The present invention relates to a recombinant microorganism characterized by: a) having an inactivation of the gene(s) encoding phosphofructokinase or showing a reduced phosphofructokinase activity as compared to a non-modified microorganism; b) having an inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or showing a reduced glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism; and c) having a genetic modification to have an increased expression of the gene(s) encoding deoxy-D-ribose-5- phosphate aldolase (EC 4.1.2.4) and/or an increased deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) activity as compared to a non-modified microorganism. Moreover, the present invention relates to the use of this recombinant microorganism for the conversion of acetyl-CoA into acetone, isobutene, propene and/or isopropanol. Further, the present invention relates to a method for producing acetone, isobutene, propene and/or isopropanol from acetyl-CoA comprising the steps of culturing the recombinant microorganism of the present invention in a suitable medium.

For the past several decades, practitioners of metabolic engineering have endeavoured to provide biological solutions for the production of chemicals, thus, providing alternatives to more traditional chemical processes. In general, biological solutions allow for the utilization of renewable feedstocks (e.g. sugars) and compete with existing petrochemical based processes. A multi-step, biological solution for the production of a chemical typically comprises a microorganism as the catalyst for the conversion of feedstock to a target molecule. A complete set of enzyme reactions for the production of a particular target molecule can be grouped into those belonging to central carbon pathways and those belonging to the product specific pathway. The reactions belonging to central carbon and product specific pathways are linked in that redox (typically, NAD(P)FI) and energetic (typically, ATP) constraints of each and every enzyme reaction must be accounted for in an overall balance contributing to the competitiveness of the process. Historically, central carbon pathways of heterotrophs growing on sugars 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, 2 ^nd Edition, Springer-Verlag, New York). Each central pathway or combinations of central pathways offer advantages and disadvantages with respect to a specific target molecule. In order to provide competitive bioprocesses, 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); C. T. Trinh et al., Appl. Environ. Microbiol. 74:3634 (2008)). More recently, recombinant microorganisms with modifications involving the PKP have been described (see Sonderegger et al. Appl. Environ. Microbiol. 70 (2004), 2892-2897, US Patent 7,253,001, Chinen et al. J. Biosci. Bioeng. 103 (2007), 262-269, US Patent 7,785,858; Fleige et al., Appl. Microbiol. Cell Physiol. 91 (2011), 769-776).

The EMPP converts 1 mol glucose to 2 mol pyruvate (PYR). When acetyl-CoA is desired, 1 mol PYR can be converted to 1 mol of acetyl-CoA with the concomitant generation of 1 mol C0 ₂ and 1 mol NADH. The sum of the reactions is given in Equation 1.

The PPP provides a means to convert 1 mol glucose to 1 mol C0 ₂ and 2 mol NADPH, with the concomitant generation of 0.67 mol fructose-6-phosphat (F6P) and 0.33 mol glyceraldehyde-3-phosphate (GAP). The F6P and GAP thus formed must be metabolized by other reaction pathways, e.g. by the EMPP. The EDP converts 1 mol glucose to 1 mol GAP and 1 mol PYR with the concomitant generation of 1 mol NADPH. As with the PPP, the GAP thus formed must be metabolized by other reaction pathways. The PKP provides a means to convert 1 mol glucose to 1 mol GAP and 1.5 mol acetyl phosphate (AcP). When acetyl-CoA is desired, 1 equivalent of AcP plus 1 equivalent coenzyme A (CoA) can be converted to 1 equivalent acetyl- CoA and 1 equivalent inorganic phosphate (Pi) by the action of phosphotransacetylase.

For specific target molecules derived from AcCoA moieties generated through the PKP and near redox neutrality to the AcCoA moieties, there exists a deficiency in the overall energy balance. The PKP (and, similarly, the PPP and EDP) does 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. through the EMPP. Recycling GAP through the PKP exacerbates the issue, particularly when the product specific pathway provides little ATP. Sonderegger (loc. cit.) and US Patent 7,253,001 disclose recombinant Saccharomyces cerevisiae strains comprising native or overexpressed phosphoketolase activity together with overexpressed phosphotransacetylase to increase the yield in the conversion of glucose/xylose mixtures to ethanol. These strains feature PEP-independent glucose uptake with both the EMPP and the PPP operative.

Chinen (loc. cit.) and US Patent 7,785,858 disclose a recombinant bacterium selected from the group consisting of the Enterobacteriaceae familiy, Coryneform bacterium, and Bacillus bacterium comprising increased phosphoketolase activity for the conversion of glucose to target molecules which are produced via the intermediate acetyl-CoA, including the group consisting of L-glutamic acid, L- glutamine, L-proline, L-arginine, L-leucine, L-cysteine, succinate and polyhydroxybutyrate. These strains feature PEP-dependent glucose uptake with the EMPP operative. Notably, the activity of phosphofructokinase in the bacterium of US Patent 7,785,858 is reduced compared to that of a wild-type or non-modified strain (see page 33).

WO 2013/007786 describes a recombinant microorganism which has phosphoketolase activity and in which the EMPP is deactivated or diminished by abolishing or reducing phosphofructokinase and in which the oxidative branch of the PPP is deactivated or diminished by abolishing or reducing glucose-6-phosphate dehydrogenase.

This is schematically illustrated in Figure 1 wherein the metabolic steps starting from glucose into fructose-6-phosphate are shown. In a first step, glucose is converted into glucose-6-phosphate by a glucose kinase (EC 2.7.1.2) and/or an N- acetylmannosamine kinase (EC 2.7.1.60). Said glucose-6-phosphate is further converted into fructose-6-phosphate by a glucose-6-phosphate isomerase (EC 5.3.1.9). No conversion from glucose-6-phosphate into 6-phosphate-gluconolacetate occurs when abolishing or reducing glucose-6-phosphate dehydrogenase (EC 1.1.1.49). No conversion from fructose-6-phosphate into fructose-1 ,6-bisphosphate occurs when abolishing or reducing phosphofructokinase (EC 2.7.1.11 ).

These measures lead to an increase in the yield of acetyl-CoA from glucose.

The pathways leading from glucose via fructose-6-phosphate to acetyl-CoA are schematically illustrated in Figure 2, 3, 4 and 5. Figures 2, 3 and 4 also illustrate the overall pathway from glucose via fructose-6-phosphate into D-ribose-1 -phosphate. As illustrated in Figure 2 and Figure 3, respectively, phosphoketolase may catalyze a step acting on D-xylulose-5-phosphate (EC 4.1.2.9) or on fructose-6-phosphate activities (EC 4.1.2.22), or both. Herein, phosphoketolase refers to any protein having at least one of these activities (EC 4.1.2.9 and/or EC 4.1.2.22).

In the overall pathway shown in Figure 2, fructose-6-phosphate and glyceraldehyde- 3-phosphate may be converted into erythrose-4-phosphate and xylulose-5-phosphate by a transketolase (EC 2.2.1.1) (step A as illustrated in Figure 2). Xylulose-5-phosphate may be converted into acetyl-phosphate and glyceraldehyde- 3-phosphate by a phosphoketolase (EC 4.1.2.9 or EC 4.1.2.22) (step E as illustrated in Figure 2).

Fructose-6-phosphate and erythrose-4-phosphate may be converted into glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate by a transaldolase (EC 2.2.1.2) (step C as illustrated in Figure 2).

Glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate may be converted into xylulose-5-phosphate and ribose-5-phosphate by a transketolase (EC 2.2.1.1) (step D as illustrated in Figure 2).

Xylulose-5-phosphate may be converted into ribulose-5-phosphate by a ribulose- phosphate-3-epimerase (EC 5.1.3.1) (step F as illustrated in Figure 2). Ribulose-5-phosphate may be converted into ribose-5-phosphate by a ribose-5- phosphate isomerase (EC 5.3.1.6) (step G as illustrated in Figure 2). Ribose-5-phosphate may be converted into ribose-1 -phosphate by a phosphopentomutase (EC 5.4.2.7) (step H as illustrated in Figure 2). Acetyl-phosphate may be converted into acetyl-CoA by a phosphate acetyltransferase (EC 2.3.1.8) (step I as illustrated in Figure 2).

In this overall pathway as shown in Figure 3, fructose-6-phosphate may be converted into acetyl-phosphate (which is then further converted into acetyl-CoA by a phosphate acetyltransferase (EC 2.3.1.8)) and erythrose-4-phosphate by a phosphoketolase activity (EC 4.1.2.9 and/or EC 4.1.2.22) (step B as illustrated in Figure 3).

Fructose-6-phosphate and erythrose-4-phosphate may be converted into glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate by a transaldolase (EC 2.2.1.2) (step C as illustrated in Figure 3).

Glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate may be converted into xylulose-5-phosphate and ribose-5-phosphate by a transketolase (EC 2.2.1.1) (step D as illustrated in Figure 3).

Xylulose-5-phosphate may be converted into ribulose-5-phosphate by a ribulose- phosphate-3-epimerase (EC 5.1.3.1) (step F as illustrated in Figure 3). Ribulose-5-phosphate may be converted into ribose-5-phosphate by a ribose-5- phosphate isomerase (EC 5.3.1.6) (step G as illustrated in Figure 3). Ribose-5-phosphate may be converted into ribose-1 -phosphate by a phosphopentomutase (EC 5.4.2.7) (step H as illustrated in Figure 3). Acetyl-phosphate may be converted into acetyl-CoA by a phosphate acetyltransferase (EC 2.3.1.8) (step I as illustrated in Figure 3).

A phosphoketolase does not necessarily have to be present.

