved by utilizing phosphomannose mutase (also known as phosphoglucomutase) (EC 5.4.2.8). Phosphomannose mutase (EC 5.4.2.8) has been reported to naturally catalyze the following reaction:

[0162]

[0163] and

[0164]

[0165] This enzyme occurs in a large variety of organisms, including eukaryotic and prokaryotic organisms, such as animals, plants, fungi and bacteria. In principle, any phosphomannose mutase (EC 5.4.2.8) can be used in the method of the present invention, as long as it can convert fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P). In a preferred embodiment, the enzyme is an enzyme encoded by the AHA_2903 gene of Aeromonas hydrophila, preferably Aeromonas hydrophila subsp. hydrophila, such as a protein having an amino acid sequence as shown in UniProt accession number A0KMA6 (SEQ ID NO: 30; encoded by the nucleotide sequence of SEQ ID NO: 63 of gene AHA 2903).

[0166] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO:30 or an amino acid sequence showing at least x% homology to SEQ ID NO:30 and has phosphomannose mutase (EC 5.4.2.8) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) as described above.

[0167] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:30.

[0168] In another embodiment, the conversion of fructose-1-phosphate (F1P) to fructose-6-phosphate (F6P) according to step (c') of the method of the invention is achieved by utilizing β-phosphoglucomutase (also known as phosphomannose mutase) (EC 5.4.2.6). β-phosphoglucomutase (EC 5.4.2.6) has been reported to naturally catalyze the following reaction:

[0169]

[0170] This enzyme occurs in a large variety of organisms, including prokaryotes, such as bacteria. In principle, any β-phosphoglucomutase (EC 5.4.2.6) can be used in the method of the present invention, as long as it can convert fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P). In a preferred embodiment, the enzyme is an enzyme encoded by the pgm gene of Escherichia coli (strain K12), such as a protein having an amino acid sequence as shown in UniProt accession number P36938 (SEQ ID NO: 32 encoded by the gene called pgm); or an enzyme encoded by the ycjU gene of Escherichia coli (strain K12), such as a protein having an amino acid sequence as shown in UniProt accession number P77366 (SEQ ID NO: 33 encoded by the gene called YcjU).

[0171] In a preferred embodiment, the enzyme has an amino acid sequence as shown in SEQ ID NO:32 or SEQ ID NO:33, or an amino acid sequence showing at least x% homology to SEQ ID NO:32 or SEQ ID NO:33 and has β-phosphoglucomutase (EC 5.4.2.6) activity, wherein x is an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, wherein the enzyme is capable of converting fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) as described above.

[0172] Preferably, the degree of identity is determined by comparing the corresponding sequence with the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33.

[0173] The method of the present invention can be implemented in vitro or in vivo. An in vitro reaction is understood to be a reaction in which no cells are used, i.e. a cell-free reaction. Therefore, in vitro preferably means in a cell-free system. In one embodiment, the term "in vitro" means in the presence of an isolated enzyme (or enzyme system, optionally comprising cofactors that may be required). In one embodiment, the enzyme used in the method is used in a purified form.

[0174] In order to carry out the method in vitro, the substrate and enzyme of the reaction are incubated under conditions that allow the enzyme to be active and allow the enzymatic conversion to occur (buffer, temperature, co-substrate, cofactor, etc.). The reaction is allowed to proceed for a time sufficient to produce the corresponding product. The production of the respective product can be measured by methods known in the art, such as gas chromatography, which may be connected to mass spectrometry detection.

[0175] The enzyme may be in any suitable form that allows the enzymatic reaction to occur. They may be purified or partially purified or in the form of a crude cell extract or a partially purified extract. The enzyme may also be immobilized on a suitable carrier.

[0176] In another embodiment, the method of the present invention is implemented in a culture mode in the presence of an organism, preferably a microorganism, to produce the enzyme described in the conversion method of the present invention as described above. The method of implementing the method of the present invention using a microorganism is called an "in vivo" method. A microorganism that naturally produces the enzyme described in the conversion method of the present invention above or a microorganism that has been genetically modified so that it expresses (including overexpression) one or more of such enzymes can be used. Therefore, the microorganism can be an engineered microorganism that expresses the above-mentioned enzyme for conversion of the method of the present invention, i.e., has a nucleotide sequence encoding such enzyme in its genome and has been modified to overexpress them. Expression can occur in a constitutive manner or in an induced or regulated manner.

[0177] In another embodiment, the microorganism can be a microorganism that has been genetically modified by introducing one or more nucleic acid molecules that contain the nucleotide sequence of one or more enzymes that are used for the method of the present invention to transform. This nucleic acid molecule can be stably integrated into the genome of this microorganism or can exist in an extrachromosomal manner, for example, on a plasmid.

[0178] Such a genetically modified microorganism may be, for example, a microorganism that does not naturally express the enzyme used for the conversion according to the method of the invention and has been genetically modified to express such an enzyme, or the microorganism naturally expresses such an enzyme and has been genetically modified, for example, by transformation with a nucleic acid (e.g., a vector encoding the corresponding enzyme) and / or by inserting a promoter in front of the endogenous nucleotide sequence encoding the enzyme to increase the corresponding activity in the microorganism.

[0179] However, the present invention preferably excludes naturally occurring microorganisms as found in nature, which express enzymes as described above at levels found in nature. In contrast, the microorganisms of the present invention and used in the methods of the present invention are preferably non-naturally occurring microorganisms, whether or not they have been genetically modified to express (including overexpression) an exogenous enzyme of the present invention that is not normally present in their genome or whether or not they have been engineered to overexpress an exogenous enzyme.

[0180] Therefore, the enzyme and (micro) organism used in the present invention are preferably non-natural enzymes or (micro) organisms, that is, they are significantly different from naturally occurring enzymes or microorganisms and do not exist in nature enzymes or microorganisms. With regard to these enzymes, they are preferably variants of naturally occurring enzymes, and the variant itself does not exist in nature. Such variants, for example, include mutants, especially mutants prepared by molecular biological methods, which show improved characteristics, such as higher enzyme activity, higher substrate specificity, higher temperature tolerance, etc. With regard to (micro) organisms, they are preferably genetically modified organisms as described above that are different from naturally occurring organisms because of genetic modification. Genetically modified organisms are not naturally occurring, that is, they cannot exist in nature and are significantly different from naturally occurring organisms because of the introduction of foreign nucleic acid molecules.

[0181] By overexpressing an exogenous enzyme or endogenous enzyme as described above, the enzyme concentration is substantially higher than that present in nature, which can then unexpectedly force the reaction of the invention utilizing the corresponding non-natural enzyme to proceed. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.

[0182] A "non-natural" substrate is understood to be a molecule that is not acted upon by the corresponding enzyme in nature, even though it may actually coexist in the microorganism together with the endogenous enzyme. Such a "non-natural" substrate is not converted by the microorganism in nature, since other substrates are preferred (e.g. "natural substrates"). Therefore, the present invention contemplates the use of non-natural substrates with the enzymes described above in environments that do not exist in nature.

[0183] Therefore, in the context of the present invention, it is also possible that the microorganism is a microorganism that does not naturally have the corresponding enzyme activity, but is genetically modified to contain a nucleotide sequence that allows the expression of the corresponding enzyme. Similarly, the microorganism may also be a microorganism that naturally has the corresponding enzyme activity, but is genetically modified to enhance this enzyme activity (e.g., by introducing an exogenous nucleotide sequence encoding the corresponding enzyme or by introducing a promoter for an endogenous gene encoding the enzyme to increase endogenous production to an overexpression (non-natural) level).

[0184] If a microorganism that naturally expresses the corresponding enzyme is used, this microorganism can be modified so that the corresponding activity is overexpressed in the microorganism. This can be achieved, for example, by implementing a mutation or introducing a high-expression promoter in the promoter region of the corresponding gene, resulting in a promoter that ensures a higher expression of the gene. Alternatively, it may also be that the gene is mutated so as to result in an enzyme that shows a higher activity.

[0185] By using a microorganism expressing the enzyme described above for the conversion method of the present invention, the method of the present invention can be carried out directly in a culture medium without isolating or purifying the enzyme.

[0186] In one embodiment, the organism used in the method of the present invention is a microorganism that has been genetically modified to contain an exogenous nucleic acid molecule encoding at least one enzyme used for the method of the present invention. In this context, the term "exogenous" or "exogenous" means that the nucleic acid molecule does not naturally exist in the microorganism. This means that it does not appear in the same structure or at the same position in the microorganism. In a preferred embodiment, the exogenous nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the corresponding enzyme, wherein the promoter driving the expression of the coding sequence is heterologous relative to the coding sequence. In this context, "heterologous" means that the promoter is not a promoter that naturally drives the expression of the coding sequence, but a promoter that naturally drives the expression of different coding sequences, that is, it is derived from another gene, or a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the microorganism, that is, a promoter that does not naturally exist in the corresponding microorganism. Even more preferably, the promoter is an inducible promoter. Promoters that drive expression in different types of organisms, especially in microorganisms, are well known to those skilled in the art.

[0187] In another embodiment, the nucleic acid molecule is foreign to the microorganism in that the encoded enzyme is not endogenous to the microorganism, i.e., it is not naturally expressed by the microorganism when the microorganism is not genetically modified. In other words, the encoded enzyme is heterologous to the microorganism. The foreign nucleic acid molecule can exist in the microorganism in an extrachromosomal form, for example as a plasmid, or stably integrated in the chromosome. Preferably, stable integration. Therefore, the genetic modification can, for example, consist in integrating the corresponding gene encoding the enzyme into the chromosome, or in expressing the enzyme from a plasmid containing a promoter upstream of the enzyme coding sequence, the promoter and the coding sequence preferably being derived from different organisms; or any other method known to those skilled in the art.

[0188] In the context of the present invention, the term "microorganism" refers to bacteria, as well as fungi, such as yeast, and also algae and archaea. In a preferred embodiment, the microorganism is a bacterium. In principle, any bacterium can be used. Preferred bacteria to be used in the inventive method are bacteria of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment, the bacterium belongs to Escherichia and even more preferably to Escherichia coli species. In another preferred embodiment, the bacterium belongs to Pseudomonas putida species, or belongs to Zymomonas mobilis species, or belongs to Corynebacterium glutamicum species or belongs to Bacillus subtilis species.

[0189] It is also possible to use extremely thermophilic bacteria such as Thermus thermophilus or anaerobic bacteria from the family Clostridiae.

[0190] In another preferred embodiment, the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Trichoderma, Kluyveromyces or Pichia, and even more preferably a species of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris, Pichia torula or Pichia utilis.

[0191] In another embodiment, the method of the present invention utilizes a photosynthetic microorganism that expresses at least one enzyme for the conversion of the present invention as described above. Preferably, the microorganism is a photosynthetic bacterium or a microalgae. In yet another embodiment, the microorganism is an algae, more preferably an algae belonging to the class of diatoms.

[0192] It is also conceivable to use a combination of microorganisms in the method of the invention, wherein different microorganisms express different enzymes as described above. The genetic modification of microorganisms for expressing the enzyme of interest will also be described in further detail below.

[0193] In another embodiment, the method of the present invention comprises the steps of providing an organism, preferably a microorganism carrying the activity of the corresponding enzyme or (cell) culture form, preferably a liquid cell culture form, and the subsequent step is to cultivate the organism, preferably a microorganism, under suitable conditions that allow the expression of the corresponding enzyme in a fermenter (often also referred to as a bioreactor), and further comprising the step of realizing the enzymatic conversion of the method of the present invention as described above herein. Suitable fermenters or bioreactor devices and fermentation conditions are known to those skilled in the art. A bioreactor or fermenter refers to any manufacturing or engineering device or system known in the art that supports a biologically active environment. Therefore, a bioreactor or fermenter can be a container in which a chemical / biochemical process (such as the method of the present invention) is implemented, and the method covers organisms, preferably microorganisms and / or biochemically active substances, that is, such organisms carrying the above-mentioned enzymes or the above-mentioned enzymes derived from such organisms. In a bioreactor or fermenter, this process can be aerobic or anaerobic. These bioreactors are commonly cylindrical and can have a range of specifications from several liters to several cubic meters, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in such a way that it is suitable for culturing organisms, preferably microorganisms, in batch culture, fed-batch culture, perfusion culture or chemostat culture, all culturing methods being generally known in the art.

[0194] The culture medium may be any medium suitable for culturing the corresponding organisms or microorganisms.

