Enzymes and methods for production of acetyl phosphate

Abstract

The invention relates to a method for production of acetyl phosphate from renewable or next generation feedstocks, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase. In embodiments, the phosphoketolase comprises at least one mutation in its active site preferably enabling an increased affinity and / or increased phosphoketolase activity for a non-phosphorylated carbohydrate, such as i) D-fructose, (ii) D-erythrulose or (iii) glycolaldehyde, compared to a wild- type phosphoketolase sequence. The invention further relates to a phosphoketolase enzyme comprising in its active site the mutation(s) H548N, N549D, H256Y, T2A:16T:H260Y:H548N, H548Y, H260Y:H548Y, H256Y:H260Y:H548Y, H142N:E153D and / or T2A:16T:H260Y:H548Y and its use for producing acetyl phosphate from a non-phosphorylated carbohydrate. The invention further relates to non-human organisms, preferably a microorganism, comprising a gene encoding and optionally expressing said phosphoketolase enzyme and compositions comprising the same.

Description

[0001] ENZYMES AND METHODS FOR PRODUCTION OF ACETYL PHOSPHATE

[0002] DESCRIPTION

[0003] The invention is in the fields of chemistry and biotechnology, in particular biotechnological process engineering, natural product synthesis and enzyme cascades.

[0004] The invention relates to a method for production of acetyl phosphate, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, using a phosphoketolase.

[0005] In embodiments, the invention relates to a method for production of acetyl phosphate, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase, wherein the phosphoketolase comprises at least one mutation in its active site, wherein preferably the mutation enables an increased affinity and / or increased phosphoketolase activity for a non-phosphorylated carbohydrate, such as i) D-fructose, (ii) D- erythrulose or (iii) glycolaldehyde, compared to a wild-type phosphoketolase sequence.

[0006] The invention further relates to a phosphoketolase enzyme comprising in its active site a mutation(s) such as H548N, N549D, H256Y, T2A:16T:H260Y:H548N, H548Y, H260Y:H548Y, H256Y:H260Y:H548Y, H142N:E153D and / or T2A:16T:H260Y:H548Y and its use for producing acetyl phosphate from a non-phosphorylated carbohydrate.

[0007] The invention further relates to an organism, preferably a non-human organism, such as a microorganism, comprising a gene encoding and optionally expressing said phosphoketolase enzyme and compositions comprising the same.

[0008] BACKGROUND OF THE INVENTION

[0009] Adenosine triphosphate (ATP) is an essential cofactor of many enzymes for various cell-free product syntheses, such as the production of phosphorylated chemicals or cell-free protein synthesis. Due to the very high price of ATP (~1400 € / mol), the transfer of such ATP-dependent, in vitro product syntheses to an industrial scale requires a low-cost system for the regeneration of ATP, starting from inexpensive substrates.

[0010] Until today, established ATP regeneration systems are based on expensive and energy-rich compounds such as polyphosphate, acetyl phosphate or phosphoenolpyruvate [10, 11], Only few ATP regeneration systems based on less expensive substrates, such as glutamate or pyruvate, have been described

[0013] ,

[0011] One disadvantage of prior at methods is that ATP regeneration starting from polyphosphate, acetyl phosphate or phosphoenolpyruvate causes on one site high substrate costs and on the other site phosphate emission, which often leads to inhibition of product synthesis. The disposal of phosphate-rich process wastewater results in environmental challenges and further increased costs. In addition, ATP regeneration starting from glutamate or pyruvate requires gassing of the reactor, thereby causing higher energy costs and strong foam formation, which renders the implementation of the reaction in a larger scale quite difficult. Further, phosphoketolase (PKT) naturally only possesses a significant catalytic activity for phosphorylated sugars. Towards non- phosphorylated substrates, such as fructose or glycolaldehyde, PKT naturally possesses only a very low affinity and catalytic activity. Only one system is presently available

[0014] employing a wild-type PKT enzyme to produce ATP from carbohydrates which achieves only low reaction rates and is therefore not suitable for industrial applications.