Hence, the above conversions of the overall pathway may also take place in the absence of a phosphoketolase (i.e., a phosphoketolase classified as EC 4.1.2.9 and/or a phosphoketolase classified as EC 4.1.2.22) (as illustrated in Figure 4). Indeed, the conversion of fructose-6-phosphate and glyceraldehyde-3-phosphate (the origin of glyceraldehyde-3-phosphate is illustrated in Figures 4 and 5) into erythrose-4-phosphate and xylulose-5-phosphate by a transketolase (EC 2.2.1.1) from the pentose phosphate pathway (step A as illustrated in Figure 4) occurs naturally in organisms with a functional pentose phosphate pathway.

In microorganisms wherein the first enzyme leading to the pentose phosphate pathway, i.e., the glucose-6-phosphate dehydrogenase encoding gene is inactivated or the glucose-6-phosphate dehydrogenase shows a reduced activity (see step C in Figure 1) the overall pathway is still functional via the conversion of fructose-e- phosphate and glyceraldehyde-3-phosphate into erythrose-4-phosphate and xylulose-5-phosphate by a transketolase (EC 2.2.1.1).

Fructose-6-phosphate and erythrose-4-phosphate may be converted into glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate by a transaldolase (EC 2.2.1.2) (step C as illustrated in Figure 4).

Glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate may be converted into xylulose-5-phosphate and ribose-5-phosphate by a transketolase (EC 2.2.1.1) (step D as illustrated in Figure 4).

Xylulose-5-phosphate may be converted into ribulose-5-phosphate by a ribulose- phosphate-3-epimerase (EC 5.1.3.1) (step F as illustrated in Figure 4). Ribulose-5-phosphate may be converted into ribose-5-phosphate by a ribose-5- phosphate isomerase (EC 5.3.1.6) (step G as illustrated in Figure 4). Ribose-5-phosphate may be converted into ribose-1 -phosphate by a phosphopentomutase (EC 5.4.2.7) (step H as illustrated in Figure 4). Acetyl-phosphate may be converted into acetyl-CoA by a phosphate acetyltransferase (EC 2.3.1.8) (step I as illustrated in Figure 4). The overall pathways from glucose via fructose-6-phosphate into D-ribose-1- phosphate (illustrated in Figures 2, 3 and 4) are not mutually exclusive and may concomitantly be performed in a microorganism.

As also illustrated in Figures 2, 3 and 4, ribose-1 -phosphate is then fed into naturally occurring metabolic nucleoside and nucleotide degradation super-pathways well- known and described in the prior art which ultimately lead to the production of 2- deoxy-D-ribose 1 -phosphate (see also Figure 5).

As acetyl-CoA is a central metabolite and can be further converted into useful substances like acetone, isobutene, propene and isopropanol, there is still a need to further improve efficiency and effectiveness of the production of acetyl-CoA in order to increase its yield and to thereby make the production of substances like acetone, isobutene, propene and isopropanol more commercially attractive.

The present invention is based on the fact that the efficiency of converting glucose into acetyl-CoA could be further improved in a recombinant microorganism by making use of an increased expression of the gene(s) encoding deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) and/or an increased deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) activity as compared to a non-modified microorganism.

Thus, the present invention meets this demand by providing a recombinant microorganism characterized by: a) having an inactivation of the gene(s) encoding phosphofructokinase or showing a reduced phosphofructokinase activity as compared to a non- modified microorganism; b) having an inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or showing a reduced glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism; and c) having a genetic modification to have an increased expression of the gene(s) encoding deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) and/or an increased deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) activity as compared to a non-modified microorganism.

The genetic modification of the present invention to have an increased expression of the gene(s) encoding deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) and/or an increased deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) activity as compared to a non-modified microorganism (described in more detail further below) is present in a microorganism which is furthermore characterized in that: (a) it has a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP), preferably by having an inactivation of the gene(s) encoding phosphofructokinase or showing a reduced phosphofructokinase activity as compared to a non-mod ified microorganism; and b) it has a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP), preferably by having an inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or showing a reduced glucose-6- phosphate dehydrogenase activity as compared to a non-modified microorganism.

In a preferred embodiment, such a microorganism is further characterized by: having phosphoketolase activity or having a genetic modification to have an increased expression of a gene encoding a phosphoketolase and/or an increased phosphoketolase activity as compared to a non-modified microorganism; or having no phosphoketolase activity.

Accordingly, in a preferred embodiment, such a microorganism is characterised by having phosphoketolase activity, so as to increase the flux of acetyl-CoA produced. Usually, a microorganism converts glucose via the Embden-Meyerhof-Parnas pathway into pyruvate which can then be converted into acetyl-CoA by the enzyme pyruvate dehydrogenase. However, this conversion is accompanied by the release of C0 ₂ and, thus, one carbon atom is lost which might have been used in the production of useful metabolites. In order to increase the amount of acetyl-CoA in a microorganism it is therefore desirable that acetyl-CoA is formed via a different pathway to avoid the loss of carbon atoms. By using a microorganism having phosphoketolase activity, phosphate and fructose-6-phosphate are converted to erythrose-4-phosphate and acetylphosphate and the phosphotransacetylase further converts acetylphosphate into acetyl-CoA without loss of a carbon atom. Thus, in the end, the yield of acetyl-CoA can be increased by using a microorganism having phosphoketolase activity. Such a microorganism is capable of converting glucose into acetyl-CoA without loss of a carbon atom. Recombinant microorganisms in which a phosphoketolase is naturally or heterologously expressed are disclosed in US 7,785,858 and US 7,253,001.

The term “phosphoketolase activity” as used herein means an enzymatic activity that is capable of converting D-xylulose-5-phosphate into D-glyceraldehyde-3-phosphate according to the following reaction:

D-xylulose-5-phosphate + phosphate - D-glyceraldehyde-3-phosphate + acetyl phosphate + water or that is capable to catalyze the above shown reaction and that is also able to convert D-fructose-6-phosphate to D-erythrose-4-phosphate according to the following reaction:

D-Fructose 6-phosphate + phosphate - acetyl phosphate + D-erythrose 4- phosphate + water

The former phosphoketolases are usually classified in EC 4.1.2.9 and the latter in EC 4.1.2.22. Both types of phosphoketolases can be employed in the scope of the present invention. Figures 2 and 3 show schemes for the overall reactions using the two options of the phosphoketolase as described herein (see step E in Figure 2 and step B in Figure 3).

As mentioned above, in accordance with the present invention, phosphoketolase may catalyze a step acting on D-xylulose-5-phosphate (EC 4.1.2.9) or on fructose-e- phosphate activities (EC 4.1.2.22), or both. Accordingly, in the context of the present invention, phosphoketolase refers to any protein having at least one of these activities (EC 4.1.2.9 and/or EC 4.1.2.22).

This enzymatic activity can be measured by assays known in the art.

In the context of the present invention, a microorganism which has phosphoketolase activity can, e.g., be a microorganism which naturally has phosphoketolase activity or a microorganism that does not naturally have phosphoketolase activity and has been genetically modified to express a phosphoketolase or a microorganism which naturally has phosphoketolase activity and which has been genetically modified, e.g. transformed with a nucleic acid, e.g. a vector, encoding a phosphoketolase in order to increase the phosphoketolase activity in said microorganism.

Microorganisms that inherently, i.e. naturally, have phosphoketolase activity are known in the art and any of them can be used in the context of the present invention.

It is also possible in the context of the present invention that the microorganism is a microorganism which naturally does not have phosphoketolase activity but which is genetically modified so as to comprise a nucleotide sequence allowing the expression of a phosphoketolase. Similarly, the microorganism may also be a microorganism which naturally has phosphoketolase activity but which is genetically modified so as to enhance the phosphoketolase activity, e.g. by the introduction of an exogenous nucleotide sequence encoding a phosphoketolase.

The genetic modification of microorganisms to express an enzyme of interest will be described in detail below. The phosphoketolase expressed in the microorganism can be any phosphoketolase, in particular a phosphoketolase from prokaryotic or eukaryotic organisms. Prokaryotic phosphoketolases are described, e.g., from Lactococcus lactis.

The phosphoketolase expressed in the microorganism can be a naturally occurring phosphoketolase or it can be a phosphoketolase which is derived from a naturally occurring phosphoketolase, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

As mentioned above, WO 2013/007786 describes a recombinant microorganism which has, inter alia, phosphoketolase activity. Thus, in a preferred embodiment, the microorganism of the present invention having the capabilities as described herein is implemented in a microorganism as described in WO 2013/007786.

In another embodiment, as already mentioned above, the present invention relates to such a microorganism which is further characterized by having no phosphoketolase activity and, accordingly, has also no genetic modification to have an increased expression of a gene encoding a phosphoketolase and/or an increased phosphoketolase activity as compared to a non-modified microorganism.

In such microorganisms, as already explained above, fructose-6-phosphate can nevertheless be converted into erythrose-4-phosphate via the conversion of fructose- e-phosphate and glyceraldehyde-3-phosphate into erythrose-4-phosphate and xylulose-5-phosphate by a transketolase (EC 2.2.1.1) from the pentose phosphate pathway (step A as illustrated in Figure 4).

As mentioned above, the recombinant microorganism is characterized by having a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP), preferably by having an inactivation of the gene(s) encoding phosphofructokinase or showing a reduced phosphofructokinase activity as compared to a non-modified microorganism or by not possessing phosphofructokinase activity. Thus, the microorganism is either a microorganism which naturally has an EMPP including phosphofructokinase activity but which has been modified, in particular genetically modified, so that the phosphofructokinase activity is either completely abolished or so that it is reduced compared to the corresponding non-modified microorganism, or the microorganism is a microorganism which naturally does not possess a phosphofructokinase activity.