[0195] As mentioned above, the method of the present invention may be particularly useful and advantageous when implemented in a microorganism as described in WO 2013 / 007786. The document describes a recombinant microorganism having phosphoketolase activity and wherein EMPP is inactivated or weakened by eliminating or reducing phosphofructokinase and wherein the oxidation branch of PPP is inactivated or weakened by eliminating or reducing glucose-6-phosphate dehydrogenase. These measures lead to an increase in the amount of fructose-6-phosphate (F6P) converted by phosphoketolase and transported to the non-oxidative branch of PPP. In this case, glyceraldehyde-3-phosphate (G3P) produced from the non-oxidative branch of PPP is recycled to fructose-1,6-bisphosphate (FBP) by condensation with dihydroxyacetone phosphate (DHAP). The reaction is catalyzed by fructose-bisphosphate aldolase (EC 4.1.2.13). Fructose-1,6-bisphosphate (FBP) is subsequently converted to fructose-6-phosphate (F6P) by the action of fructose bisphosphatase (EC 3.1.3.11). The method of the present invention circumvents the regulatory effects of fructose-bisphosphate aldolase (EC 4.1.2.13) and fructose bisphosphatase (EC 3.1.3.11) and their inhibitory effects at higher glucose or sucrose levels in the fermentation medium. Thus, the method of the present invention allows the conversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) in such microorganisms even under conditions of high glucose concentration or sucrose concentration in the culture medium or in the reaction vessel. Thus, in a preferred embodiment, the above method of the present invention for producing fructose-6-phosphate is implemented in a microorganism as described in WO 2013 / 007786.

[0196] Therefore, in a preferred embodiment, the method according to the invention is carried out in a microorganism which is not only characterized by the recombinant expression of the enzyme described above in connection with the method, but is further characterized in that the microorganism:

[0197] a) having phosphoketolase activity;

[0198] b) (i) having an attenuated or inactivated Embden-Meyerhof-Parnas pathway (EMPP) due to inactivation of the gene encoding phosphofructokinase or due to reduction of phosphofructokinase activity compared to an unmodified microorganism; or

[0199] (ii) no phosphofructokinase activity;

[0200] and

[0201] c) (i) having a weakened or inactivated pentose phosphate pathway oxidation branch (PPP) due to inactivation of the gene encoding glucose-6-phosphate dehydrogenase or due to reduced glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism; or

[0202] (ii) No glucose-6-phosphate dehydrogenase activity.

[0203] This microorganism is characterized in that it has phosphoketolase activity, thereby increases the flux of the acetyl-CoA produced.Usually, microorganisms make glucose conversion into pyruvic acid by Embden-Meyerhof-Parnas approach, and the pyruvic acid can be converted into acetyl-CoA by pyruvate dehydrogenase subsequently.However, this conversion is accompanied by CO2 release, and therefore, the loss may be used to produce a carbon atom in the useful metabolite.In order to increase the amount of acetyl-CoA in the microorganism, it is therefore desirable to form acetyl-CoA by a different approach to avoid carbon atom loss.By using a microorganism with phosphoketolase activity, phosphate and fructose-6-phosphate are converted into erythrose-4-phosphate and acetyl phosphate, and phosphotransacetylase further converts acetyl phosphate into acetyl-CoA, without losing carbon atom.Therefore, finally, the productive rate of acetyl-CoA can be increased by using a microorganism with phosphoketolase activity.This microorganism can convert glucose into acetyl-CoA without losing carbon atom. Recombinant microorganisms in which phosphoketolase is expressed natively or heterologously are disclosed in US 7,785,858 and US 7,253,001.

[0204] The term "phosphoketolase activity" as used herein means an enzyme activity capable of converting D-xylulose-5-phosphate into D-glyceraldehyde-3-phosphate according to the following reaction:

[0205] D-xylulose-5-phosphate + phosphate → D-glyceraldehyde-3-phosphate + acetyl-phosphate + water

[0206] Or an enzyme activity that is capable of catalyzing the reaction shown above and is also capable of converting D-fructose-6-phosphate into D-erythrose-4-phosphate according to the following reaction:

[0207] D-fructose 6-phosphate + phosphate → acetyl phosphate + D-erythrose 4-phosphate + water

[0208] The former phosphoketolases are generally classified as EC 4.1.2.9 and the latter as EC 4.1.2.22. Both types of phosphoketolases can be used within the scope of the present invention. Figure 1 Scheme showing the overall reaction of phosphoketolase as described herein using two options.

[0209] This enzyme activity can be measured by assays known in the art. An example of such an assay is given in the Examples section below.

[0210] In the context of the present invention, a microorganism having phosphoketolase activity may, for example, be a microorganism that naturally has phosphoketolase activity or a microorganism that does not naturally have phosphoketolase activity and has been genetically modified to express phosphoketolase or a microorganism that naturally has phosphoketolase activity and has been genetically modified (e.g., transformed with a nucleic acid (e.g., a vector) encoding phosphoketolase) in order to increase the phosphoketolase activity in the microorganism.

[0211] Microorganisms which intrinsically, ie naturally, possess phosphoketolase activity are known in the art, and any of them may be used in the context of the present invention.

[0212] In the context of the present invention, the microorganism may also be a microorganism that does not naturally have phosphoketolase activity, but is genetically modified to contain a nucleotide sequence that allows phosphoketolase expression. Similarly, the microorganism may also be a microorganism that naturally has phosphoketolase activity, but is genetically modified to enhance phosphoketolase activity (e.g., by introducing an exogenous nucleotide sequence encoding phosphoketolase).

[0213] The genetic modification of microorganisms to express the enzyme of interest is described in detail below.

[0214] The phosphoketolase expressed in the microorganism may be any phosphoketolase, in particular a phosphoketolase from a prokaryotic or eukaryotic organism. For example a prokaryotic phosphoketolase from Lactococcus lactis is described.

[0215] The phosphoketolase expressed in the microorganism may be a naturally occurring phosphoketolase or it may be a phosphoketolase derived from a naturally occurring phosphoketolase (e.g. by introducing mutations or other changes that, e.g., alter or improve enzyme activity, stability, etc.).

[0216] The microorganism is preferably further characterized by having an attenuated or inactivated Embden-Meyerhof-Parnas pathway (EMPP) or having no phosphofructokinase activity due to inactivation of the gene encoding phosphofructokinase or due to reduced phosphofructokinase activity compared to an unmodified microorganism. Thus, the microorganism is a microorganism that naturally has an EMPP including phosphofructokinase activity, but has been modified, in particular genetically modified, such that the phosphofructokinase activity is completely eliminated or such that the activity is reduced compared to a corresponding unmodified microorganism, or the microorganism is a microorganism that naturally has no phosphofructokinase activity.

[0217] As mentioned above, when glucose is processed into acetyl-CoA through EMPP, a carbon atom is lost due to the release of CO2 in the last step. By introducing phosphoketolase, this loss can be avoided. Since fructose-6-phosphate is the substrate of phosphoketolase, it is necessary to maintain the fructose-6-phosphate pool in microorganisms at a high level to increase the productive rate of acetyl-CoA. Since fructose-6-phosphate is also the substrate of the enzyme (i.e., phosphofructokinase) of the Embden-Meyerhof-Parnas approach, compared with unmodified microorganisms, the recombinant microorganism has reduced phosphofructokinase activity or the gene encoding phosphofructokinase has been inactivated. This ensures that the flux of fructose-6-phosphate points to phosphoketolase and points to the generation of acetyl-CoA, without CO2 loss, because fructose-6-phosphate or most of fructose-6-phosphate can no longer be processed through the Embden-Meyerhof-Parnas approach. Recombinant microorganisms in which phosphoketolase is natively or heterologously expressed and which have reduced phosphofructokinase activity are disclosed in US Pat. No. 7,785,858.

[0218] "Phosphofructokinase activity" means the enzyme activity (EC 2.7.1.11) that converts ATP and fructose-6-phosphate into ADP and fructose-1,6-bisphosphate. This enzyme activity can be measured by assays known in the art (e.g., as described by Kotlarz et al. (Methods Enzymol. (1982) 90, 60-70)).

[0219] The term "a microorganism characterized by having an attenuated or inactivated Embden-Meyerhof-Parnas pathway (EMPP) due to inactivation of a gene encoding phosphofructokinase or due to a reduction in phosphofructokinase activity compared to an unmodified microorganism" preferably refers to a microorganism in which the inactivation of a gene encoding phosphofructokinase or the reduction in phosphofructokinase activity compared to an unmodified microorganism is achieved by a genetic modification of the microorganism resulting in said inactivation or reduction.

[0220] In a preferred embodiment, the recombinant microorganism is a recombinant microorganism with an inactivated Embden-Meyerhof-Parnas pathway (EMPP) due to the inactivation of the gene encoding phosphofructokinase. In the context of the present invention, the inactivation of the gene encoding phosphofructokinase means the inactivation of the genes encoding phosphofructokinase present in the microorganism, so that they are no longer expressed and / or do not lead to the synthesis of functional phosphofructokinase. Inactivation can be achieved in many different ways known in the art. Inactivation can be achieved, for example, by destroying the gene encoding phosphofructokinase or by completely deleting the gene by introducing a selection marker. Alternatively, the promoter of the gene encoding phosphofructokinase can be mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating the gene encoding phosphofructokinase known in the art are: expressing a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the phosphofructokinase gene, so that the mRNA is no longer likely to be translated into protein; expressing a polynucleotide encoding RNA that suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding RNA that has the activity of specifically cutting the transcript of the gene; or expressing a polynucleotide encoding RNA that inhibits the expression of the gene through co-suppression. These polynucleotides can be incorporated into a vector, wherein the vector can be introduced into a microorganism by transformation to achieve the inactivation of the gene encoding phosphofructokinase.

[0221] In the context of the present invention, the term "inactivated" preferably means complete inactivation, ie the microorganism does not show phosphofructokinase activity. This means in particular that the microorganism does not show phosphofructokinase activity independently of the growth conditions used.

[0222] Preferably, "inactivation" means that the gene encoding the phosphofructokinase present in the microorganism is genetically modified in such a way that the expression of the enzyme is prevented. This can be achieved, for example, by deleting the gene or a part thereof, wherein the deletion of the part prevents the expression of the enzyme, or by disrupting the gene in the coding region or in the promoter region, wherein the disruption has the effect that no protein is expressed or that a functionally abnormal protein is expressed.

[0223] In a preferred embodiment, the recombinant microorganism is a recombinant microorganism with a weakened Embden-Meyerhof-Parnas pathway (EMPP) due to reduced phosphofructokinase activity compared to an unmodified microorganism. Preferably, this reduction is achieved by genetically modifying the microorganism. This can be achieved, for example, by random mutagenesis or site-directed mutagenesis of promoters and / or enzymes and subsequent selection of promoters and / or enzymes with the desired properties or by complementary nucleotide sequences or RNAi effects as described above.

[0224] In the context of the present invention, "reduced activity" means that the expression and / or activity of an enzyme, in particular phosphofructokinase, in a genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferably at least 90% or 100% lower than in a corresponding unmodified microorganism. Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. Assays for measuring the reduced enzymatic activity of phosphofructokinase are known in the art.

[0225] In another embodiment, the microorganism is a microorganism without phosphofructokinase activity. This preferably means that the microorganism naturally has no phosphofructokinase activity. This means that the microorganism naturally does not contain a nucleotide sequence encoding an enzyme with phosphofructokinase activity in its genome. Examples of such microorganisms are Zymomonas mobilis (JS Suo et al., Nat. Biotechnol. 23: 63 (2005)) and Ralstonia eutropha (C. Fleige et al., Appl. Microb. Cell Physiol. 91: 769 (2011)).

[0226] The microorganism is further characterized as follows: due to the inactivation of the gene encoding glucose-6-phosphate dehydrogenase or due to the reduction of glucose-6-phosphate dehydrogenase activity compared with an unmodified microorganism, there is a weakened or inactivated pentose phosphate pathway oxidation branch (PPP) or there is no glucose-6-phosphate dehydrogenase activity. Thus, the microorganism is preferably such a microorganism, the microorganism naturally has a PPP including glucose-6-phosphate dehydrogenase activity, but has been modified, particularly genetically modified, so that glucose-6-phosphate dehydrogenase activity is completely eliminated, or thereby compared with a corresponding unmodified microorganism, the activity is reduced, or the microorganism is a microorganism that does not naturally have glucose-6-phosphate dehydrogenase activity.