[0012] To date, no cell-free enzyme systems are available for cost-efficient, large-scale, simple and rapid ATP regeneration that also lack potential negative effects on the synthesis of the target product. Hence, there exists an unmet need for improved means for producing and / or regenerating ATP, preferably in cell-free systems, in a low-cost and industrial large-scale approach.

[0013] SUMMARY OF THE INVENTION

[0014] In light of the prior art the technical problem underlying the present invention is to provide alternative or improved means for producing and / or regenerating ATP from sustainable and low- cost substrates, such as non-phosphorylated carbohydrates, suitable for large-scale applications and industrial use.

[0015] Another object of the invention is to provide means for producing and / or regenerating adenosine triphosphate (ATP) that do not exhibit the disadvantages of the prior art. In this regard, a further object of the invention is to provide improved means for regenerating ATP from ADP in suitable in vitro systems.

[0016] Another object of the invention is to provide improved phosphoketolase (PKT) enzymes suitable for producing and / or regenerating ATP.

[0017] In light of the prior art the technical problem underlying the present invention is to provide alternative or improved means for (cell-free) production of acetyl phosphate.

[0018] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0019] The invention therefore relates to a method for production of acetyl phosphate, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate (C2H5O5P) and a product, with a phosphoketolase (PKT), wherein the phosphoketolase comprises at least one mutation, preferably in its active site / catalytic center.

[0020] In preferred embodiments the at least one mutation comprised by the phosphoketolase enables an increased affinity and / or increased phosphoketolase activity for a non-phosphorylated carbohydrate compared to a wild-type phosphoketolase sequence. In preferred embodiments, the at least one mutation comprised by the phosphoketolase enables an increased affinity and / or increased phosphoketolase activity for at least one non-phosphorylated carbohydrate compared to a wild-type phosphoketolase sequence.

[0021] In embodiments, the at least one mutation comprised by the phosphoketolase is with respect to the wild type PKT enzyme of Bifidobacterium adolescentis (SEQ ID NO 14) and is preferably located within the amino acid sequence position 142 and 605 of the PKT amino acid sequence of Bifidobacterium adolescentis (SEQ ID NO 14). In preferred embodiments the non-phosphorylated carbohydrate is selected from (i) D-fructose (CeH^Oe), (ii) D-erythrulose (C4H8O4) and (iii) glycolaldehyde (C2H4O2), and the product is selected from (i) D-erythrose (C4H8O4), (ii) glycolaldehyde (C2H4O2) and (iii) water (H2O), respectively. In embodiments, further non-phosphorylated sugars may be selected from D- ribulose (C5H10O5), D-xylulose (C5H10O5), D-tagatose (CeH^Oe), D-sorbose (CeH^Oe) and D- psicose (CeH^Oe).

[0022] In embodiments, the non-phosphorylated carbohydrate is selected from (i) D-fructose, (ii) D- erythrulose and (iii) glycolaldehyde, and / or the product is selected from (i) D-erythrose, (ii) glycolaldehyde and (iii) water (H2O), respectively.

[0023] In embodiments, the non-phosphorylated carbohydrate is selected from (i) D-fructose, and (ii) glycolaldehyde, and / or the product is selected from (i) D-erythrose, and (ii) water (H2O), respectively.

[0024] In embodiments the non-phosphorylated carbohydrate is D-fructose (CeH^Oe), and the product is D-erythrose (C4H8O4). In embodiments the non-phosphorylated carbohydrate is D-erythrulose (C4H8O4) and the product is glycolaldehyde (C2H4O2). In embodiments the non-phosphorylated carbohydrate is glycolaldehyde (C2H4O2), and the product is water (H2O).

[0025] In embodiments, each of the PKT-catalyzed reactions according to the present invention also release water, e.g., those starting from (i) fructose or (ii) erythrulose, in addition to products, like (i) erythrose or (ii) glycolaldehyde (see e.g., also Figure 8 and 9).

[0026] In embodiments the method for production of acetyl phosphate, comprises (optionally as a first step) enzymatically converting D-fructose into acetyl phosphate and D-erythrose with a PKT comprising at least one mutation.