As mentioned above, WO 2013/007786 describes a recombinant microorganism which in which, inter alia, the EMPP is deactivated or diminished by abolishing or reducing phosphofructokinase. Thus, in a preferred embodiment, the microorganism of the present invention having the capabilities as described herein is implemented in a microorganism as described in WO 2013/007786.

As already mentioned above, when glucose is processed via the EMPP to acetyl- CoA, one carbon atom is lost by the release of CO2 in the last step. By introducing the phosphoketolase, this loss can be avoided. Since fructose-6-phosphate is a substrate for the phosphoketolase, it is desirable that the pool of fructose-e- phosphate is kept at a high level in the microorganism in order to increase the yield in acetyl-CoA.

Since fructose-6-phosphate is also a substrate for an enzyme of the Embden- Meyerhof-Parnas pathway, i.e. the phosphofructokinase, the recombinant microorganism has a reduced phosphofructokinase activity as compared to a non- mod if ied microorganism or the gene(s) encoding a phosphofructokinase has/have been inactivated. This ensures the flux of fructose-6-phosphate is directed to the phosphoketolase and to the production of acetyl-CoA without loss of CO2 because fructose-6-phosphate or most of fructose-6-phosphate can no longer be processed via the Embden-Meyerhof-Parnas pathway. Recombinant microorganisms in which a phosphoketolase is naturally or heterologously expressed and which have reduced phosphofructokinase activity are disclosed in US 7,785,858.

The term “phosphofructokinase” and the term “phosphofructokinase activity” means an enzyme and an enzymatic activity, respectively that converts ATP and fructose-6- phosphate to ADP and fructose-1 ,6-bisphosphate (EC 2.7.1.11). This enzymatic activity can be measured by assays known in the art as, for example, described by Kotlarz et al. (Methods Enzymol. (1982) 90, 60-70).

The term “a microorganism which is characterised by having a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP), preferably by having an inactivation of the gene(s) encoding phosphofructokinase or showing a reduced phosphofructokinase activity as compared to a non-modified microorganism” preferably refers to a microorganism in which the inactivation of the gene(s) encoding a phosphofructokinase or the reduction of the phosphofructokinase activity as compared to a non-modified microorganism is achieved by a genetic modification of the microorganism which leads to said inactivation or reduction.

In a preferred embodiment, the recombinant microorganism is a recombinant microorganism that has an inactivated Embden-Meyerhof-Parnas pathway (EMPP), preferably by having an inactivation of the gene(s) encoding phosphofructokinase. The inactivation of the gene(s) encoding a phosphofructokinase in the context of the present invention means that the gene(s) coding for phosphofructokinase which are present in the microorganism is (are) inactivated so that they are no longer expressed and/or do not lead to the synthesis of functional phosphofructokinase. Inactivation can be achieved by many different ways known in the art. The inactivation can, e.g., be achieved by the disruption of the gene(s) encoding the phosphofructokinase or by clean deletion of said gene(s) through the introduction of a selection marker. Alternatively, the promoter of the gene(s) encoding the phosphofructokinase can be mutated in a way that the gene is no longer transcribed into mRNA. Other ways to inactivate the gene(s) encoding the phosphofructokinase known in the art are: to express a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the phosphofructokinase gene(s) so that the mRNA can no longer be translated into a protein, to express a polynucleotide encoding RNA that suppresses the expression of said gene(s) through RNAi effect; to express a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of said gene(s); or to express a polynucleotide encoding RNA that suppresses expression of said gene(s) through co-suppression effect. These polynucleotides can be incorporated into a vector, which can be introduced into the microorganism by transformation to achieve the inactivation of the gene(s) encoding the phosphofructokinase.

The term “inactivation” in the context of the present invention preferably means complete inactivation, i.e. that the microorganism does not show phosphofructokinase activity. This means in particular that the microorganism does not show phosphofructokinase activity independent from the used growth conditions. Preferably, “inactivation” means that the gene(s) encoding phosphofructokinase which are present in the microorganism are genetically modified so as to prevent the expression of the enzyme. This can be achieved, e.g., by deletion of the gene or parts thereof wherein the deletion of parts thereof prevents expression of the enzyme, or by disruption of the gene either in the coding region or in the promoter region wherein the disruption has the effect that no protein is expressed or a dysfunctional protein is expressed.

In a preferred embodiment, the recombinant microorganism is a recombinant microorganism that has a diminished Embden-Meyerhof-Parnas pathway (EMPP) by reducing the phosphofructokinase activity as compared to a non-modified microorganism. Preferably, this reduction is achieved by a genetic modification of the microorganism. This can be achieved e.g., by random mutagenesis or site-directed mutagenesis of the promoter and/or the enzyme and subsequent selection of promoters and/or enzymes having the desired properties or by complementary nucleotide sequences or RNAi effect as described above. In the context of the present invention, a “reduced activity” means that the expression and/or the activity of an enzyme, in particular of the phosphofructokinase, 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 preferred at least 90% or 100% lower than in the corresponding non- mod ified microorganism. Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. Assays for measuring the reduced enzyme activity of a phosphofructokinase are known in the art.

In another embodiment the microorganism is a microorganism which does not possess a phosphofructokinase activity. This preferably means that such a microorganism naturally does not possess a phosphofructokinase activity. This means that such a microorganism does naturally not contain in its genome a nucleotide sequence encoding an enzyme with phosphofructokinase activity. Examples for such microorganisms are Zymomonas mobilis (J. S. Suo et al., Nat. Biotechnol. 23:63 (2005)) and Ralstonia eutropha (C. Fleige et al., Appl. Microb. Cell Physiol. 91:769 (2011)).

The recombinant microorganism may be further characterised by having a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP), preferably by having an inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or showing a reduced glucose-6-phosphate dehydrogenase activity as compared to a non-mod ified microorganism, or by not possessing glucose-e- phosphate dehydrogenase activity.

Thus, the microorganism is preferably either a microorganism which naturally has a PPP including glucose-6-phosphate dehydrogenase activity but which has been modified, in particular genetically modified, so that the glucose-6-phosphate dehydrogenase activity is either completely abolished or so that it is reduced compared to the corresponding non-modified microorganism, or the microorganism is a microorganism which naturally does not possess a glucose-6-phosphate dehydrogenase activity.

Diminishing or inactivating the oxidative branch of the pentose phosphate pathway further increases the yield in acetyl-CoA since glucose-6-phosphate will no longer be drawn through the pentose phosphate cycle. All or almost all glucose-6-phosphate in the microorganism will be converted into fructose-6-phosphate which will then be further converted into acetyl-CoA.

The term “glucose-6-phosphate dehydrogenase” and the term “glucose-6-phosphate dehydrogenase activity” means and enzyme and an enzymatic activity, respectively, that converts glucose-6-phosphate and NADP ⁺ to 6-phosphoglucono-6-lactone and NADPH (EC 1.1.1.49). This enzymatic activity can be measured by assays known in the art as, for example, described by Noltmann et al. (J. Biol. Chem. (1961) 236, 1225-1230).

The term “a microorganism which is characterised by having a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP), preferably by having an inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase as compared to a non-mod ified microorganism” preferably refers to a microorganism in which the inactivation of the gene(s) encoding a glucose-6-phosphate dehydrogenase or the reduction of the glucose-6-phosphate dehydrogenase activity as compared to a non-mod ified microorganism is achieved by a genetic modification of the microorganism which leads to said inactivation or reduction.

As mentioned above, WO 2013/007786 describes a recombinant microorganism in which, inter alia, the oxidative branch of the PPP is deactivated or diminished by abolishing or reducing glucose-6-phosphate dehydrogenase.. Thus, in a preferred embodiment, the microorganism of the present invention having the capabilities as described herein is implemented in a microorganism as described in WO 2013/007786.

In a preferred embodiment, the recombinant microorganism is a recombinant microorganism that has an inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivation of the gene(s) encoding a glucose-6-phosphate dehydrogenase. The inactivation of the gene(s) encoding a glucose-6-phosphate dehydrogenase in the context of the present invention means that the gene(s) coding for glucose-6-phosphate dehydrogenase which is (are) present in the microorganism is (are) inactivated so that they are no longer expressed and/or do not lead to the synthesis of functional glucose-6-phosphate dehydrogenase. Inactivation can be achieved by many different ways known in the art. The inactivation can, e.g., be achieved by the disruption of the gene(s) encoding the glucose-6-phosphate dehydrogenase or by clean deletion of said gene(s) through the introduction of a selection marker. Alternatively, the promoter of the gene(s) encoding the glucose-e- phosphate dehydrogenase can be mutated in a way that the gene(s) is/are no longer transcribed into mRNA. Other ways to inactivate the gene(s) encoding the glucose-e- phosphate dehydrogenase known in the art are: to express a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the glucose-6-phosphate dehydrogenase gene(s) so that the mRNA can no longer be translated into a protein, to express a polynucleotide encoding RNA that suppresses the expression of said gene(s) through RNAi effect; to express a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of said gene(s); or to express a polynucleotide encoding RNA that suppresses expression of said gene(s) through co-suppression effect. These polynucleotides can be incorporated into a vector, which can be introduced into the microorganism by transformation to achieve the inactivation of the gene(s) encoding the glucose-6-phosphate dehydrogenase.

The term “inactivation” in the context of the present invention preferably means complete inactivation, i.e. that the microorganism does not show glucose-e- phosphate dehydrogenase activity. This means in particular that the microorganism does not show glucose-6-phosphate dehydrogenase activity independent from the used growth conditions.

Preferably, “inactivation” means that the gene(s) encoding glucose-6-phosphate dehydrogenase which are present in the microorganism are genetically modified so as to prevent the expression of the enzyme. This can be achieved, e.g., by deletion of the gene or parts thereof wherein the deletion of parts thereof prevents expression of the enzyme, or by disruption of the gene either in the coding region or in the promoter region wherein the disruption has the effect that no protein is expressed or a dysfunctional protein is expressed.