[0227] Weakening or inactivating the pentose phosphate pathway oxidation branch further increases the yield of acetyl-CoA, because glucose-6-phosphate will no longer be pumped out through the pentose phosphate cycle. All or nearly all glucose-6-phosphate in the microorganism will be converted to fructose-6-phosphate, which will then be further converted to acetyl-CoA.

[0228] Glucose-6-phosphate dehydrogenase activity refers to the activity of glucose-6-phosphate dehydrogenase +Enzyme activity that converts to 6-phosphoglucono-δ-lactone and NADPH (EC 1.1.1.49). This enzyme activity can be measured by assays known in the art (eg, as described by Noltmann et al. (J. Biol. Chem. (1961) 236, 1225-1230)).

[0229] The term "a microorganism characterized by having a weakened or inactivated pentose phosphate pathway (PPP) oxidation branch due to inactivation of a gene encoding glucose-6-phosphate dehydrogenase or due to reduced glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism" preferably refers to a microorganism in which the inactivation of a gene encoding glucose-6-phosphate dehydrogenase or the reduction of glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism is achieved by genetic modification of the microorganism resulting in said inactivation or reduction.

[0230] In a preferred embodiment, the recombinant microorganism is a recombinant microorganism with an inactivated pentose phosphate pathway (PPP) oxidation branch due to the gene of the inactivated glucose-6-phosphate dehydrogenase. In the context of the present invention, the inactivation of the gene encoding the glucose-6-phosphate dehydrogenase means the gene inactivation of the glucose-6-phosphate dehydrogenase present in the microorganism, so that they no longer express and / or do not cause the synthesis of functional glucose-6-phosphate dehydrogenase. Inactivation can be achieved by many different ways known in the art. Inactivation can be achieved, for example, by destroying the gene encoding the glucose-6-phosphate dehydrogenase or by importing a selective marker to thoroughly lack the gene. Alternatively, the promoter of the gene encoding the glucose-6-phosphate dehydrogenase can be mutated in a way that the gene is no longer transcribed into mRNA. Other ways of inactivating the gene encoding glucose-6-phosphate dehydrogenase known in the art are: expressing a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the glucose-6-phosphate dehydrogenase gene, so that the mRNA is no longer likely to be translated into protein; expressing a polynucleotide encoding RNA, the RNA suppresses the gene expression through the RNAi effect; expressing a polynucleotide encoding RNA, the RNA has the activity of specifically cutting the transcript of the gene; or expressing a polynucleotide encoding RNA, the RNA suppresses the expression of the gene through co-suppression. These polynucleotides can be incorporated into a vector, wherein the vector can be introduced into a microorganism by transformation to achieve the inactivation of the gene encoding glucose-6-phosphate dehydrogenase.

[0231] In the context of the present invention, the term "inactivated" preferably means complete inactivation, ie the microorganism does not show glucose-6-phosphate dehydrogenase activity. This means in particular that the microorganism does not show glucose-6-phosphate dehydrogenase activity regardless of the growth conditions used.

[0232] Preferably, "inactivation" means that the gene encoding glucose-6-phosphate dehydrogenase present in the microorganism is genetically modified so as to prevent the expression of the enzyme. This can be achieved, for example, by deleting the gene or a part thereof, wherein deleting the part prevents the expression of the enzyme, or by disrupting the gene in the coding region or in the promoter region, wherein the disruption has the effect of no protein expression or expression of a dysfunctional protein.

[0233] In a preferred embodiment, the recombinant microorganism is a recombinant microorganism that has a weakened pentose phosphate pathway (PPP) oxidation branch due to reducing glucose-6-phosphate dehydrogenase activity compared to unmodified microorganism. Preferably, this reduction is achieved by genetic modification of this microorganism. This can be achieved, for example, by random mutagenesis or site-directed mutagenesis promoter and / or enzyme and subsequent selection of promoter and / or enzyme with desired characteristics or by complementary nucleotide sequence as described above or RNAi effect.

[0234] In the context of the present invention, "reduced activity" means that the expression and / or activity of enzyme, especially glucose-6-phosphate dehydrogenase in the genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferably at least 90% or 100% lower than in the corresponding unmodified microorganism. Those skilled in the art are familiar with the method for measuring the expression level of a given protein in the cell. The assay for measuring the reduced enzymatic activity of glucose-6-phosphate dehydrogenase is known in the art.

[0235] In another embodiment, the microorganism is a microorganism without glucose-6-phosphate dehydrogenase activity. This preferably means that the microorganism naturally has no glucose-6-phosphate dehydrogenase activity. This means that the microorganism naturally does not contain a nucleotide sequence encoding an enzyme with glucose-6-phosphate dehydrogenase activity in its genome. Examples of such microorganisms are Acinetobacter baylyi (Barbe et al., Nucl. Acids Res. 32 (2004), 5766-5779), extreme thermophilic archaea such as Sulfolobus solfataricus (Nunn et al., J. Biol. Chem. 285 (2010), 33701-33709), Thermoproteus tenax, Thermoplasma acidophilum and Picrophilus torridus (Reher and FEBS Lett. 580(2006), 1198-1204).

[0236] The microorganism can be characterized in principle by having fructose-1,6-bisphosphate phosphatase activity. However, as described above, since this enzyme can be inhibited by high levels of glucose, it is preferred that its action is replaced by the method of the present invention in which fructose-6-phosphate (F6P) is produced from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). Fructose-6-phosphate can then be converted again into acetyl-CoA via the phosphoketolase pathway. In fact, acetyl phosphate, the product of phosphoketolase, is back-converted into acetyl-CoA by the action of phosphate acetyltransferase EC 2.3.1.8. Therefore, the recombinant microorganism is able to produce acetyl-CoA from glucose in a stoichiometry close to 3:1. The sum of the reactions is given in equation 2:

[0237] Glucose + ATP + 3CoA → 3-acetyl-CoA + ADP + H3PO4 + 2H2O (Equation 2)

[0238] In another embodiment, the microorganism is further characterized in that: EMPP is further weakened or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified microorganism. Further weakening EMPP by weakening or inactivating glyceraldehyde 3-phosphate dehydrogenase at a further downstream step ensures that glyceraldehyde 3-phosphate that can be produced in the microorganism will not or almost not be processed into acetyl-CoA by glycolysis, and one of the carbon atoms will be lost by releasing CO2 in the last step catalyzed by pyruvate dehydrogenase. Therefore, blocking EMPP by weakening or inactivating glyceraldehyde 3-phosphate dehydrogenase activity further ensures that the total flux is directed to phosphoketolase.

[0239] "Glyceraldehyde 3-phosphate dehydrogenase activity" means the activity of glyceraldehyde 3-phosphate, phosphate and NAD + Converted into 3-phospho-D-glyceroyl phosphate and NADH+H + Enzyme activity of (EC 1.2.1.12). This activity can be measured by assays known in the art (eg, as described by D'Alessio et al. (J. Biol. Chem. (1971) 246, 4326-4333)).

[0240] The term "a microorganism characterized by having an attenuated or inactivated additional Embden-Meyerhof-Parnas pathway (EMPP) due to inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase or due to a reduction in glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified microorganism" preferably refers to a microorganism in which the inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase or the reduction in glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified microorganism is achieved by genetic modification of the microorganism resulting in said inactivation or reduction.

[0241] In a preferred embodiment, the recombinant microorganism is a recombinant microorganism in which the EMPP is further attenuated or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified microorganism. In the context of the present invention, inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase means inactivation of genes encoding glyceraldehyde 3-phosphate dehydrogenase present in the microorganism so that they are no longer expressed and / or do not result in the synthesis of a functional glyceraldehyde 3-phosphate dehydrogenase. Inactivation can be achieved in many different ways known in the art. Inactivation can be achieved, for example, by destroying a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by completely deleting the gene by introducing a selection marker. Alternatively, the promoter of a gene encoding glyceraldehyde 3-phosphate dehydrogenase can be mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating the gene encoding glyceraldehyde 3-phosphate dehydrogenase known in the art are: expressing a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the glyceraldehyde 3-phosphate dehydrogenase gene, so that the mRNA is no longer likely to be translated into protein; expressing a polynucleotide encoding RNA, the RNA suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding RNA, the RNA has the activity of specifically cutting the transcript of the gene; or expressing a polynucleotide encoding RNA, the RNA inhibits the expression of the gene through co-suppression. These polynucleotides can be incorporated into a vector, wherein the vector can be introduced into a microorganism by transformation to achieve the inactivation of the gene encoding glyceraldehyde 3-phosphate dehydrogenase.

[0242] In the context of the present invention, the term "inactivated" preferably means complete inactivation, ie the microorganism does not show glyceraldehyde 3-phosphate dehydrogenase activity. This means in particular that the microorganism does not show glyceraldehyde 3-phosphate dehydrogenase activity regardless of the growth conditions used.

[0243] Preferably, "inactivation" means that the gene encoding glyceraldehyde 3-phosphate dehydrogenase present in the microorganism is genetically modified in such a way that the expression of the enzyme is prevented. This can be achieved, for example, by deleting the gene or a part thereof, wherein the deletion of the part prevents the expression of the enzyme, or by disrupting the gene in the coding region or in the promoter region, wherein the disruption has the effect that no protein is expressed or that a functionally abnormal protein is expressed.

[0244] In a preferred embodiment, the recombinant microorganism is a recombinant microorganism with an impaired EMPP due to a reduction in glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified microorganism. Preferably, this reduction is achieved by genetically modifying the microorganism. This can be achieved, for example, by random or site-directed mutagenesis of promoters and / or enzymes and subsequent selection of promoters and / or enzymes with the desired properties or by complementary nucleotide sequences or RNAi effects as described above.

[0245] In the context of the present invention, "reduced activity" means that the expression and / or activity of an enzyme, especially glyceraldehyde 3-phosphate dehydrogenase in a genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferably at least 90% or 100% lower than in a corresponding unmodified microorganism. Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. Determination of the enzymatic activity of the reduction of glyceraldehyde 3-phosphate dehydrogenase is known in the art.

[0246] In another embodiment in which the recombinant microorganism is a bacterium, the genes encoding the PEP-dependent PTS transporter have been inactivated. In the context of the present invention, inactivation means that the genes encoding the PEP-dependent PTS transporter present in the microorganism are inactivated so that they are no longer expressed and / or do not lead to the synthesis of a functional PEP-dependent PTS transporter. The inactivation of the gene encoding the PEP-dependent PTS transporter should be such that the bacterium is no longer able to transport glucose via the PEP-dependent PTS transporter.

[0247] PEP-dependent PTS transporters are known in the art (eg from E. coli, B. subtilis). Examples of inactivated PEP-dependent PTS transporters are shown in the Examples section below.

[0248] Inactivation can be achieved in many different ways known in the art. Inactivation can be achieved, for example, by destroying the gene encoding the PEP-dependent PTS transporter or completely deleting the gene by introducing a selection marker. Alternatively, the promoter of the gene encoding the PEP-dependent PTS transporter can be mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating genes encoding PEP-dependent PTS transporters known in the art are: expressing a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the PEP-dependent PTS transporter gene, so that the mRNA is no longer likely to be translated into protein; expressing a polynucleotide encoding RNA that suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding RNA that has the activity of specifically cutting the transcript of the gene; or expressing a polynucleotide encoding RNA that suppresses the expression of the gene through co-suppression. These polynucleotides can be incorporated into a vector, wherein the vector can be introduced into a microorganism by transformation to achieve inactivation of the gene encoding the PEP-dependent PTS transporter.

[0249] In a preferred embodiment, the method of the invention utilizes an organism, preferably a microorganism, capable of consuming glucose.

[0250] In another preferred embodiment, the method of the invention utilizes an organism, preferably a microorganism, capable of consuming fructose.

[0251] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, capable of consuming xylose.

[0252] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, capable of consuming mannose.

[0253] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, that can consume more than one sugar. Preferably, the more than one sugar includes sucrose, glucose, mannose and / or xylose. In a more preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, that can consume two or more sugars selected from sucrose, glucose, mannose and xylose. The organism and / or microorganism that can consume glucose, fructose, xylose and / or mannose does exist naturally and is known in the art.

[0254] In another embodiment, the organism and / or microorganism is genetically modified to be able to consume glucose, fructose, xylose and / or mannose and / or is genetically modified to increase the ability of the organism and / or microorganism to consume glucose, fructose, xylose and / or mannose. Corresponding genetic modifications are known in the art.

[0255] In a preferred embodiment, the method of the invention utilizes an organism, preferably a microorganism, that is capable of consuming sugars via a phosphotransferase transport system (PTS).

[0256] In another embodiment, the methods of the invention utilize organisms, preferably microorganisms, that are capable of consuming sugars via a non-phosphotransferase transport system (non-PTS).

[0257] Organisms and / or microorganisms capable of consuming sugars by a phosphotransferase transport system (PTS) and / or by a non-phosphotransferase transport system (non-PTS) are known in the art.

[0258] In another embodiment, the organism and / or microorganism is genetically modified to be able to consume sugars by a phosphotransferase transport system (PTS) or by a non-phosphotransferase transport system (non-PTS). In another preferred embodiment, the organism and / or microorganism is genetically modified to increase the ability of the organism and / or microorganism to consume sugars by a phosphotransferase transport system (PTS) or by a non-phosphotransferase transport system (non-PTS). Corresponding genetic modifications are known in the art.

[0259] In another preferred embodiment, the method of the invention utilizes an organism, preferably a microorganism, having an impaired or inactivated phosphotransferase transport system (PTS).

[0260] Without being bound by theory, the organism, preferably microorganism, may be genetically modified, preferably by deletion or inactivation of the gene for the phosphotransferase transport system (PTS).

[0261] Corresponding genetic modifications are known in the art.

[0262] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, with an enhanced non-phosphotransferase transport system (non-PTS) for uptake of sugars.

[0263] Without being bound by theory, such an organism, preferably a microorganism, may be genetically modified to uptake sugars, preferably by overexpressing genes for said non-phosphotransferase transport system (non-PTS).

[0264] Corresponding genetic modifications are known in the art.

[0265] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, having an impaired or inactivated phosphotransferase transport system (PTS) and an enhanced non-phosphotransferase transport system (non-PTS) for uptake of sugars.

[0266] In another preferred embodiment, the method of the invention utilizes an organism, preferably a microorganism, capable of consuming sucrose via a non-phosphotransferase transport system (non-PTS).

[0267] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, capable of consuming sucrose, wherein the organism, preferably the microorganism, has been genetically modified by introducing at least one gene for a non-phosphotransferase transport system (non-PTS). Without being bound by theory, such an organism and / or microorganism has been genetically modified by introducing a gene selected from cscA, cscB and cscK from Escherichia coli W (M. Bruschi et al., Biotechnology Advances 30 (2012) 1001-1010).

[0268] In another preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, which has been genetically modified to have an impaired or inactivated phosphotransferase transport system (PTS) and overexpression of at least one gene selected from galP, glk and glf.

[0269] In a preferred embodiment, the method of the present invention utilizes an organism, preferably a microorganism, that has been genetically modified to avoid leakage of acetyl-CoA, thereby increasing the intracellular concentration of acetyl-CoA. Genetic modifications that result in increased intracellular concentrations of acetyl-CoA are known in the art. Without being bound by theory, such an organism, preferably a microorganism, may be genetically modified, preferably by deleting or inactivating one or more of the following genes:

[0270] ΔackA (acetate kinase), Δldh (lactate dehydrogenase), ΔadhE (alcohol dehydrogenase), ΔfrdB and / or ΔfrdC (fumarate reductase and fumarate dehydrogenase), ΔpoxB (pyruvate oxidase), Δpgk (phosphoglycerate kinase), ΔiclR (DNA-binding transcriptional repressor protein IclR).

[0271] Additionally, deletions that may be advantageous in the context of the present invention are deletions in the gene encoding 6-phosphogluconate dehydratase (e.g. the edd gene in E. coli) and / or in the gene encoding 2-keto-3-deoxy-6-phosphogluconate aldolase (e.g. the eda gene in E. coli).

[0272] Alternatively or in addition to any of the above mentioned deletions, the organism or microorganism may be genetically modified by overexpressing the gene panK / coaA encoding pantothenate kinase, thereby increasing the CoA / acetyl-CoA intracellular pool.

[0273] These modifications to avoid acetyl-CoA leakage are known in the art, and corresponding modified organisms have been used in a method for the bioconversion of exogenous isopentanol into isopentanyl acetate by an E. coli strain expressing ATF2 (Metab. Eng. 6 (2004), 294-309).

[0274] Other genes that can be overexpressed in an organism or microorganism include the following:

[0275] ○pckA (phosphoenolpyruvate carboxykinase)

[0276] ○tktA (transketolase 1)

[0277] ○tktB (transketolase 2)

[0278] ○talA (transaldolase A)

[0279] ○talB (transaldolase B)

[0280] ○rpiA (ribose-5-phosphate isomerase A)

[0281] ○rpiB (ribose-5-phosphate isomerase B)

[0282] ○rpE (ribulose-phosphate 3-epimerase)

[0283] ○pgi (glucose-6-phosphate isomerase)

[0284] ○galP (galactose: H + Symporter

[0285] ○glk (glucokinase)

[0286] ○glf (glucose-facilitated diffusion protein)

[0287] ○pta (phosphate acetyltransferase)

[0288] The recombinant microorganism may also be further characterized in that it is capable of converting acetyl-CoA into acetone. Methods for providing such recombinant microorganisms are disclosed, for example, in EP 2 295 593. In the context of the present invention, the term "capable of converting acetyl-CoA into acetone" means that the organism / microorganism has the ability to produce acetone inside the cell due to the presence of the following enzyme, which provides an enzymatic activity that allows acetone to be produced from acetyl-CoA.

[0289] Acetone is produced by certain microorganisms, such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa and Pseudomonas putida. The synthesis of acetone is best characterized in Clostridium acetobutylicum. It begins with a reaction (reaction step 1) in which two molecules of acetyl-CoA condense to form acetoacetyl-CoA. The reaction is catalyzed by acetyl-CoA acetyltransferase (EC 2.3.1.9). Subsequently, acetoacetyl-CoA is converted into acetoacetate by a reaction (reaction step 2) in which acetic acid or butyric acid also results in the production of acetyl-CoA or butyryl-CoA. The reaction is catalyzed by, for example, acetoacetyl-CoA transferase (EC 2.8.3.8). Acetoacetyl CoA transferase is known from various organisms, such as from Escherichia coli, where the enzyme is encoded by the atoAD gene, or from Clostridium acetobutylicum, where the enzyme is encoded by the ctfAB gene. However, other enzymes can also catalyze this reaction, such as 3-oxoacid CoA transferase (EC 2.8.3.5) or succinate CoA ligase (EC 6.2.1.5).

[0290] Finally, acetoacetate is converted to acetone via a decarboxylation step (reaction step 3) catalyzed by acetoacetate decarboxylase (EC 4.1.1.4).

[0291] The above reaction steps 1 and 2 and the enzymes catalyzing them are not specific to acetone synthesis and can be present in a variety of organisms. In contrast, reaction step 3, catalyzed by acetoacetate decarboxylase (EC 4.1.1.4), is present only in those organisms that are able to produce acetone.

[0292] In a preferred embodiment, the recombinant microorganism is a microorganism that naturally has the ability to produce acetone. Therefore, preferably, the microorganism belongs to the genus Clostridium, Bacillus or Pseudomonas, more preferably belongs to the following species: Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa or Pseudomonas putida.

[0293] In another preferred embodiment, the recombinant microorganism is a microorganism derived from an organism / microorganism that does not produce acetone naturally, but has been genetically modified to produce acetone, i.e., by introducing genes necessary to allow the production of acetone in the microorganism. In principle, any microorganism can be genetically modified in this manner. The enzyme responsible for synthesizing acetone has been described above. The genes encoding the corresponding enzymes are known in the art and can be used to genetically modify a given microorganism to produce acetone. As described above, reaction steps 1 and 2 of acetone synthesis are naturally present in most organisms. However, reaction step 3 is characteristic and essential for acetone synthesis. Therefore, in a preferred embodiment, a genetically modified microorganism derived from a microorganism that does not produce acetone naturally is modified to contain a nucleotide sequence encoding an enzyme (e.g., acetoacetate decarboxylase (EC 4.1.1.4)) that catalyzes the conversion of acetoacetate into acetone by decarboxylation. Nucleotide sequences from several organisms encoding such an enzyme are known in the art, for example the adc gene from Clostridium acetobutylicum (Uniprot Accession Nos. P23670 and P23673), Clostridium beijerinckii (Clostridium MP; Q9RPK1), Clostridium pasteurianum (Uniprot Accession No. P81336), Bradyrhizobium sp. (strain BTAi1 / ATCC BAA-1182; Uniprot Accession No. A5EBU7), Burkholderia mallei (ATCC 10399 A9LBS0), Burkholderia mallei (Uniprot Accession No. A3MAE3), Burkholderia mallei FMH A5XJB2, Burkholderia cepacia (ATCC 10399 A9LBS0 ... cenocepacia) (Uniprot accession number A0B471), Burkholderia ambifaria (Uniprot accession number Q0b5P1), Burkholderia phytofirmans (Uniprot accession number B2T319), Burkholderia spec.)(Uniprot accession number Q38ZU0), Clostridium botulinum (Uniprot accession number B2TLN8), Ralstonia pickettii (Uniprot accession number B2UIG7), Streptomyces nogalater (Uniprot accession number Q9EYI7), Streptomyces avermitilis (Uniprot accession number Q82NF4), Legionella pneumophila (Uniprot accession number Q5ZXQ9), Lactobacillus salivarius (Uniprot accession number Q1WVG5), Rhodococcus spec (Uniprot accession number Q0S7W4), Lactobacillus plantarum (Uniprot accession number Q0S7W4), plantarum) (Uniprot accession number Q890G0), Rhizobium leguminosarum (Uniprot accession number Q1M911), Lactobacillus casei (Uniprot accession number Q03B66), Francisella tularensis (Uniprot accession number Q0BLC9), Saccharopolyspora erythreae (Uniprot accession number A4FKR9), Korarchaeum cryptofilum (Uniprot accession number B1L3N6), Bacillus amyloliquefacins (Uniprot accession number A7Z8K8), Cochliobolus heterostrophus (Uniprot accession number Q8NJQ3), Sulfolobus icelandica (Uniprot accession number Q0BLC9), and Saccharopolyspora erythreae (Uniprot accession number A4FKR9). islandicus) (Uniprot accession number C3ML22) and Francisella tularensis subsp. holarctica (strain OSU18).

[0294] More preferably, the microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing step 2 of the acetone synthesis reaction mentioned above, ie, the conversion of acetoacetyl-CoA to acetoacetate.

[0295] Even more preferably, the microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing step 1 of the above-mentioned acetone synthesis reaction, ie the condensation of two molecules of acetyl CoA to form acetoacetyl CoA.

[0296] In a particularly preferred embodiment, the microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 1 and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 2, or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 1 and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 3, or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 2 and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 3, or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 1 and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 2 and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-mentioned acetone synthesis reaction step 3.

[0297] Methods for preparing the above-mentioned genetically modified microorganisms are well known in the art. Thus, generally, the microorganism is transformed with a DNA construct that allows the expression of the corresponding enzyme in the microorganism. Such a construct normally comprises the coding sequence in question connected to regulatory sequences (e.g., promoters and / or enhancers and / or transcription terminators and / or ribosome binding sites, etc.) that allow transcription and translation in the corresponding host cell. The prior art has described microorganisms that have been genetically modified so as to be able to produce acetone. In particular, genes from, for example, Clostridium acetobutylicum have been introduced into Escherichia coli, thus allowing the synthesis of acetone in Escherichia coli (a bacterium that does not naturally produce acetone) (Bermejo et al., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818). Specifically, Hanai et al. (see above citations) showed that the introduction of a nucleic acid sequence encoding an acetoacetate decarboxylase (such as the acetoacetate decarboxylase from Clostridium acetobutylicum) was sufficient to achieve acetone production in E. coli, indicating that the endogenous enzymes in E. coli catalyzing steps 1 and 2 of the above reaction (i.e., the expression products of the E. coli atoB and atoAD genes) are sufficient to provide substrates for acetone production.

[0298] In another aspect, the recombinant microorganism is further characterized in that: it is capable of converting acetyl-CoA into acetone and converting acetone into isobutylene. For example, methods for providing such recombinant microorganisms are disclosed in EP-A 2295 593 (EP 09 17 0312), WO 2011 / 032934, WO 2015 / 101493, WO 2014 / 086780, WO 2010 / 001078, WO 2012 / 052427, WO 2017 / 071124, WO 2015 / 004211, WO 2014 / 064198 and WO 2014 / 086781.