In a preferred embodiment, the recombinant microorganism is a recombinant microorganism that has a diminished oxidative branch of the pentose phosphate pathway (PPP) by reducing the glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism. Preferably, this reduction is achieved by a genetic modification of the microorganism. This can be achieved e.g., by random mutagenesis or site-directed mutagenesis of the promoter and/or the enzyme and subsequent selection of promoters and/or enzymes having the desired properties or by complementary nucleotide sequences or RNAi effect as described above.

In the context of the present invention, a “reduced activity” means that the expression and/or the activity of an enzyme, in particular of the 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 preferred at least 90% or 100% lower than in the corresponding non-modified microorganism. Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. Assays for measuring the reduced enzyme activity of a glucose-6-phosphate dehydrogenase are known in the art. In another embodiment the microorganism is a microorganism which does not possess a glucose-6-phosphate dehydrogenase activity. This preferably means that such a microorganism naturally does not possess a glucose-6-phosphate dehydrogenase activity. This means that such a microorganism does naturally not contain in its genome a nucleotide sequence encoding an enzyme with glucose-6- phosphate dehydrogenase activity. Examples for such microorganisms are Acinetobacter baylyi (Barbe et al., Nucl. Acids Res. 32 (2004), 5766-5779), archae of the hyperthermophilic phylum such as Sulfolobus solfataricus (Nunn et al., J. Biol. Chem. 285 (2010), 33701-33709), Thermoproteus tenax, Thermoplasma acidophilum and Picrophilus torridus (Reherand Schonheit, FEBS Lett. 580 (2006), 1198-1204).

As mentioned above, the present invention is based on the fact that the efficiency of converting glucose into acetyl-CoA could be further improved in a recombinant microorganism by making use of an increased expression of the gene(s) encoding deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) and/or an increased deoxy-D- ribose-5-phosphate aldolase (EC 4.1.2.4) activity as compared to a non-modified microorganism.

Accordingly, the recombinant microorganism of the present invention is characterized by: having a genetic modification to have an increased expression of the gene(s) encoding deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) and/or an increased deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) activity as compared to a non- modified microorganism.

Deoxy-D-ribose-5-phosphate aldolases (EC 4.1.2.4), also termed deoxyribose- phosphate aldolase are enzymes which catalyze the following reversible aldol reaction:

2-deoxy-D-ribose 5-phosphate D-glyceraldehyde 3-phosphate + acetaldehyde

In the context of the present invention, the above reaction takes place in the direction towards the cleavage of 2-deoxy-D-ribose 5-phosphate into D-glyceraldehyde 3- phosphate and acetaldehyde as also schematically illustrated in Figure 5.

This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, algae, animals, fungi and bacteria. The enzyme has, e.g., been described in Aciduliprofundum boonei (UniProt Accession Number B5IEU6), Aeropyrum pernix (UniProt Accession Number Q9Y648, Bacillus cereus, Bacillus subtilis, Bacteriovorax stolpii, Bdellovibrio bacteriovorus, Bos taurus, Equus asinus, Equus caballus, Escherichia coli, Gallus gallus, Homo sapiens, Hyperthermus butylicus, Klebsiella pneumoniae, Lactobacillus plantarum, Mycoplasma mycoides, Oryctolagus cuniculus, Ovis aries, Paenibacillus sp. (UniProt Accession Number C7E719), Pyrobaculum aerophilum, Rattus norvegicus, Rhodococcus erythropolis (UniProt Accession Number JN665070; SwissProt Accession Number C0ZUQ6), Salmonella enterica subsp. enterica serovar Typhimurium, Streptococcus mutans (UniProt Accession Number Q9AIP7), Streptococcus uberis, Sus scrota, Thermococcus kodakarensis, Thermotoga maritima, Thermus thermophilus, Toxoplasma gondii, Ureaplasma urealyticum and Yersinia sp. EA015 (UniProt Accession Number C0LSK9).

Preferably, the deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) is encoded by the gene deoC which is also termed dra or thyR or tlr.

In a preferred embodiment, the recombinant microorganism of the present invention described herein is a microorganism which is implemented in a microorganism which is capable of converting glucose into acetyl-CoA and D-ribose-1 -phosphate as schematically illustrated in Figures 1, 2, 3 and 4.

Accordingly, the recombinant microorganism of the present invention is a microorganism which naturally or endogenously expresses any of the enzymes disclosed in Figures 1, 2, 3 and 4 and/or is a microorganism which has a genetic modification to have an increased expression of the gene(s) encoding these enzymes and/or an increased respective enzymatic activity as compared to a non- modified microorganism.

These enzymes are:

- a glucose kinase (glk) (EC 2.7.1.2) and/or N-acetylmannosamine kinase (nanK) (EC 2.7.1.60) capable of converting glucose into glucose-6-phosphate (step A as illustrated in Figure 1)

- a glucose-6-phosphate isomerase (pgi) (EC 5.3.1.9) capable of converting glucose-6-phosphate into fructose-6-phosphate (step B as illustrated in Figure 1 )

- a transketolase (tkt) EC 2.2.1.1 capable of converting fructose-6-phosphate and D-glyceraldehyde-3-phosphate into erythrose-4-phosphate and xylulose- 5-phosphate (step A as illustrated in Figures 2 and 4)

- a phosphoketolase (EC 4.1.2.9 and/or EC 4.1.2.22) capable of converting fructose-6-phosphate into erythrose-6-phosphate and acetyl-phosphate (step B as illustrated in Figure 3) - a phosphate acetyltransferase (pta) (EC 2.3.1.8) capable of converting acetyl- phosphate and Co ASH into acetyl-CoA (step I as illustrated in Figures 2 and

3)

- a phosphoketolase (EC 4.1.2.9 and/or EC 4.1.2.22) capable of converting xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate by a phosphoketolase (step E as illustrated in Figure 2)

- a transaldolase (talB) (EC 2.2.1.2) capable of converting erythrose-4- phosphate and fructose-6-phosphate into sedoheptulose-7-phosphate and D- glyceraldehyde-3-phosphate (step C as illustrated in Figures 2, 3 and 4)

- a transketolase (EC 2.2.1.1) capable of converting D-glyceraldehyde-3- phosphate and sedoheptulose-7-phosphate into D-xylulose-5-phosphate and D-ribose-5-phosphate (step D as illustrated in Figures 2, 3 and 4)

- a ribulose-phosphate-3-epimerase (rpe) (EC 5.1.3.1) capable of converting D- xylulose-5-phosphate into D-ribulose-5-phosphate (step F as illustrated in Figures 2, 3 and 4)

- a ribose-5-phosphate isomerase (rpiA, rpiB) (EC 5.3.1.6) capable of converting D-ribulose-5-phosphate into D-ribose-5-phosphate (step G as illustrated in Figures 2, 3 and 4)

- a phospho pentomutase (deoB) (EC 5.4.2.7) capable of converting D-ribose-5- phosphate into D-ribose-1 -phosphate (step H as illustrated in Figures 2, 3 and

4)

In a preferred embodiment, the recombinant microorganism of the present invention described herein is a microorganism which is implemented in a microorganism which is capable of converting glucose into acetyl-CoA and D-ribose-1 -phosphate as schematically illustrated in Figures 2, 3 and 4 and as described above and which is capable of converting 2-deoxy-D-ribose-1 -phosphate into acetyl-CoA as schematically illustrated in Figure 5 and as described in more detail herein above and below.

Accordingly, the recombinant microorganism of the present invention is a microorganism which naturally or endogenously expresses any of the enzymes disclosed in Figure 5 and/or is a microorganism which has a genetic modification to have an increased expression of the gene(s) encoding one or more of these enzymes and/or an increased respective enzymatic activity as compared to a non- mod if ied microorganism.

These enzymes are described in more detail herein above and below.

The enzyme which is capable of converting the first step of the overall pathway from 2-deoxy-D-ribose-1 -phosphate into acetyl-CoA as illustrated in Figure 5, i.e., the enzyme capable of converting 2-deoxy-D-ribose-1 -phosphate into 2-deoxy-D-ribose- 5-phosphate, is a phospho-pentomutase (deoB) (EC 5.4.2.7).

The conversion of glucose into acetyl-CoA and D-ribose-1 -phosphate as schematically illustrated in Figures 2, 3 and 4 and the conversion of 2-deoxy-D- ribose-1 -phosphate into acetyl-CoA as schematically illustrated in Figure 5 is connected by metabolic nucleoside and nucleotide degradation super-pathways (well-known and described in the prior art) into which D-ribose-1 -phosphate is fed and which ultimately lead to the production of 2-deoxy-D-ribose 1 -phosphate (see also Figure 5).

Accordingly, in a preferred embodiment, the recombinant microorganism of the present invention described herein is a microorganism which is not only implemented in a microorganism which is capable of converting glucose into acetyl-CoA and D- ribose-1 -phosphate as schematically illustrated in Figures 2, 3 and 4 and as described above and which is capable of converting 2-deoxy-D-ribose-1 -phosphate into acetyl-CoA as schematically illustrated in Figure 5 and as described in more detail herein above and below, but is also a microorganism in which these metabolic nucleoside and nucleotide degradation super-pathways naturally occur or, preferably, wherein such microorganism has a genetic modification to have an increased expression of the gene(s) encoding one or more of these enzymes and/or an increased respective enzymatic activity as compared to a non-mod ified microorganism.

In another preferred embodiment, the recombinant microorganism of the present invention is further characterized by being capable of converting the acetaldehyde produced by the above deoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) into acetyl-CoA.

The conversion of acetaldehyde into acetyl-CoA may occur via different routes as schematically illustrated by “A”, “B” and/or “C” in Figure 5.