[0299] In another aspect, the recombinant microorganism is further characterized in that it is capable of converting acetyl-CoA to isobutylene using a metabolic pathway that does not include an acetone intermediate. For example, methods for providing such recombinant microorganisms are disclosed in the following documents: WO2016042012, WO2017 / 085167, WO2018 / 206262, WO2013 / 186215, WO2016 / 034691, WO2017 / 191239, US2019 / 0100742, WO 2016 / 042011, WO 2017 / 162738, WO2015082447, WO2010 / 001078, WO 2012 / 052427, WO 2017 / 071124, WO 2015 / 004211, WO 2014 / 064198 and WO2014 / 086781.

[0300] In another aspect, the recombinant microorganism is characterized in that it is capable of converting acetyl-CoA into acetone and converting acetone into propylene. Methods for providing such recombinant microorganisms are disclosed, for example, in Hanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818.

[0301] In another aspect, the recombinant microorganism is characterized in that it is capable of converting acetyl-CoA into acetone and converting acetone into isopropanol.

[0302] The conversion of acetone to isopropanol requires a secondary alcohol dehydrogenase that converts acetone to isopropanol in an NADPH-dependent reaction (Chen, J.-S., FEMS Microbiol. Rev. 17: 263-273 (1995)).

[0303] Thus, in another aspect, the recombinant microorganism is characterized in that it is capable of converting acetone into isopropanol by (over)expressing a secondary alcohol dehydrogenase.

[0304] To increase NADPH availability, expression of transhydrogenase enzymes such as PntAB and UdhA (SthA) can be modified (Jan et al., Biotechnol Prog. 29(5): 1124-30 (2013)).

[0305] Thus, in another aspect, the recombinant microorganism is characterized by increased availability of NADPH, eg by (over)expression of one or more transhydrogenase enzymes, preferably PntAB and UdhA (SthA).

[0306] In a preferred embodiment, it is contemplated to implement further gene deletions to increase acetone production and thus isopropanol production. In this case, for example, deletion of one or more, preferably all, of the following genes may be advantageous: fsaA (encoding fructose-6-phosphate aldolase 1) and fsaB (encoding fructose-6-phosphate aldolase 2).

[0307] Preferably, other genes that may be modified to be overexpressed in order to further increase isopropanol production are the pntAB (pyridine nucleotide transhydrogenase subunits alpha and beta, Uniprot P07001 and P0AB67, NCBI reference sequences: NP_416120.1 and NP_416119.1) genes, preferably from E. coli.

[0308] In a more preferred embodiment, the organism or microorganism is characterized in that it overexpresses one or more of the following genes for converting acetyl-CoA into acetone and / or isopropanol:

[0309] o For step 1 above: thlA (acetyl-CoA transferase; NCBI reference number WP_010966157.1; UniProt accession number P45359); preferably the thlA gene from Clostridium acetobulyticum

[0310] o For step 2 above: atoD, atoA (acetate CoA-transferase; NCBI reference numbers NP_416725.1 and NP_416726.1; UniProt accession numbers P76458 and P76459, respectively); preferably atoD, atoA genes from Escherichia coli

[0311] o For step 3 above: adc (acetoacetate decarboxylase; NCBI reference number NP_149328.1; UniProt accession number P23670); preferably the adc gene from Clostridium acetobutylicum

[0312] o For step 4: adh (NADP-dependent isopropanol dehydrogenase; NCBI reference AF_157307.2; UniProt accession P25984); preferably the adh gene from Clostridium beijerinckii.

[0313] Those skilled in the art will recognize that further genetic modification of the microorganisms of the present invention may result in improved efficacy of the microorganisms of the present invention in converting raw materials into products. For example, natural microorganisms often produce products such as formate, acetate, lactate, succinate, ethanol, glycerol, 2,3-butanediol, methylglyoxal, and hydrogen; all of which will impair the production of, for example, acetone, isobutylene, or propylene from sugars. Elimination or substantial reduction of such unwanted by-products can be achieved by elimination or reduction of key enzyme activities that lead to their production. Such activities include, but are not limited to, the group consisting of:

[0314] - acetyl-CoA + formate = CoA + pyruvate (e.g., catalyzed by formate C-acetyltransferase, also known as pyruvate formate-lyase (EC 2.3.1.54); for E. coli-pflB, NCBI-GeneID: 945514);

[0315] - ATP + acetate = ADP + acetyl phosphate (eg, catalyzed by acetate kinase (EC 2.7.2.1); for E. coli - ackA, NCBI-GeneID: 946775);

[0316] -(R)-Lactate + NAD + =pyruvate + NADH + H + (e.g., catalyzed by L-lactate dehydrogenase (EC 1.1.1.28); for Escherichia coli-ldhA, NCBI-GeneID: 946315);

[0317] - succinate + acceptor = fumarate + reduced acceptor (e.g., catalyzed by succinate dehydrogenase (EC 1.3.99.1); for E. coli - comprising frdA and frdB, NCBI-GeneID: 948667 and 948666, respectively);

[0318] -2-oxocarboxylate (eg pyruvate) = aldehyde (eg acetaldehyde + CO2 (eg catalyzed by pyruvate decarboxylase (EC 4.1.1.1));

[0319] -Acetaldehyde+CoA+NAD + =Acetyl-CoA+NADH+H + (e.g., catalyzed by acetaldehyde dehydrogenase (acetylation) (EC 1.2.1.10); for Escherichia coli-adhE, NCBI-Gene ID: 945837);

[0320] -sn-Glycerol 3-phosphate + NAD(P) + = phosphoglycerol + NAD(P)H + H + (For example, catalyzed by glycerol-3-phosphate dehydrogenase [NAD(P) + ](EC 1.1.1.94); for E. coli-gpsA, NCBI-GeneID: 948125);

[0321] -2-pyruvate=2-acetolactate+CO2 (e.g., catalyzed by acetolactate synthase (EC 2.2.1.6); for E. coli - ilvH and ilvI, NCBI-GeneID: 947267 and 948793, respectively);

[0322] - phosphoglycerone = methylglyoxal + phosphate (eg, catalyzed by methylglyoxal synthase (EC 4.2.3.3); for E. coli-mgsA, NCBI-GeneID: 945574); and

[0323] -Formic acid + H + =CO2+H2 (eg, catalyzed by formate hydrogenase (EC 1.2.1.2 together with EC 1.12.1.2); for E. coli - fdhF (EC 1.2.1.2), NCBI-Gene ID: 948584).

[0324] Thus, in a preferred embodiment, the microorganism may be further characterized by the elimination or reduction of one or more of the enzyme activities listed above.

[0325] Those skilled in the art will also recognize that genetic modification of regulatory elements in the microorganisms of the present invention may result in improved efficiency of the microorganisms of the present invention in converting raw materials into products. Within E. coli, such genetic modifications include, but are not limited to, the group consisting of:

[0326] - Deletion of the fnr gene (NCBI-GeneID: 945908), a general regulator of anaerobic growth; and

[0327] - Deletion of the rpoS gene (NCBI-GeneID: 947210), RNA polymerase, sigma S (sigma 38) factor; and

[0328] - Deletion of the iclR gene (DNA-binding transcriptional repressor IclR).

[0329] Thus, in another preferred embodiment, the microorganism exhibits at least one of these deletions.

[0330] Therefore, as described above, the method of the present invention can be implemented in a recombinant microorganism that can be used to convert glucose into acetyl-CoA as described above. Acetyl-CoA (also referred to as acetyl-CoA) is a thioester between coenzyme A (sulfhydryl) and acetic acid in chemical structure and is an important precursor molecule for producing useful metabolites. Acetyl-CoA can then be further converted into useful metabolites such as L-glutamic acid, L-glutamine, L-proline, L-arginine, L-leucine, succinate and polyhydroxybutyrate by the recombinant microorganism.

[0331] The recombinant microorganism can also be used to convert acetyl-CoA into acetone.

[0332] The recombinant microorganism can also be used to convert acetyl-CoA to isobutylene.

[0333] The recombinant microorganism can also be used to convert acetyl-CoA to propylene.

[0334] The recombinant microorganism can also be used to convert acetyl-CoA to isopropanol.

[0335] In another embodiment, the method of the present invention comprises the following steps: providing an organism, preferably a microorganism carrying the corresponding enzyme activity or (cell) culture form, preferably a liquid cell culture form, the subsequent step is to cultivate the organism, preferably a microorganism, under suitable conditions allowing the expression of the corresponding enzyme in a fermenter (often also referred to as a bioreactor), and further comprising the step of realizing the enzymatic conversion of the method of the present invention as described above herein. Suitable fermenters or bioreactor devices and fermentation conditions are known to those skilled in the art. A bioreactor or fermenter refers to any manufacturing or engineering device or system known in the art that supports a biologically active environment. Therefore, a bioreactor or fermenter can be a container in which a chemical / biochemical process (such as the method of the present invention) is implemented, and the chemical / biochemical process involves an organism, preferably a microorganism and / or a biochemically active substance, that is, from such an organism carrying the above-mentioned enzyme or the above-mentioned enzyme derived from an organism. In a bioreactor or fermenter, this process can be aerobic or anaerobic. These bioreactors are commonly cylindrical and can have a range of sizes from several liters to several cubic meters, and are often made of stainless steel. In this respect, without being bound by theory, the fermentor or bioreactor may be designed in such a way that it is suitable for culturing organisms, preferably microorganisms, in batch culture, fed-batch culture, perfusion culture or chemostate-culture, all cultivation methods being generally known in the art.

[0336] The culture medium may be any medium suitable for culturing the corresponding organisms or microorganisms.

[0337] When implemented by using microorganisms, the method of the present invention can be designed as a continuous fermentation culture method or as a batch culture method or any suitable culture method known to those skilled in the art.

[0338] The present invention also relates to a method for producing acetone and / or isobutylene and / or propylene from any of glucose or other carbon sources mentioned above, wherein the above-mentioned recombinant microorganism is cultivated under conditions that allow the production of acetone and / or isobutylene and / or propylene and wherein acetone and / or isobutylene and / or propylene are separated. The microorganism is cultivated under suitable culture conditions that allow the enzymatic reaction to occur. The specific culture conditions depend on the specific microorganism used, but are well known to those skilled in the art. The culture conditions are usually selected in such a way that they allow the expression of genes encoding enzymes for the corresponding reactions. Various methods are known to those skilled in the art to improve and fine-tune the expression of certain genes at certain culture stages, such as by inducing gene expression through chemical inducers or by temperature fluctuations.

[0339] In another preferred embodiment, the method of the invention further comprises the step of collecting gaseous products, in particular isobutene or propene, degassing the reaction, i.e. recovering the product degassed, for example, from the culture. Thus, in a preferred embodiment, the method is carried out in the presence of a system for collecting isobutene or propene in gaseous form during the reaction.

[0340] As a matter of fact, short-chain olefins such as isobutylene and propylene take the gaseous state at room temperature and pressure. The process of the invention therefore does not require the extraction of the product from a liquid culture medium, a step that is always very expensive when carried out on an industrial scale. The evacuation and storage of the gaseous hydrocarbons and their possible subsequent physical separation and chemical conversion can be carried out according to any method known to those skilled in the art.

[0341] The enzyme used in the method of the invention may be a naturally occurring enzyme or an enzyme that may be derived from a naturally occurring enzyme (eg by introducing mutations or other changes that eg alter or improve enzyme activity, stability etc.).

[0342] Methods for modifying and / or improving a desired enzymatic activity of a protein are well known to the person skilled in the art and comprise, for example, random mutagenesis or site-directed mutagenesis with subsequent selection of enzymes with the desired properties, or so-called "directed evolution" approaches.

[0343] For example, for gene modification in prokaryotic cells, nucleic acid molecules encoding corresponding enzymes can be introduced into plasmids, which allow mutagenesis or sequence modification by DNA sequence recombination. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchange or addition of natural or synthetic sequences. DNA fragments can be connected by using adapters and joints complementary to the fragments. In addition, engineering measures that provide suitable restriction sites or remove redundant DNA or restriction sites can be used. In those cases where insertion, deletion or substitution may be possible, in vitro mutagenesis, "primer repair", restriction or connection can be used. Generally speaking, sequence analysis, restriction analysis and other biochemical and molecular biological methods are implemented as analytical methods. Subsequently, the enzyme variants produced are tested for the desired activity, for example, enzyme activity, and particularly the enzyme activity they increase using the assay as described above.

[0344] As mentioned above, the microorganism used in the inventive method or contained in the present composition can be a microorganism that has been genetically modified by introducing a nucleic acid molecule encoding a corresponding enzyme. Therefore, in a preferred embodiment, the microorganism is such a recombinant microorganism, and the recombinant microorganism has been genetically modified to have the activity of the increase of at least one enzyme that is used for the inventive method conversion. This can be realized, for example, by transforming this microorganism with a nucleic acid encoding a corresponding enzyme. Preferably, the nucleic acid molecule introduced into the microorganism is a heterologous nucleic acid molecule relative to the microorganism, that is, it is not naturally present in the microorganism.

[0345] In the context of the present invention, "increased activity" preferably means that the expression of enzyme in the genetically modified microorganism and / or the activity ratio in the corresponding unmodified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferably at least 90% or 100%. In even more preferred embodiments, the increase of expression and / or activity can be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments, the expression ratio is at least 10 times, more preferably at least 100 times and even more preferably at least 1000 times higher in the corresponding unmodified microorganism.

[0346] The term "increased" expression / activity also encompasses situations in which the corresponding unmodified microorganism does not express the corresponding enzyme, so that the corresponding expression / activity in the unmodified microorganism is zero. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.

[0347] Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. In one embodiment, the expression level is measured by measuring the amount of the corresponding protein. Corresponding methods are well known to those skilled in the art and include Western blotting, ELISA, etc. In another embodiment, the expression level is measured by measuring the amount of the corresponding RNA. Corresponding methods are well known to those skilled in the art and include, for example, Northern blotting.

[0348] In the context of the present invention, the term "recombinant" means that the microorganism is genetically modified so that it contains a nucleic acid molecule encoding an enzyme as defined above compared to a wild-type or unmodified microorganism. The nucleic acid molecule encoding an enzyme as defined above can be used alone or as part of a vector.

[0349] The nucleic acid molecule may also comprise an expression control sequence operably linked to the polynucleotide contained in the nucleic acid molecule. As used throughout this specification, the term "operably linked" or "operably linked" refers to a connection between one or more expression control sequences and a coding region in a polynucleotide to be expressed in such a manner that expression is achieved under conditions compatible with the expression control sequences.

[0350] Expression includes transcription of the heterologous DNA sequence, preferably, into translatable mRNA. Regulatory elements that ensure expression in fungi and in bacteria are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals, etc. Examples are further given below in connection with the explanation of the relevant vectors.

[0351] The promoter used for the nucleic acid molecule can be homologous or heterologous relative to its source and / or relative to the gene to be expressed. Suitable promoters are promoters that for example themselves lead to constitutive expression. However, promoters that are activated only at a time point determined by external influences can also be used. In this case, artificial and / or chemically inducible promoters can be used.

[0352] The vector may further comprise an expression control sequence operably linked to the polynucleotide contained in the vector. These expression control sequences may be suitable for ensuring transcription and synthesis of translatable RNA in bacteria or fungi.

[0353] In addition, different mutations can be inserted into polynucleotides by conventional methods in molecular biology (see, for example, Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), resulting in the synthesis of polypeptides that may have modified biological properties. It is conceivable to introduce point mutations at positions that modify the amino acid sequence, for example, to affect the biological activity of the polypeptide or its regulatory effect.

[0354] In addition, mutants with modified substrate or product specificity can be prepared. Preferably, such mutants show increased activity. Alternatively, mutants that eliminate their catalytic activity without losing substrate binding activity can be prepared.

[0355] Furthermore, the introduction of mutations into a polynucleotide encoding an enzyme as defined above allows increasing or decreasing the gene expression rate and / or the activity of the enzyme encoded by said polynucleotide.

[0356] In order to modify bacteria or fungi genetically, the polynucleotides encoding the enzymes defined above or parts of these molecules can be introduced into plasmids, which allow mutagenesis or sequence modification by DNA sequence recombination. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchange or addition of natural or synthetic sequences. DNA fragments can be connected by using adapters and joints for fragments. In addition, engineering measures that provide suitable restriction sites or remove redundant DNA or restriction sites can be used. In those cases where insertion, deletion or replacement may be possible, in vitro mutagenesis, "primer repair", restriction or connection can be used. Generally speaking, sequence analysis, restriction analysis and other biochemical and molecular biological methods are implemented as analytical methods.

[0357] Thus, according to the present invention, a recombinant microorganism can be produced by genetically modifying a fungus or a bacterium, comprising introducing the above-mentioned polynucleotide, nucleic acid molecule or vector into a fungus or a bacterium.

[0358] The polynucleotide encoding the corresponding enzyme is expressed, thereby resulting in the production of a polypeptide having any of the activities described above. An overview of different expression systems is for example in Methods in Enzymology 153 (1987), 385-516, Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and contained in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al. (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is given, for example, by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261-279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93), Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) and Buckholz (Bio / Technology 9 (1991), 1067-1072).

[0359] Expression vectors have been described extensively in the literature. Usually, they not only contain a selectable marker gene and a replication origin that ensures replication in the selected host, but also contain a bacterial promoter or a viral promoter, and in most cases a transcription termination signal. Between the promoter and the termination signal, there is usually at least one restriction site or a polylinker that makes it possible to insert the coding DNA sequence. If active in the selected host organism, the DNA sequence of the transcription of the natural control corresponding gene can be used as a promoter sequence. However, this sequence can also be exchanged for other promoter sequences. Promoters that ensure that the gene is expressed in a built-in manner and inducible promoters that allow artificial control of gene expression can be used. Bacterial promoters and viral promoter sequences with these characteristics are described in detail in the literature. The regulatory sequences for expression in microorganisms (e.g., Escherichia coli, Saccharomyces cerevisiae) are fully described in the literature. Promoters which allow particularly high expression of downstream sequences are, for example, the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., cited in Rodriguez and Chamberlin (eds.), Promoters, Structure and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), lp1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than constitutive promoters. In order to obtain an optimal polypeptide amount, a two-stage method is often used. First, the host cells are cultured under optimal conditions to a relatively high cell density. In a second step, transcription is induced depending on the type of promoter used. In this respect, the tac promoter is particularly suitable, which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

[0360] Transformation of host cells with polynucleotides or vectors as described above can be carried out by standard methods such as described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. Host cells are cultured in a nutrient medium that meets the requirements of the specific host cell used, especially with respect to pH, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements, etc.

[0361] The present invention further relates to a recombinant microorganism which has been transformed with the nucleotide sequence

[0362] (a) a nucleotide sequence encoding a phosphomonoester hydrolase (EC 3.1.3.-); and

[0363] (b) a nucleotide sequence encoding an enzyme selected from

[0364] (i) aldehyde lyase (EC 4.1.2.-); and / or

[0365] (ii) Transaldolase (EC 2.2.1.2).

[0366] In a preferred embodiment, the phosphomonoester hydrolase (EC 3.1.3.-) encoded by the corresponding nucleotide sequence is heterologous to the microorganism, which means that it does not naturally occur in this microorganism. More preferably, the encoded enzyme is derived from another microorganism, especially from a microorganism of a different genus or species. In another embodiment, the enzyme is artificial in that it does not exist in nature. This includes improved enzyme variants that have been prepared by mutagenesis protocols or genetic engineering methods.

[0367] In another preferred embodiment, the aldehyde lyase (EC 4.1.2.-) or transaldolase (EC 2.2.1.2) encoded by the corresponding nucleotide sequence is heterologous with respect to the microorganism, which means that it does not naturally occur in this microorganism. More preferably, the encoded enzyme originates from another microorganism, especially from a microorganism of a different genus or a different species. In another embodiment, the enzyme is artificial in that it does not exist in nature. This includes improved enzyme variants that have been prepared by mutagenesis protocols or genetic engineering methods.

[0368] In a particularly preferred embodiment, the enzymes mentioned in both (a) and (b) above are heterologous with respect to the microorganism.

[0369] The present invention also relates to the use of the microorganism of the present invention for first converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) by the enzyme mentioned in (a), and then further converting the produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) by the enzyme mentioned in (b).

[0370] The present invention further relates to a recombinant microorganism which has been transformed with the nucleotide sequence

[0371] (a) a nucleotide sequence encoding a phosphomonoester hydrolase (EC 3.1.3.-); and

[0372] (b) a nucleotide sequence encoding fructose bisphosphate aldolase (EC 4.1.2.13);

[0373] The microorganism further has phosphoglucomutase (EC 5.4.2.2) activity or phosphomannose mutase (EC 5.4.2.8) activity.

[0374] In a preferred embodiment, the phosphomonoester hydrolase (EC 3.1.3.-) encoded by the corresponding nucleotide sequence is heterologous to the microorganism, which means that it does not naturally occur in this microorganism. More preferably, the encoded enzyme is derived from another microorganism, especially from a microorganism of a different genus or species. In another embodiment, the enzyme is artificial in that it does not exist in nature. This includes improved enzyme variants that have been prepared by mutagenesis protocols or genetic engineering methods.

[0375] In another preferred embodiment, the fructose bisphosphate aldolase (EC 4.1.2.13) encoded by the corresponding nucleotide sequence is heterologous to the microorganism, which means that it does not naturally occur in this microorganism. More preferably, the encoded enzyme is derived from another microorganism, especially from a microorganism of a different genus or species. In another embodiment, the enzyme is artificial in that it does not exist in nature. This includes improved enzyme variants that have been prepared by mutagenesis protocols or genetic engineering methods.

[0376] In a particularly preferred embodiment, the enzymes mentioned in both (a) and (b) above are heterologous with respect to the microorganism.

[0377] In yet another preferred embodiment, this microorganism has been further transformed with the nucleotide sequence

[0378] (c) a nucleotide sequence encoding an enzyme selected from the group consisting of:

[0379] (i) phosphoglucomutase (EC 5.4.2.2); or

[0380] (ii) Phosphomannose mutase (EC 5.4.2.8).

[0381] In a preferred embodiment, the phosphoglucomutase (EC 5.4.2.2) or phosphomannose mutase (EC 5.4.2.8) encoded by the corresponding nucleotide sequence is heterologous to the microorganism, which means that it does not naturally occur in this microorganism. More preferably, the encoded enzyme is derived from another microorganism, especially from a microorganism of a different genus or species. In another embodiment, the enzyme is artificial in that it does not exist in nature. This includes improved enzyme variants that have been prepared by mutagenesis protocols or genetic engineering methods.

[0382] In a particularly preferred embodiment, all three enzymes mentioned above in (a), (b) and (c) are heterologous with respect to the microorganism.

[0383] The present invention also relates to the use of the microorganism of the present invention for first converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde by the enzyme mentioned in (a), and then further converting the produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) by the enzyme mentioned in (b), and then further converting the produced fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) by the enzyme mentioned in (c).

[0384] The recombinant microorganism of the invention may also exhibit one or more of the characteristics as described above for the microorganisms in which the methods of the invention can be performed.

[0385] Thus, in a preferred embodiment, the microorganism is a recombinant microorganism that has been transformed with the nucleotide sequence

[0386] (a) a nucleotide sequence encoding a phosphomonoester hydrolase (EC 3.1.3.-); and

[0387] (b) a nucleotide sequence encoding an enzyme selected from

[0388] (i) aldehyde lyase (EC 4.1.2.-); and / or

[0389] (ii) transaldolase (EC 2.2.1.2);

[0390] and / or

[0391] has been transformed with the following nucleotide sequence

[0392] (a) a nucleotide sequence encoding a phosphomonoester hydrolase (EC 3.1.3.-); and

[0393] (b) a nucleotide sequence encoding fructose bisphosphate aldolase (EC 4.1.2.13);

[0394] The microorganism also has glucose phosphomutase (EC 5.4.2.2) or phosphomannose mutase (EC 5.4.2.8) activity

[0395] and is further characterized in that it:

[0396] a) having phosphoketolase activity;

[0397] b) (i) having an attenuated or inactivated Embden-Meyerhof-Pamas pathway (EMPP) due to inactivation of the gene encoding phosphofructokinase or due to reduction of phosphofructokinase activity compared to an unmodified microorganism; or

[0398] (ii) no phosphofructokinase activity;

[0399] and

[0400] c) (i) having a weakened or inactivated pentose phosphate pathway oxidation branch (PPP) due to inactivation of the gene encoding glucose-6-phosphate dehydrogenase or due to reduced glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism; or

[0401] (ii) No glucose-6-phosphate dehydrogenase activity.