According to “A", acetaldehyde is converted into acetate which is further converted into acetyl phosphate which is then further converted into acetyl-CoA.

According to “B”, acetaldehyde is converted into acetate which is then directly further converted into acetyl-CoA.

According to “C”, acetaldehyde is directly converted into acetyl-CoA.

Accordingly, in another preferred embodiment, the recombinant microorganism of the present invention is further characterized by having any one of “A”) to “C”):

“A”) having a genetic modification to have an increased expression of the gene(s) encoding an oxidoreductase acting on the aldehyde or oxo group of donors with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.-), preferably encoding an aldehyde dehydrogenase with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.4) and/or an increased oxidoreductase acting on the aldehyde or oxo group of donors with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.-) activity, preferably and increased aldehyde dehydrogenase with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.4) activity, as compared to a non-modified microorganism; and having a genetic modification to have an increased expression of the gene(s) encoding an acetate kinase (EC 2.7.2.1) and/or an increased acetate kinase (EC 2.7.2.1) activity as compared to a non-modified microorganism; and/or having a genetic modification to have an increased expression of the gene(s) encoding a propionate kinase (EC 2.7.2.15) and/or an increased propionate kinase (EC 2.7.2.15) activity as compared to a non-modified microorganism; and having a genetic modification to have an increased expression of the gene(s) encoding a phosphate acetyltransferase (EC 2.3.1.8) and/or an increased phosphate acetyltransferase (EC 2.3.1.8) activity as compared to a non- modified microorganism; and/or

“B”) having a genetic modification to have an increased expression of the gene(s) encoding an oxidoreductase acting on the aldehyde or oxo group of donors with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.-), preferably encoding an aldehyde dehydrogenase with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.4) and/or an increased oxidoreductase acting on the aldehyde or oxo group of donors with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.-) activity, preferably and increased aldehyde dehydrogenase with NAD ⁺ or NADP ⁺ as acceptor (EC 1.2.1.4) activity, as compared to a non-modified microorganism; and having a genetic modification to have an increased expression of the gene(s) encoding an acetate-CoA ligase (EC 6.2.1.1) and/or encoding a propionate- CoA ligase (EC 6.2.1.17) and/or an increased acetate-CoA ligase (EC 6.2.1.1) activity and/or an increased propionate-CoA ligase (EC 6.2.1.17) activity as compared to a non-modified microorganism; and/or “C”) having a genetic modification to have an increased expression of the gene(s) encoding an acetaldehyde dehydrogenase (acylating) (EC1.2.1.10) and/or an increased acetaldehyde dehydrogenase (acylating) (EC 1.2.1.10) activity as compared to a non-modified microorganism.

In a preferred embodiment, the recombinant microorganism of the present invention is capable of consuming glucose.

In a preferred embodiment, the recombinant microorganism of the present invention is capable of consuming sucrose. In another preferred embodiment, the recombinant microorganism of the present invention is capable of consuming fructose.

In another preferred embodiment, the recombinant microorganism of the present invention is capable of consuming xylose.

In another preferred embodiment, the recombinant microorganism of the present invention is capable of consuming mannose.

In a preferred embodiment, the recombinant microorganism of the present invention is capable of consuming arrabinose.

In another preferred embodiment, the recombinant microorganism of the present invention is capable of consuming more than one sugar. Preferably, said more than one sugar comprises sucrose, glucose, mannose, arrabinose and/or xylose. In a more preferred embodiment, the recombinant microorganism of the present invention is capable of consuming two or more sugars selected from the group consisting of sucrose, glucose, mannose, arrabinose and xylose. Organisms and/or microorganisms which are capable of consuming glucose, fructose, xylose, arrabinose and/or mannose do naturally occur and are known in the art.

In another embodiment, said microorganism is genetically modified in order to be capable of consuming glucose, fructose, xylose and/or mannose and/or genetically modified in order to increase the microorganism’s capability of consuming glucose, fructose, xylose, arrabinose and/or mannose. Corresponding genetic modifications are known in the art.

In one embodiment, the recombinant microorganism of the present invention is capable of consuming sugar through a Phosphotransferase Transport System (PTS). In another embodiment, the recombinant microorganism of the present invention is capable of consuming sugar through a non-Phosphotransferase Transport System (non-PTS).

Microorganisms which are capable of consuming sugar through a Phosphotransferase Transport System (PTS) and/or through a non- Phosphotransferase Transport System (non-PTS) are known in the art.

In another embodiment, said microorganism is genetically modified in order to be capable of consuming sugar through a Phosphotransferase Transport System (PTS) or through a non-Phosphotransferase Transport System (non-PTS). In another preferred embodiment, said microorganism is genetically modified in order to increase the microorganism’s capability of consuming sugar through a Phosphotransferase Transport System (PTS) or through a non-Phosphotransferase Transport System (non-PTS). Corresponding genetic modifications are known in the art. In another preferred embodiment, the recombinant microorganism of the present invention has a diminished or inactivated Phosphotransferase Transport System (PTS).

Without being bound to theory, such a microorganism may preferably be genetically modified by deleting or inactivating (a) gene(s) of said Phosphotransferase Transport System (PTS).

Corresponding genetic modifications are known in the art.

In another preferred embodiment, the recombinant microorganism of the present invention has an enhanced non-Phosphotransferase Transport System (non-PTS) for sugar uptake.

Without being bound to theory, such a microorganism may preferably be genetically modified by overexpressing (a) gene(s) of said non-Phosphotransferase Transport System (non-PTS) for sugar uptake.

Corresponding genetic modifications are known in the art.

In another preferred embodiment, the recombinant microorganism of the present invention has a diminished or inactivated Phosphotransferase Transport System (PTS) and an enhanced non Phosphotransferase Transport System (non-PTS) for sugar uptake.

In another preferred embodiment, the recombinant microorganism of the present invention is capable of consuming sucrose through a non-Phosphotransferase Transport System (non-PTS).

In another preferred embodiment, the recombinant microorganism of the present invention is consuming sucrose, wherein said microorganism has genetically been modified by the introduction of at least one gene of a non-Phosphotransferase Transport System (non-PTS). Without being bound to theory, such an organism and/or microorganism has genetically been modified by introducing a gene selected from the group consisting of cscA, cscB, and cscK from Escherichia coli W (M. Bruschi et al., Biotechnology Advances 30 (2012) 1001-1010).

In another preferred embodiment, the recombinant microorganism of the present invention has genetically been modified to have a diminished or inactivated Phosphotransferase Transport System (PTS) and an overexpression of at least one gene selected from the group consisting of galP, glk and gif. In a preferred embodiment, the recombinant microorganism of the present invention is genetically modified in order to avoid the leakage of acetyl-CoA, thereby increasing the intracellular concentration of acetyl-CoA. Genetic modifications leading to an increase in the intracellular concentration of acetyl-CoA are known in the art. Without being bound to theory, such a microorganism may preferably be genetically modified by deleting or inactivating one or more of the following genes: AackA (acetate kinase), Aldh (lactate dehydrogenase), AadhE (alcohol dehydrogenase), AfrdB and/or AfrdC (fumarate reductase and fumarate dehydrogenase), ApoxB (pyruvate oxidase), Apgk (phosphoglycerate kinase), A icIR (DNA-binding transcriptional repressor IcIR).

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

These modifications which avoid the leakage of acetyl-CoA are known in the art and corresponding modified microorganisms have been used in methods for the bioconversion of exogenous isoamyl alcohol into isoamyl acetate by an E. coli strain expressing ATF2 (Metab. Eng. 6 (2004), 294-309).

Further genes which may be overexpressed in the microorganism include the following: o pckA (phosphoenolpyruvate carboxykinase) o tktA (transketolase 1 ) o tktB (transketolase 2) o talA (transaldolase A) o talB (transaldolase B) o rpiA (ribose-5-phosphate isomerase A) o rpiB (ribose-5-phosphate isomerase B) o rpE (ribulose-phosphate 3-epimerase) o pgi (glucose-6-phosphate isomerase) o galP (galactose: H ⁺ symporter) o glk (glucokinase) o gif (glucose facilitated diffusion protein) o pta (phosphate acetyltransferase)

In a preferred embodiment, as will be outlined in more detail further below, the recombinant organism of the present invention is a fungus or a yeast. In a preferred embodiment, as will be outlined in more detail further below, the recombinant organism of the present invention is a bacterium.

In a preferred embodiment, the recombinant organism of the present invention is further characterized in that it: is capable of converting acetyl-CoA into acetone.

Methods for providing such a recombinant microorganism are for instance disclosed in EP 2 295 593. The term “which is capable of converting acetyl-CoA into acetone” in the context of the present invention means that the organism/microorganism has the capacity to produce acetone within the cell due to the presence of enzymes providing enzymatic activities allowing the production of acetone from acetyl-CoA. 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 starts out with a reaction (reaction step 1) in which two molecules of acetyl-CoA are condensed into acetoacetyl-CoA. This reaction is catalyzed by acetyl-CoA acetyltransferase (EC 2.3.1.9). Acetoacetyl-CoA is then converted into acetoacetate by a reaction with acetic acid or butyric acid resulting also in the production of acetyl-CoA or butyryl-CoA (reaction step 2). This reaction is catalyzed e.g. by acetoacetylCoA transferase (EC 2.8.3.8). AcetoacetylCoA transferase is known from various organisms, e.g. from E. coli in which it is encoded by the atoAD genes or from Clostridium acetobutylicum in which it is encoded by the ctfAB genes. However, also other enzymes can catalyze this reaction, e.g. 3-oxoacid CoA transferase (EC 2.8.3.5) or succinate CoA ligase (EC 6.2.1.5).

Finally, acetoacetate is converted into acetone by a decarboxylation step (reaction step 3) catalyzed by acetoacetate decarboxylase (EC 4.1.1.4).

The above described reaction steps 1 and 2 and the enzymes catalyzing them are not characteristic for the acetone synthesis and can be found in various organism. In contrast, reaction step 3 which is catalyzed by acetoacetate decarboxylase (EC 4.1.1.4) is only found in those organisms which are capable of producing acetone.

In a preferred embodiment the recombinant microorganism is a microorganism, which naturally has the capacity to produce acetone. Thus, preferably the microorganism belongs to the genus Clostridium, Bacillus or Pseudomonas, more preferably to the species Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa or Pseudomonas putida. In another preferred embodiment, the recombinant microorganism is a microorganism, derived from an organism/microorganism which naturally does not produce acetone but which has been genetically modified so as to produce acetone, i.e. by introducing the gene(s) necessary for allowing the production of acetone in the microorganism. In principle any microorganism can be genetically modified in this way. The enzymes responsible for the synthesis of acetone have been described above. Genes encoding corresponding enzymes are known in the art and can be used to genetically modify a given microorganism so as to produce acetone. As described above, the reaction steps 1 and 2 of the acetone synthesis occur naturally in most organisms. However, reaction step 3 is characteristic and crucial for acetone synthesis. Thus, in a preferred embodiment, a genetically modified microorganism derived from a microorganism which naturally does not produce acetone is modified so as to contain a nucleotide sequence encoding an enzyme catalyzing the conversion of acetoacetate into acetone by decarboxylation, e.g. an acetoacetate decarboxylase (EC 4.1.1.4). Nucleotide sequences from several organisms encoding this enzyme are known in the art, e.g. the adc gene from Clostridium acetobutylicum (Uniprot accession number P23670), Clostridium beijerinckii (Clostridium MP; Q9RPK1), Clostridium pasteurianum (Uniprot accession number P81336), Bradyrhizobium sp. (strain BTAil / ATCC BAA-1182; Uniprot accession number A5EBU7), Burkholderia mallei, Burkholderia mallei (Uniprot accession number A3MAE3), Burkholderia 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, Rhizobium leguminosarum (Uniprot accession number Q1M911), Lactobacillus casei (Uniprot accession number Q03B66), Francisella tularensis, Saccharopolyspora erythreae (Uniprot accession number A4FKR9), Korarchaeum cryptofilum (Uniprot accession number B1L3N6), Bacillus amyloliquefaciens (Uniprot accession number A7Z8K8), Cochliobolus heterostrophus (Uniprot accession number Q8NJQ3), Sulfolobus islandicus (Uniprot accession number C3ML22) and Francisella tularensis subsp. holarctica.

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

Even more preferably, the microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis, i.e. the condensation of two molecules of acetyl CoA into acetoacetatyl CoA.

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 reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis.

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 allowing expression of the respective enzyme in the microorganism. Such a construct normally comprises the coding sequence in question linked to regulatory sequences allowing transcription and translation in the respective host cell, e.g. a promoter and/enhancer and/or transcription terminator and/or ribosome binding sites etc. The prior art already describes microorganisms which have been genetically modified so as to be able to produce acetone. In particular, genes from, e.g., Clostridium acetobutylicum have been introduced into E. coli thereby allowing the synthesis of acetone in E. coli, a bacterium which naturally does not produce acetone (Bermejo et al., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818). In particular Hanai et al. (loc. cit.) shows that it is sufficient to introduce a nucleic acid sequence encoding an acetoacetate decarboxylase (such as that from Clostridium acetobutylicum) in order to achieve acetone production in E. coli indicating that the endogenous enzymes in E. coli catalyzing the above-mentioned reaction steps 1 and 2 (i.e. the expression products of the E. coli atoB and atoAD genes) are sufficient to provide substrate for the acetone production.

In another preferred embodiment, the recombinant organism of the present invention is further characterized in that it: is capable of converting acetyl-CoA into isobutene.

Accordingly, the recombinant microorganism is further characterized in that it is capable of converting acetyl-CoA into acetone and converting acetone into isobutene. Methods for providing such a recombinant microorganism are for instance disclosed in EP-A 2295593 (EP 09 170312), WO 2010/001078 and EP 10 188001.

In another preferred embodiment, the recombinant organism of the present invention is further characterized in that it: is capable of converting acetyl-CoA into propene.

In another aspect, the recombinant microorganism is further characterized in that it is capable of converting acetyl-CoA into acetone and converting acetone into isobutene. Methods for providing such a recombinant microorganism are for instance disclosed in EP-A 2 295 593 (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.

In another aspect, the recombinant microorganism is further characterized in that it is capable of converting acetyl-CoA into isobutene using a metabolic route that does not include an acetone intermediate. Methods for providing such a recombinant microorganism are for instance disclosed in WO2016042012, WO2017/085167, WO201 8/206262, WO2013/186215, WO201 6/034691 , WO2017/191239,

US2019/0100742, WO 2016/042011, WO 2017/162738, WO2015082447, WO 2010/001078, WO 2012/052427, WO 2017/071124, WO 2015/004211, WO 2014/064198 and WO 2014/086781.

In another preferred embodiment, the recombinant organism of the present invention is further characterized in that it: is capable of converting acetyl-CoA into isopropanol.

Accordinly, the recombinant microorganism is characterized in that it is capable of converting acetyl-CoA into acetone and converting acetone into isopropanol. One skilled in the art would recognize that further genetic modifications to the microorganisms of the present invention could lead to improvements in the efficacy by which the microorganisms of the present invention convert feedstock to product. For example, natural microorganisms commonly produce products such as formate, acetate, lactate, succinate, ethanol, glycerol, 2,3-butanediol, methylglyoxal and hydrogen; all of which would be deleterious to the production of, e.g., acetone, isobutene or propene from sugars. Elimination or substantial reduction of such unwanted by-products may be achieved by elimination or reduction of key enzymes activities leading their production. Such activities include, but are not limited to, the group consisting of:

- acetyl-CoA + formate = CoA + pyruvate (for example, catalyzed by formate C- acetyltransferase, also known as pyruvate formate-lyase (EC 2.3.1.54); for E. coli- pfIB, NCBI-GenelD: 945514);

- ATP + acetate = ADP + acetyl phosphate (for example, catalyzed by acetate kinase (EC 2.7.2.1); for E. coll· ackA, NCBI-GenelD: 946775);

- (R)-lactate + NAD ⁺ = pyruvate + NADH + tC (for example, catalyzed by L- lactate dehydrogenase (EC 1.1.1.28); for E. coll- IdhA, NCBI-GenelD: 946315);

- succinate + acceptor = fumarate + reduced acceptor (for example, catalyzed by succinate dehydrogenase (EC 1.3.99.1); for E. coll· comprising frdA and frdB, NCBI-GenelD: 948667 and 948666, respectively);

- a 2-oxo carboxylate (e.g. pyruvate) = an aldehyde (e.g. acetaldehyde) + C0 ₂ (for example, catalyzed by pyruvate decarboxylase (EC 4.1.1.1 ));

- acetaldehyde + CoA + NAD ⁺ = acetyl-CoA + NADH + H ⁺ (for example, catalyzed by acetaldehyde dehydrogenase (acetylating) (EC 1.2.1.10); for E. coll· adhE, NCBI-GenelD: 945837);

- sn-glycerol 3-phosphate + NAD(P = glycerone phosphate + NAD(P)H + H ⁺ (for example, catalyzed by glycerol-3-phosphate dehydrogenase [NAD(P) ⁺ ]

(EC 1.1.1.94); for E. coll· gpsA, NCBI-GenelD: 948125);

- 2 pyruvate + hC = 2-acetolactate + C0 ₂ (for example, catalyzed by acetolactate synthase (EC 2.2.1.6); for £. coli- ilvH and ilvl, NCBI-GenelD: 947267 and 948793, respectively);

- glycerone phosphate = methylglyoxal + phosphate (for example, catalyzed by methylglyoxal synthase (EC 4.2.3.3); for E. coli- mgsA, NCBI-GenelD:

945574); and

- formate + H ⁺ = C0 ₂ + H ₂ (for example, catalyzed by formate hydrogenlyase (EC 1.2.1.2 together with EC 1.12.1.2); for E. coli- fdhF ( EC 1.2.1.2), NCBI- GenelD: 948584). Thus, in a preferred embodiment, the microorganism may further be characterized in that one or more of the above listed enzyme activities are eliminated or reduced.

One skilled in the art would further recognize that genetic modifications to regulatory elements in the microorganisms of the present invention could lead to improvements in the efficacy by which the microorganisms of the present invention convert feedstock to product. Within E. coli, such genetic modifications include, but are not limited to, the group consisting of:

- deleting the fnr gene (NCBI-GenelD: 945908), a global regulator of anaerobic growth; and

- deleting the rpoS gene (NCBI-GenelD: 947210), a RNA polymerase, sigma S (sigma 38) factor; and

- deleting the icIR gene (DNA-binding transcriptional repressor IcIR).

Thus, in another preferred embodiment the microorganism shows at least one of these deletions.

Thus, as described above, the recombinant microorganism of the present invention can be used for the conversion of glucose into acetyl-CoA. Acetyl CoA (also known as acetyl Coenzyme A) in chemical structure is the thioester between coenzyme A (a thiol) and acetic acid and is an important precursor molecule for the production of useful metabolites. Acetyl-CoA can then be further converted by the recombinant microorganism into useful metabolites such as L-glutamic acid, L-glutamine, L- proline, L-arginine, L-leucine, succinate and polyhydroxybutyrate.

The recombinant microorganism can also be used for converting acetyl-CoA into acetone.