[0402] With regard to the enzymes which can be expressed by the microorganism and the preferred embodiments, the same applies as has been described above in connection with the process according to the invention and the microorganism according to the invention.

[0403] The present invention further relates to an enzyme combination comprising

[0404] (a) phosphomonoester hydrolases (EC 3.1.3.-); and

[0405] (b) an enzyme selected from the group consisting of

[0406] (i) aldehyde lyase (EC 4.1.2.-); and / or

[0407] (ii) Transaldolase (EC 2.2.1.2).

[0408] The present invention further relates to an enzyme combination comprising

[0409] (a) phosphomonoester hydrolases (EC 3.1.3.-); and

[0410] (b) fructose bisphosphate aldolase (EC 4.1.2.13); and

[0411] (c) an enzyme selected from the group consisting of

[0412] (i) phosphoglucomutase (EC 5.4.2.2); or

[0413] (ii) Phosphomannose mutase (EC 5.4.2.8).

[0414] The present invention also relates to a composition comprising the microorganism of the present invention or the enzyme combination of the present invention.

[0415] The present invention further relates to the use of the enzyme combination or microorganism or composition of the present invention for converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) first by the enzyme mentioned in (a) as described above, and subsequently further converting the produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) by the enzyme mentioned in (b), or for converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde first by the enzyme mentioned in (a) as described above, and subsequently further converting the produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) by the enzyme mentioned in (b), and subsequently further converting the produced fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) by the enzyme mentioned in (c).

[0416] For the enzymes and microorganisms listed above, the same applies as has been described above in connection with the process according to the invention, in particular with regard to the preferred embodiments.

[0417] Figure 1 Shown is the effect of AMP concentration on fructose bisphosphatase activity.

[0418] Figure 2 Shown is the effect of AMP concentration on FsaA129S.

[0419] Figure 3 Shown are the specific productivity of acetone and isopropanol for a strain overexpressing the enzyme responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose-6-phosphate (F6P) (GBI 17553, solid line) and a strain not overexpressing the enzyme responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose-6-phosphate (GBI 15847, dotted line).

[0420] In this specification, many documents including patent applications are cited. The disclosures of these documents, although not considered relevant to the patentability of the present invention, are fully incorporated herein by reference. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0421] The present invention will now be described with reference to the following examples, which are merely illustrative and are not to be construed as limiting the scope of the present invention. Example

[0422] General methods and materials

[0423] Ligation and transformation methods are well known in the art. Techniques suitable for use in the following examples can be found in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY, 1989, and Sambrook J., supra.

[0424] Materials and methods suitable for maintaining and growing bacterial cultures are well known in the art. Techniques suitable for use in the following examples can be found in Manual of Methods for General Bacteriology (Eds. Philipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg, and G. Briggs Philips).

[0425] Unless otherwise stated, all reagents and materials used for bacterial cell growth and maintenance were obtained from Sigma-Aldrich (St. Louis, MO).

[0426] Enzyme overexpression and purification

[0427] a) Enzymes from E. coli

[0428] Plasmids from the ASKA collection (Kitagawa, M et al. DNA Res. 12: 291-299 (2005)) have been used to overexpress the enzyme from E. coli.

[0429] In LB medium, strain BL21 (DE3) cells (Novagen) were cultivated and made into electrocompetent state. Electrocompetent state BL21 cells were transformed with corresponding plasmids (see Table 1) expressing the required enzymes, and then planted on LB plates containing chloramphenicol (25ug / ml). The plates were incubated overnight at 30°C.

[0430] Transformed cells were incubated at 30°C for 20 hours with shaking (160 rpm) using ZYM-5052 autoinduction medium (Studier FW, Prot. Exp. Pur. 41: 207-234 (2005)). Cells were harvested by centrifugation at 4°C, 4,000 rpm for 20 minutes and the pellets were stored at -80°C.

[0431]

[0432]

[0433]

[0434]

[0435]

[0436] After purification of the recombinant protein using His trap (Protino Ni-IDA 1000 kit, Macherey Nagel), the expression of the recombinant enzyme was checked on a protein gel. Purification was performed according to the manufacturer's recommendations.

[0437] b) Enzymes from organisms other than E. coli

[0438] For optimal expression in E. coli, (Invitrogen) codon optimized target genes from several organisms (see Table 2). In addition, a His tag was added at the 5' end of the gene and an additional stop codon was added at the 3' end of the gene. The gene construct was flanked by NdeI and EcoRI restriction sites and provided internally in plasmid pET25b+ (Merckmillipore).

[0439] Competent E. coli BL21 (DE3) cells (Novagen) were transformed with these vectors according to a standard heat shock method. The transformed cells were cultured at 30°C for 20 hours with shaking (160 rpm) using ZYM-5052 autoinduction medium (Studier FW, Prot. Exp. Pur. 41: 207-234 (2005)). The cells were collected by centrifugation at 4°C, 4,000 rpm for 20 minutes and the pellets were stored at -80°C.

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446] After purification of the recombinant protein using His trap (Protino Ni-IDA 1000 kit, Macherey Nagel), the expression of the recombinant enzyme was checked on a protein gel. Purification was performed according to the manufacturer's recommendations.

[0447] Example 1: Fructose-6-phosphate aldolase and fructose diphosphate Enzymes Activity inhibition test

[0448] A series of tests were performed to determine whether AMP had an inhibitory effect on the enzyme activity of fructose-6-phosphate aldolase and / or fructose bisphosphatase. The method used to test the enzyme activity was adapted from C. Guérard-Hélaine, V. De Berardinis, M. Besnard-Gonnet, E. Darii, M. Debacker et al., Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. ChemCatChem, Wiley, 7: 1871-1879 (2015).

[0449] a) Effect of AMP concentration on fructose bisphosphatase activity

[0450] Each kinetic assay system contained 120 μl of Tris HCl buffer (50 mM; pH 7.5), 20 mM NaCl, 10 mM MgCl2, 1 mM NADP + , AMP (several tested concentrations), 1 mM fructose 1,6-bisphosphate (F1,6bisP), 0.2 mg / ml FBP enzyme and auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP + The mixture was incubated at 30°C for up to 20 minutes and the reaction was monitored spectrophotometrically at 340 nm (NADPH formation was measured), assuming that 1 reduced NADPH molecule was generated per fructose-6-phosphate molecule. Figure 1 The results are shown in . A strong inhibitory effect of AMP on fructose bisphosphatase activity was observed.

[0451] b) Effect of AMP concentration on fructose-6-phosphate aldolase activity of FsaA A129S

[0452] 120 μl of each kinetic assay system contained Tris HCl buffer (50 mM; pH 8.5), 1 mM NADP + , AMP (several tested concentrations), 200 mM DHA, 3 mM D,L-G3P, 0.4 mg / ml FsaA A129S and auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP + dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg / ml each).

[0453] The mixture was incubated at 30°C for up to 20 minutes and the reaction was monitored spectrophotometrically at 340 nm (NADPH formation was measured), assuming that 1 reduced NADPH molecule was generated per fructose-6-phosphate molecule. Figure 2 The results are shown in. No AMP inhibition was observed with FSAA A129S.

[0454] Example 2: In vitro conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) into fructose-6-phosphate (F6P) via dihydroxyacetone (DHA) intermediate.

[0455] A series of tests were performed to determine the best enzyme combinations for the conversion of G3P and DHAP to F6P. These enzyme combinations should perform 2 steps:

[0456] 1) DHAP → DHA

[0457] 2) DHA+G3P→F6P

[0458] a) Enzyme that catalyzes the conversion of DHA and G3P into F6P

[0459] The method used to test enzyme activity was adapted from C. Guérard-Hélaine, V. De Berardinis, M. Besnard-Gonnet, E. Darii, M. Debacker et al., Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. ChemCatChem, Wiley, 7: 1871-1879 (2015).

[0460] Each kinetic assay system of 120 μl contained Tris HCl buffer (50 mM; pH 8.5), 3 mM D,L-G3P, 200 mM DHA, 1 mM NADP +, 0.4 mg / ml enzyme and auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP + The mixture was incubated at 30°C and the reaction was monitored spectrophotometrically at 340 nm (NADPH formation was measured), assuming that 1 reduced NADPH molecule was generated per F6P molecule. The results are shown in Table 3.

[0461]

[0462] Table 3: Production of F6P from DHA and G3P using different enzymes

[0463] a) Enzyme that catalyzes the conversion of DHAP to DHA

[0464] The method used to test enzyme activity was adapted from C. Guérard-Hélaine, V. De Berardinis, M. Besnard-Gonnet, E. Darii, M. Debacker et al., Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. ChemCatChem, Wiley, 7: 1871-1879 (2015).

[0465] Each kinetic assay system contained 120 μl of Tris HCl buffer (50 mM; pH 8.5), 10 mM MgCl2, 100 mM DHAP, 0.8 mM NADP + , 0.6 mg / ml enzyme, 0.8 mg / ml fructose-6-phosphate aldolase 1 (mutant FSAA A129S) and auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP + The mixture was incubated at 30°C and the reaction was monitored spectrophotometrically at 340 nm (NADPH formation was measured), assuming that 1 reduced NADPH molecule was generated per F6P molecule. The results are shown in Table 3.

[0466]

[0467] Table 4: Production of DHA from DHAP using different enzymes

[0468] Example 3: In vitro conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose-6-phosphate (F6P) via a glyceraldehyde intermediate.

[0469] A series of assays were performed to determine the best enzyme combination for the conversion of G3P and DHAP to F6P. The best enzyme combination should perform 3 steps:

[0470] 1) G3P → Glyceraldehyde

[0471] 2) Glyceraldehyde + DHAP → F1P

[0472] 3) F1P → F6P

[0473] a) Enzyme that catalyzes the conversion of G3P to glyceraldehyde

[0474] 200 μl of each kinetic assay system contained Tris HCl buffer (50 mM pH 7.5), 100 mM NaCl, 10 mM MgCl2, G3P (1-10-50 mM) and 2 mg / ml of the tested enzyme (see Table 7). The mixture was incubated overnight at 30°C and the reaction was quenched with 1 volume of acetonitrile. The final product was analyzed by LCMS. LC-MS analysis was performed on an Ultimate 3000 (Dionex, Thermo Fisher Scientific) coupled to a Q-Orbitrap mass spectrometer (ThermoFisher Scientific), which was equipped with an electrospray (ESI) source and operated in negative ion mode. Chromatographic separation was performed using a HILIC amide (1.9 μm, 2.1×150 mm) column (Waters) maintained at 25°C, run under gradient elution, as follows. The mobile phase was: (A) 10 mM ammonium formate pH 9.45 (adjusted with ammonium hydroxide), and the mobile phase (B) was 100% acetonitrile, and the flow rate was 500 μL / min. Elution started with a 1.5 minute isocratic step at 95% B, followed by a linear gradient from 95% to 55% phase B over 7 minutes. The chromatographic system was then eluted at 55% B for 2 minutes, and the run ended with an 8.5 minute equilibration step.

[0475]

[0476] Table 5: Enzymes catalyzing the conversion of G3P to glyceraldehyde.

[0477] a) An enzyme that catalyzes the conversion of glyceraldehyde and DHAP into F1P and further catalyzes the conversion of F1P into F6P

[0478] Each kinetic assay system contained 200 μl of Tris HCl buffer (50 mM; pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM NADP +, 10 mM DHAP, 10 mM glyceraldehyde, 1 mg / ml AldoB, 1 mg / ml PGM and PMM and auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP + The mixture was incubated at 30°C and the reaction was monitored spectrophotometrically at 340 nm (NADPH formation was measured), assuming that 1 reduced NADPH molecule was generated per F6P molecule. The results are shown in Table 6.

[0479]

[0480] Table 6: Enzymes catalyzing the conversion of glyceraldehyde and DHAP to F1P and further catalyzing the conversion of F1P to F6P. The enzyme encoded by ALDOB was incubated together with the enzyme encoded by pgm (Assay 1), with the enzyme encoded by AHA_2903 (Assay 2), or with both (Assay 3).

[0481] Example 4: Construction of a new E. coli prototype that produces acetone and isopropanol

[0482] Similar to most organisms, E. coli converts glucose to acetyl-CoA. A modified E. coli prototype in which acetyl-CoA production efficiency was optimized has been described previously (WO 2013 / 007786). A bacterial prototype with the following genotype was constructed, Strain A :

[0483] MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB

[0484] Strain A Plasmid-based overexpression of the PKT gene of the phosphoketolase YP 003354041.1 from Lactococcus lactis produces Strain B , i.e., a strain with a rearranged central carbon metabolism in which a new carbon dissimilatory pathway based on phosphoketolase replaces the inactivated Embden-Meyerhoff-Parnas pathway (EMPP), pentose phosphate pathway (PPP), and Entner-Doudoroff pathway (EDP). Compared with the wild-type MG1655 strain expressing the same acetone pathway, once Strain B When the acetone pathway was introduced into the acetone reaction, superior acetone yields were observed.