The recombinant microorganism can also be used for converting acetyl-CoA into isobutene.

The recombinant microorganism can also be used for converting acetyl-CoA into propene.

The recombinant microorganism can also be used for converting acetyl-CoA into isopropanol.

The present invention also relates to the use of the recombinant microorganism of the present invention as described herein for the conversion of acetyl-CoA into acetone.

The present invention also relates to the use of the recombinant microorganism of the present invention as described herein for the conversion of acetyl-CoA into isobutene. The present invention also relates to the use of the recombinant microorganism of the present invention as described herein for the conversion of acetyl-CoA into propene.

The present invention also relates to the use of the recombinant microorganism of the present invention as described herein for the conversion of acetyl-CoA into isopropanol.

The present invention also relates to a method for producing acetone from acetyl- CoA comprising the steps of: culturing the microorganism of the present invention as described herein in a suitable medium; and optionally recovering said acetone from the culture medium.

The present invention also relates to a method for producing isobutene from acetyl- CoA comprising the steps of: culturing the microorganism of the present invention as described herein in a suitable medium; and optionally recovering said isobutene.

The present invention also relates to a method for producing propene from acetyl- CoA comprising the steps of: culturing the microorganism of the present invention as described herein in a suitable medium; and optionally recovering said propene.

The present invention also relates to a method for producing isopropanol from acetyl- CoA comprising the steps of: culturing the microorganism of the present invention in a suitable medium; and optionally recovering said isopropanol.

As regards the enzymes and the microorganism recited in the above uses and methods, the same applies as has been set forth above in connection with the recombinant microorganism according to the present invention, in particular as regards the preferred embodiments.

In another embodiment, the use or the method of the invention comprises the step of providing the organism, preferably the microorganism carrying the respective enzyme activity or activities in the form of a (cell) culture, preferably in the form of a liquid cell culture, a subsequent step of cultivating the organism, preferably the microorganism in a fermenter (often also referred to a bioreactor) under suitable conditions allowing the expression of the respective enzyme and further comprising the step of effecting an enzymatic conversion of a method of the invention as described herein above. Suitable fermenter or bioreactor devices and fermentation conditions are known to the person skilled in the art. A bioreactor or a fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or a fermenter may be a vessel in which a chemical/biochemical like the method of the present invention is carried out which involves organisms, preferably microorganisms and/or biochemically active substances, i.e., the enzyme(s) described above derived from such organisms or organisms harbouring the above described enzyme(s). In a bioreactor or a fermenter, this process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, and may range in size from litres to cubic metres, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in a way that it is suitable to cultivate the organisms, preferably microorganisms, in, e.g., a batch-culture, feed-batch-culture, perfusion culture or chemostate-culture, all of which are generally known in the art.

The culture medium can be any culture medium suitable for cultivating the respective organism or microorganism.

When carried out by making use of a microorganism, the method according to the present invention may, e.g. be designed as a continuous fermentation culturing method or as a batch culture or any suitable culture method known to the person skilled in the art.

The present invention also relates to the above uses or the method for the production of acetone and/or isobutene and/or propene and/or isopropanol from glucose or any of the other above-mentioned carbon sources in which the above-described recombinant microorganism is cultivated under conditions allowing for the production of acetone and/or isobutene and/or propene and/or isopropanol and in which the acetone and/or isobutene and/or propene and/or isopropanol is isolated. The microorganisms are cultivated under suitable culture conditions allowing the occurrence of the enzymatic reaction(s). The specific culture conditions depend on the specific microorganism employed but are well known to the person skilled in the art. The culture conditions are generally chosen in such a manner that they allow the expression of the genes encoding the enzymes for the respective reactions. Various methods are known to the person skilled in the art in order to improve and fine-tune the expression of certain genes at certain stages of the culture such as induction of gene expression by chemical inducers or by a temperature shift. In another preferred embodiment the method according to the invention furthermore comprises the step of collecting gaseous products, in particular isobutene or propene, degassing out of the reaction, i.e. recovering the products which degas, e.g., out of the culture. Thus in a preferred embodiment, the method is carried out in the presence of a system for collecting isobutene or propene under gaseous form during the reaction.

As a matter of fact, short alkenes such as isobutene and propene adopt the gaseous state at room temperature and atmospheric pressure. The method according to the invention therefore does not require extraction of the product from the liquid culture medium, a step which is always very costly when performed at industrial scale. The evacuation and storage of the gaseous hydrocarbons and their possible subsequent physical separation and chemical conversion can be performed according to any method known to one of skill in the art.

The enzymes of the microorganisms and/or used in the method according to the invention can be a naturally occurring enzymes or enzymes which are derived from a naturally occurring enzymes, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and include, e.g., random mutagenesis or site-directed mutagenesis and subsequent selection of enzymes having the desired properties or approaches of the so-called “directed evolution”.

For example, for genetic modification in prokaryotic cells, a nucleic acid molecule encoding a corresponding enzyme can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be ligated by using adapters and linkers complementary to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, “primer repair”, restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods. The resulting enzyme variants are then tested for the desired activity, e.g., enzymatic activity, with an assay as described above and in particular for their increased enzyme activity.

As described above, the microorganism of the present invention and the microorganism employed in a use or method of the invention may be a microorganism which has been genetically modified by the introduction of a nucleic acid molecule encoding a corresponding enzyme. Thus, in a preferred embodiment, the microorganism is a recombinant microorganism which has been genetically modified to have an increased activity of at least one enzyme described above for the conversions according to the present invention. This can be achieved e.g. by transforming the microorganism with a nucleic acid encoding a corresponding enzyme. Preferably, the nucleic acid molecule introduced into the microorganism is a nucleic acid molecule which is heterologous with respect to the microorganism, i.e., it does not naturally occur in said microorganism.

In the context of the present invention, an “increased activity” preferably means that the expression and/or the activity of an enzyme 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 preferred at least 90% or 100% higher than in the corresponding non-modified microorganism. In even more preferred embodiments the increase in expression and/or activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-modified microorganism.

The term “increased” expression/activity also covers the situation in which the corresponding non-modified microorganism does not express a corresponding enzyme so that the corresponding expression/activity in the non-modified 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.

Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot.

In the context of the present invention the term “recombinant” means that the microorganism is genetically modified so as to contain a nucleic acid molecule encoding an enzyme as defined above as compared to a wild-type or non-modified microorganism. A nucleic acid molecule encoding an enzyme as defined above can be used alone or as part of a vector. The nucleic acid molecules can further comprise expression control sequences operably linked to the polynucleotide comprised in the nucleic acid molecule. The term “operatively linked” or "operably linked", as used throughout the present description, refers to a linkage between one or more expression control sequences and the coding region in the polynucleotide to be expressed in such a way that expression is achieved under conditions compatible with the expression control sequence.

Expression comprises transcription of the heterologous DNA sequence, preferably into a translatable mRNA. Regulatory elements ensuring expression in fungi as well as in bacteria, are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals and the like. Examples are given further below in connection with explanations concerning vectors.

Promoters for use in connection with the nucleic acid molecule may be homologous or heterologous with regard to its origin and/or with regard to the gene to be expressed. Suitable promoters are for instance promoters which lend themselves to constitutive expression. However, promoters which are only activated at a point in time determined by external influences can also be used. Artificial and/or chemically inducible promoters may be used in this context.

The vectors can further comprise expression control sequences operably linked to said polynucleotides contained in the vectors. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in bacteria or fungi.

In addition, it is possible to insert different mutations into the polynucleotides by methods usual in molecular biology (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), leading to the synthesis of polypeptides possibly having modified biological properties. The introduction of point mutations is conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide.

Moreover, mutants possessing a modified substrate or product specificity can be prepared. Preferably, such mutants show an increased activity. Alternatively, mutants can be prepared the catalytic activity of which is abolished without losing substrate binding activity.

Furthermore, the introduction of mutations into the polynucleotides encoding an enzyme as defined above allows the gene expression rate and/or the activity of the enzymes encoded by said polynucleotides to be reduced or increased.

For genetically modifying bacteria or fungi, the polynucleotides encoding an enzyme as defined above or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning; A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, “primer repair”, restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.

Thus, in accordance with the present invention a recombinant microorganism can be produced by genetically modifying fungi or bacteria comprising introducing the above-described polynucleotides, nucleic acid molecules or vectors into a fungus or bacterium.

The polynucleotide encoding the respective enzyme is expressed so as to lead to the production of a polypeptide having any of the activities described above. An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and 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 for instance given 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).

Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., 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), Ip1, 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 do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-¾-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

The transformation of the host cell with a polynucleotide or vector as described above can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSFI Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.

A method according to the present invention may carried out in vivo. Thus, according to the invention, the method is carried out in culture, in the presence of a microorganism producing the enzymes described above for the conversions according to the present invention as described herein above. A method which employs a microorganism for carrying out a method according to the invention is referred to as an “in vivo” method. It is possible to use a microorganism which naturally produces the enzymes described above for the conversions of the method according to the present invention or a microorganism which had been genetically modified so that it expresses (including overexpresses) one or more of such enzymes. Thus, the microorganism can be an engineered microorganism which expresses enzymes described above for the conversions of the method according to the present invention, i.e. which has in its genome a nucleotide sequence encoding such enzymes and which has been modified to overexpress them. The expression may occur constitutively or in an induced or regulated manner. In another embodiment the microorganism can be a microorganism which has been genetically modified by the introduction of one or more nucleic acid molecules containing nucleotide sequences encoding one or more enzymes described above for the conversions of the methods according to the present invention. The nucleic acid molecule can be stably integrated into the genome of the microorganism or may be present in an extrachromosomal manner, e.g. on a plasmid.