[0485] In order to construct a strain that possesses the PKT pathway and can grow robustly on sucrose as a carbon source, Strain A Further engineered as described below.

[0486] The PKT gene was introduced into the kdgk locus (kdgK::P1_RBST7_pkt). Strain A The resulting strain had the following genotype:

[0487] MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB kdgK::P1_RBST7_pkt

[0488] This strain was passaged for several months on minimal medium supplemented with glucose as the carbon source with continuous selection for clones or populations with the highest growth rate until a doubling time of less than 5 hours was achieved.

[0489] Several gene deletions were made to increase acetone and isopropanol production: ΔhemAΔfsaAΔfsaB.

[0490] To further increase isopropanol production, pntAB (pyridine nucleotide transhydrogenase subunits α and β, Uniprot P07001 and P0AB67, NCBI reference sequences: NP_416120.1 and NP_416119.1) genes from E. coli were overexpressed by inserting a strong constitutive promoter at the pntAB locus.

[0491] The resulting strain is hereinafter referred to as Strain C .

[0492] Example 5: Construction of E. coli strains producing acetone and isopropanol from acetyl-CoA

[0493] This working example shows the production of acetone and isopropanol by a recombinant E. coli strain expressing the genes that constitute the acetone and isopropanol pathways.

[0494] Table 7 lists the enzymes used in this study to convert acetyl-CoA to acetone and isopropanol.

[0495]

[0496]

[0497] Table 7: Enzymes catalyzing the conversion of acetyl-CoA to acetone and isopropanol

[0498] Expression of the acetone / isopropanol biosynthetic pathway in Escherichia coli.

[0499] Use the method described in Example 4 Strain C as host microorganisms.

[0500] All listed genes were codon optimized for expression in E. coli and (Thermofisher), with the exception of the genes atoD and atoA. The last two genes were amplified directly from genomic DNA of Escherichia coli MG1655.

[0501] The expression vector containing the replication origin pSC and the spectinomycin resistance marker was used to express the genes thlA, atoD, atoA, adc and adh. The constructed vector was named pGB5344.

[0502] The enzyme responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose-6-phosphate (F6P) is expressed in E. coli.

[0503] A modified version of pUC18 (New England Biolabs) containing a modified multiple cloning site (pUC18 MCS) (WO 2013 / 007786) and an ampicillin resistance gene (plasmid pGB 271) was used to overexpress the genes listed in Table 8.

[0504]

[0505] Table 8: Enzymes catalyzing the conversion of DHAP and G3P to F6P.

[0506] Different combinations of plasmids were transformed into Strain C The strains generated in this way are summarized in Table 9.

[0507]

[0508]

[0509] Table 9: Strains generated for in vivo conversion of glucose to acetone + isopropanol.

[0510] Example 6: E. coli strain growth and production of acetone / isopropanol from acetyl-CoA

[0511] Pre-culture conditions

[0512] The transformed cells were then plated on LB plates supplemented with ampicillin (100 μg / ml) and spectinomycin (100 μg / ml). The plates were incubated at 30°C for 2 days. The isolated colonies were used to inoculate LB medium supplemented with ampicillin and spectinomycin. These precultures were cultivated at 30°C to achieve an optical density of 0.6.

[0513] Growing Conditions:

[0514] Fermentation was performed in a 1 liter bioreactor (Multifors 2, Infors HT) with pH and temperature control. Cells from the preculture were used to inoculate 500 ml of fermentation medium (Table 10) supplemented with ampicillin (100 μg / ml), spectinomycin (100 μg / ml), thiamine (0.6 mM), glucose (1 g / l) and glycerol (5 g / L) to achieve an initial optical density (OD) of 0.05. 600 During the growth phase, the temperature (T = 32°C), pH = 6.5 and pO2 = 5% were maintained constant. The glucose feed was increased from 0.1 g / g DCW / hour to 0.35 g / g DCW / hour. When OD 600 At 2, 8 and 20, 5 g / L yeast extract was added in pulses.

[0515]

[0516] Table 10. Fermentation medium composition (derived from ZYM-5052 medium (Studier FW, Prot. Exp. Pur. 41, (2005), 207-234)).

[0517] Acetone / isopropyl alcohol production stage

[0518] During this phase, the temperature T = 34°C, pH 6.5 and pO2 = 5% were kept constant. Glucose feed was started at 0.50 g sucrose / g DCW / hour and subsequently amended according to strain consumption. Glycerol concentration was maintained above 2 g / l.

[0519] The acetone / isopropanol production of the strains was continuously analyzed using a gas chromatograph 7890A (Agilent Technology) equipped with a flame ionization detector (FID) to measure acetone and isopropanol. Volatile organic compounds were separated by chromatography using an Agilent 1260 InfinityII chromatograph on a Hi-Plex H USP L17, 100x7.7mm (Agilent). Acetone / isopropanol was quantified using a standard (Sigma).

[0520] Figure 3 Shown is a comparison between the observed specific productivity of acetone and isopropanol for a production strain expressing the enzyme responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHA) to fructose-6-phosphate (F6P) or as a control a strain not expressing the enzyme responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHA) to fructose-6-phosphate (F6P).

[0521] When the enzyme responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose-6-phosphate (F6P) was overexpressed (strain GBI 17553), the specific productivity (moles produced per unit of cell weight per unit time) of acetone and isopropanol was higher compared to strain GBI 15847.

Claims

1. A method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), comprising the steps of: (a) enzymatic conversion of dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) by a phosphomonoester hydrolase with an EC number of EC 3.1.3.-; and (b) enzymatic conversion of the dihydroxyacetone (DHA) thus produced together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) by an aldehyde lyase with an EC number of EC 4.1.2.- or a transaldolase with an EC number of EC 2.2.1.2, wherein the method is carried out in vivo in a recombinant microorganism which has been transformed with a nucleotide sequence encoding an enzyme that can catalyze the conversion process listed in step (a) and a nucleotide sequence encoding an enzyme that can catalyze the conversion process listed in step (b).

2. A method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), comprising the steps of: (a') enzymatic conversion of glyceraldehyde-3-phosphate (G3P) into glyceraldehyde by a phosphomonoester hydrolase with an EC number of EC 3.1.3.-; and (b') enzymatic conversion of the glyceraldehyde thus produced together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) by fructose bisphosphate aldolase having the EC number EC 4.1.2.13; and (c') enzymatically converting the fructose-1-phosphate (F1P) thus produced into fructose-6-phosphate (F6P) by a phosphoglucomutase with an EC number of EC 5.4.2.2 or a phosphomannose mutase with an EC number of EC 5.4.2.8, wherein the method is carried out in vivo in a recombinant microorganism that has been transformed with a nucleotide sequence encoding an enzyme that can catalyze the conversion process listed in step (a') and a nucleotide sequence encoding an enzyme that can catalyze the conversion process listed in step (b').

3. The method according to claim 1, wherein the phosphorus monoester hydrolase with EC number EC 3.1.3.- is selected from: (i) Sugar phosphatases with the EC number EC 3.1.3.23; (ii) 6-phosphogluconate phosphatase with the EC number EC 3.1.3.-; (iii) pyridoxal phosphate phosphatase with the EC number EC 3.1.3.74; (iv) fructose-1-phosphate phosphatase with EC number EC 3.1.3.-; (v) dihydroxyacetone phosphatase with the EC number EC 3.1.3.-; (vi) hexitol phosphatases with the EC number EC 3.1.3.-; (vii) acid phosphatases with the EC number EC 3.1.3.2; (viii) alkaline phosphatase with the EC number EC 3.1.3.1; (ix) glycerol-1-phosphate phosphatase having the EC number EC 3.1.3.21; and (x) 3-Phosphoglycerate phosphatase with the EC number EC 3.1.3.

38.

4. The method according to claim 2, wherein the phosphorus monoester hydrolase with EC number EC 3.1.3.- is selected from: (i) glyceraldehyde 3-phosphate phosphatase with EC number EC 3.1.3.-; (ii) alkaline phosphatase with the EC number EC 3.1.3.1; (iii) acid phosphatases with the EC number EC 3.1.3.2; (iv) sugar phosphatases with the EC number EC 3.1.3.23; and (v) Hexitol phosphatase with EC number EC 3.1.3.-.

5. The method according to claim 2, wherein the microorganism is further transformed with a nucleotide sequence encoding an enzyme that can catalyze the transformation process recited in step (c') of claim 2.

6. The method according to any one of claims 1 to 3 or 4 or 5, wherein the microorganism is further characterized in that it a) having phosphoketolase activity; b) (i) having an attenuated or inactivated Embden-Meyerhof-Parnas pathway (EMPP) due to inactivation of the gene encoding phosphofructokinase or due to reduction of phosphofructokinase activity compared to an unmodified microorganism; or (ii) no phosphofructokinase activity; and c) (i) having an impaired or inactivated pentose phosphate pathway (PPP) oxidation branch due to inactivation of the gene encoding glucose-6-phosphate dehydrogenase or due to reduced glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism; or (ii) No glucose-6-phosphate dehydrogenase activity.

7. The method of claim 6, wherein the microorganism is further characterized in that EMPP is further impaired or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified microorganism.

8. A recombinant microorganism which has been transformed with the following nucleotide sequence (a) a nucleotide sequence encoding a phosphomonoester hydrolase having an EC number of EC 3.1.3.-; and (b) a nucleotide sequence encoding an enzyme selected from (i) an aldehyde lyase with the EC number EC 4.1.2.-; and / or (ii) a transaldolase having an EC number of EC 2.2.1.2, The recombinant microorganism is further characterized in that: a) having phosphoketolase activity; b) (i) having an attenuated or inactivated Embden-Meyerhof-Parnas pathway (EMPP) due to inactivation of the gene encoding phosphofructokinase or due to reduction of phosphofructokinase activity compared to an unmodified microorganism; or (ii) no phosphofructokinase activity; and c) (i) having an impaired or inactivated pentose phosphate pathway (PPP) oxidation branch due to inactivation of the gene encoding glucose-6-phosphate dehydrogenase or due to reduced glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism; or (ii) No glucose-6-phosphate dehydrogenase activity.

9. A recombinant microorganism which has been transformed with the following nucleotide sequence (a) a nucleotide sequence encoding an enzyme having an EC number of EC 3.1.3.-phosphorus monoester hydrolase; and (b) a nucleotide sequence encoding a fructose bisphosphate aldolase having an EC number of EC 4.1.2.13; The microorganism further has a phosphoglucomutase activity with an EC number of EC 5.4.2.2 or a phosphomannose mutase activity with an EC number of EC 5.4.2.8, The recombinant microorganism is further characterized in that: a) having phosphoketolase activity; b) (i) having an attenuated or inactivated Embden-Meyerhof-Parnas pathway (EMPP) due to inactivation of the gene encoding phosphofructokinase or due to reduction of phosphofructokinase activity compared to an unmodified microorganism; or (ii) no phosphofructokinase activity; and c) (i) having an impaired or inactivated pentose phosphate pathway (PPP) oxidation branch due to inactivation of the gene encoding glucose-6-phosphate dehydrogenase or due to reduced glucose-6-phosphate dehydrogenase activity compared to an unmodified microorganism; or (ii) No glucose-6-phosphate dehydrogenase activity.

10. The recombinant microorganism according to claim 9, which has been further transformed with the following nucleotide sequence: (c) a nucleotide sequence encoding an enzyme selected from: (i) phosphoglucomutase with the EC number EC 5.4.2.2; and (ii) Phosphomannose mutase with the EC number EC 5.4.2.

8.

11. The recombinant microorganism according to claim 8 or 9, further characterized in that EMPP is further weakened or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified microorganism.

12. Use of the microorganism according to claim 8 for first converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) by the enzyme mentioned in (a) and then further converting the produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) by the enzyme mentioned in (b).

13. Use of the microorganism according to claim 9 or 10 for first converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde by the enzyme mentioned in (a) and then further converting the produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) by the enzyme mentioned in (b), and then further converting the produced fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) by the enzyme mentioned in (c).

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