Such a genetically modified microorganism can, e.g., be a microorganism that does not naturally express enzymes described above for the conversions of the method according to the present invention and which has been genetically modified to express such enzymes or a microorganism which naturally expresses such enzymes and which has been genetically modified, e.g. transformed with a nucleic acid, e.g. a vector, encoding the respective enzyme(s), and/or insertion of a promoter in front of the endogenous nucleotide sequence encoding the enzyme in order to increase the respective activity in said microorganism.

However, the invention preferably excludes naturally occurring microorganisms as found in nature expressing an enzyme as described above at levels as they exist in nature. Instead, the microorganism of the present invention and employed in a method of the present invention is preferably a non-naturally occurring microorganism, whether it has been genetically modified to express (including overexpression) an exogenous enzyme of the invention not normally existing in its genome or whether it has been engineered to overexpress an exogenous enzyme. Thus, the enzymes and microorganisms employed in the present invention are preferably non-naturally occurring enzymes or (microorganisms), i.e. they are enzymes or (micro)organisms which differ significantly from naturally occurring enzymes or microorganism and which do not occur in nature. As regards the enzymes, they are preferably variants of naturally occurring enzymes which do not as such occur in nature. Such variants include, for example, mutants, in particular prepared by molecular biological methods, which show improved properties, such as a higher enzyme activity, higher substrate specificity, higher temperature resistance and the like. As regards the microorganisms, they are preferably genetically modified organisms as described herein above which differ from naturally occurring organisms due to a genetic modification. Genetically modified organisms are organisms which do not naturally occur, i.e., which cannot be found in nature, and which differ substantially from naturally occurring organisms due to the introduction of a foreign nucleic acid molecule.

By overexpressing an exogenous or endogenous enzyme as described herein above, the concentration of the enzyme is substantially higher than what is found in nature, which can then unexpectedly force the reaction of the present invention which uses a non-natural for the respective enzyme. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.

A “non-natural" substrate is understood to be a molecule that is not acted upon by the respective enzyme in nature, even though it may actually coexist in the microorganism along with the endogenous enzyme. This “non-natural” substrate is not converted by the microorganism in nature as other substrates are preferred (e.g. the “natural substrate”). Thus, the present invention contemplates utilizing a non natural substrate with the enzymes described above in an environment not found in nature.

Thus, it is also possible in the context of the present invention that the microorganism is a microorganism which naturally does not have the respective enzyme activity but which is genetically modified so as to comprise a nucleotide sequence allowing the expression of a corresponding enzyme. Similarly, the microorganism may also be a microorganism which naturally has the respective enzyme activity but which is genetically modified so as to enhance such an activity, e.g. by the introduction of an exogenous nucleotide sequence encoding a corresponding enzyme or by the introduction of a promoter for the endogenous gene encoding the enzyme to increase endogenous production to overexpressed (non-natural) levels.

If a microorganism is used which naturally expresses a corresponding enzyme, it is possible to modify such a microorganism so that the respective activity is overexpressed in the mircroorganism. This can, e.g., be achieved by effecting mutations in the promoter region of the corresponding gene or introduction of a high expressing promoter so as to lead to a promoter which ensures a higher expression of the gene. Alternatively, it is also possible to mutate the gene as such so as to lead to an enzyme showing a higher activity.

By using microorganisms which express enzymes described above for the conversions of the methods according to the present invention, it is possible to carry out the methods according to the invention directly in the culture medium, without the need to separate or purify the enzymes.

In one embodiment the microorganism employed in a method according to the invention is a microorganism which has been genetically modified to contain a foreign nucleic acid molecule encoding at least one enzyme described above for the conversions of the methods according to the present invention. The term “foreign” or “exogenous” in this context means that the nucleic acid molecule does not naturally occur in said microorganism. This means that it does not occur in the same structure or at the same location in the microorganism. In one preferred embodiment, the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the respective enzyme in which the promoter driving expression of the coding sequence is heterologous with respect to the coding sequence. “Heterologous” in this context means that the promoter is not the promoter naturally driving the expression of said coding sequence but is a promoter naturally driving expression of a different coding sequence, i.e., it is derived from another gene, or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the microorganism, i.e. a promoter which does naturally not occur in the respective microorganism. Even more preferably, the promoter is an inducible promoter. Promoters for driving expression in different types of organisms, in particular in microorganisms, are well known to the person skilled in the art.

In a further embodiment the nucleic acid molecule is foreign to the microorganism in that the encoded enzyme is not endogenous to the microorganism, i.e. is naturally not expressed by the microorganism when it is not genetically modified. In other words, the encoded enzyme is heterologous with respect to the microorganism. The foreign nucleic acid molecule may be present in the microorganism in extrachromosomal form, e.g. as a plasmid, or stably integrated in the chromosome. A stable integration is preferred. Thus, the genetic modification can consist, e.g. in integrating the corresponding gene(s) encoding the enzyme(s) into the chromosome, or in expressing the enzyme(s) from a plasmid containing a promoter upstream of the enzyme-coding sequence, the promoter and coding sequence preferably originating from different organisms, or any other method known to one of skill in the art.

The term “microorganism” in the context of the present invention refers to bacteria, as well as to fungi, such as yeasts, and also to algae and archaea. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli. In another preferred embodiment the bacterium belongs to the species Pseudomonas putida or to the species Zymomonas mobilis or to the species Corynebacterium glutamicum or to the species Bacillus subtilis.

It is also possible to employ an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae.

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

In another embodiment, the present invention makes use of a photosynthetic microorganism expressing at least one enzyme for the conversion according to the invention as described above. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae. In a further embodiment the microorganism is an algae, more preferably an algae belonging to the diatomeae.

It is also conceivable to use in accordance with the present invention a combination of microorganisms wherein different microorganisms express different enzymes as described above. The genetic modification of microorganisms to express an enzyme of interest will also be further described in detail below.

In another embodiment, as already mentioned above, the method of the invention comprises the step of providing the microorganism carrying the respective enzyme activity or activities in the form of a (cell) culture, preferably in the form of a liquid cell culture, a subsequent step of cultivating the organism, preferably the microorganism in a fermenter (often also referred to a bioreactor) under suitable conditions allowing the expression of the respective enzyme and further comprising the step of effecting an enzymatic conversion of a method of the invention as described herein above. Suitable fermenter or bioreactor devices and fermentation conditions are known to the person skilled in the art. A bioreactor or a fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or a fermenter may be a vessel in which a chemical/biochemical like the method of the present invention is carried out which involves organisms, preferably microorganisms and/or biochemically active substances, i.e., the enzyme(s) described above derived from such organisms or organisms harboring the above described enzyme(s). In a bioreactor or a fermenter, this process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, and may range in size from litres to cubic metres, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in a way that it is suitable to cultivate the organisms, preferably microorganisms, in, e.g., a batch-culture, feed-batch-culture, perfusion culture or chemostate-culture, all of which are generally known in the art.

The culture medium can be any culture medium suitable for cultivating the respective organism or microorganism.

Figure 1: shows the individual steps of the conversion from glucose into fructose- e-phosphate. Step A is catalyzed by a glucose kinase (EC 2.7.1.2) and/or a N-acetylmannosamine kinase (EC 2.7.1.60). Step B is catalyzed by a glucose-6-phosphate isomerase (EC 5.3.1.9). The recombinant microorganism of the present invention has a deactivated or abolished glucose-6-phosphate dehydrogenase (EC 1.1.1.49), and cannot perform step C. Moreover, the recombinant microorganism of the present invention also has a deactivated or abolished phosphofructokinase (EC 2.7.1.11) and cannot perform step D.

Figure 2: shows the individual steps of the conversion from glucose into D-ribose- 1 -phosphate (comprising a step catalyzed by a phosphoketolase acting on xylulose-5-phosphate). The individual steps are catalyzed by a transketolase (EC 2.2.1.1) for steps A and D, a phosphoketolase (EC 4.1.2.9 or EC 4.1.2.22) for step E, a transaldolase (EC 2.2.1.2) for step C, a ribulose-phosphate 3-epimerase (EC 5.1.3.1) for step F, a ribose- 5-phosphate isomerase (EC 5.3.1.6) for step G, a phosphopentomutase (EC 5.4.2.7) for step H, and a phosphate acetyltransferase (EC 2.3.1.8) for step I.

Figure 3: shows the individual steps (comprising a step catalyzed by a phosphoketolase acting on fructose-6-phosphate) of the conversion from glucose into D-ribose-1 -phosphate. The individual steps are catalyzed by a transketolase (EC 2.2.1.1) for step D, a phosphoketolase (EC 4.1.2.9 or EC 4.1.2.22) for step B, a transaldolase (EC 2.2.1.2) for step C, a ribulose-phosphate 3-epimerase (EC 5.1.3.1) for step F, a ribose-5-phosphate isomerase (EC 5.3.1.6) for step G, a phosphopentomutase (EC 5.4.2.7) for step FI, and a phosphate acetyltransferase (EC 2.3.1.8) for step I.

Figure 4: shows the individual steps (without a step catalyzed by a phosphoketolase) of the conversion from glucose into D-ribose-1- phosphate. The individual steps are catalyzed by a transketolase (EC 2.2.1.1) for steps A and D, a transaldolase (EC 2.2.1.2) for step C, a ribulose-phosphate 3-epimerase (EC 5.1.3.1) for step F, a ribose-5- phosphate isomerase (EC 5.3.1.6) for step G, a phosphopentomutase (EC 5.4.2.7) for step FI, and a phosphate acetyltransferase (EC 2.3.1.8) for step I.

Figure 5: shows the individual steps of the conversion from 2-deoxy-D-ribiose-1 - phosphate into acetyl-CoA and glyceraldehyde-3-phosphate. In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. 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.