[0027] In embodiments the method for production of acetyl phosphate, comprises (optionally as a consecutive step to the afore reaction) enzymatically converting D-erythrulose, optionally obtained by enzymatically converting D-erythrose into D-erythrulose, preferably using erythrose isomerase (El), into acetyl phosphate and glycolaldehyde with a PKT comprising at least one mutation.

[0028] In embodiments the method for production of acetyl phosphate, comprises (optionally as a consecutive step to the afore reaction(s)) enzymatically converting glycolaldehyde into acetyl phosphate and water with a PKT comprising at least one mutation.

[0029] In embodiments one or more of the three afore reactions / enzymatic conversions may be combined consecutively into a reaction cascade for producing acetyl phosphate in a highly efficient manner, wherein the product of one reaction is processed by a respective next reaction. In embodiments the same or different mutant PKT enzymes according to the invention may be used in the consecutive reaction steps. Preferably each used mutant PKT is capable of performing the respective reaction with an advantageously high efficiency and / or speed, e.g., due to an increased affinity and / or enzymatic activity towards the respective non-phosphorylated carbohydrate that is processed. In embodiments the method for the production of acetyl phosphate, comprises enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase (PKT), wherein the phosphoketolase comprises at least one mutation, wherein the non-phosphorylated carbohydrate is selected from (i) D-fructose, (ii) D- erythrulose and (iii) glycolaldehyde, and the product is selected from (i) D-erythrose, (ii) glycolaldehyde and (iii) water (H2O), respectively, and wherein the method comprises:

[0030] (i) enzymatically converting D-fructose into acetyl phosphate and D-erythrose with a PKT comprising at least one mutation,

[0031] (ii) enzymatically converting D-erythrulose, (iia) preferably obtained by enzymatically converting D-erythrose obtained in (i) into D-erythrulose, preferably using erythrose isomerase (El), into acetyl phosphate and glycolaldehyde with a PKT comprising at least one mutation, and

[0032] (iii) optionally enzymatically converting glycolaldehyde into acetyl phosphate and water (H2O) with a PKT comprising at least one mutation.

[0033] In embodiments, fructose may be obtained from glucose using a glucose isomerase.

[0034] The present invention provides methods and enzymes for production of acetyl phosphate from low-cost non-phosphorylated carbohydrates into acetyl phosphate using a phosphoketolase comprising at least one mutation in its active (catalytic) site.

[0035] At the present time, the production or regeneration of adenosine triphosphate (ATP) is expensive, posing a great disadvantage for diverse cell-free synthesis applications requiring ATP as enzymatic cofactor and energy carrier. Efficient and low-cost in vitro synthesis methods for producing acetyl phosphate and / or regenerating ATP are urgently needed, as until today, ATP regeneration systems commonly require energy-rich compounds such as polyphosphate, acetyl phosphate or phosphoenolpyruvate [10, 11],

[0036] One major disadvantage of the prior at methods is that ATP regeneration starting from polyphosphate, acetyl phosphate or phosphoenolpyruvate causes high substrate costs and phosphate emission, which often leads to inhibition of product synthesis. The disposal of phosphate-rich process wastewater results in environmental challenges and further increased costs. In addition, ATP regeneration starting from more cost-effective substrates, such as glutamate or pyruvate, requires gassing of the reactor, thereby causing higher energy costs and strong foam formation, which impedes the application of such reactions in a larger scale.

[0037] Further, phosphoketolase naturally possesses a catalytic activity only towards phosphorylated sugars but shows only a very low affinity and activity towards cheap non-phosphorylated substrates, such as fructose or glycolaldehyde. Employing a wild-type PKT enzyme to regenerate ATP or produce acetyl phosphate from non-phosphorylated carbohydrates achieves only low reaction rates and is therefore not suitable for industrial applications. Therefore, it was entirely surprising and beneficial that the phosphoketolase enzymes according to the invention are able to process / convert low-cost non-phosphorylated carbohydrates, e.g., fructose, glucose, erythrulose, and glycolaldehyde, into acetyl phosphate at high reaction rates, which are suitable for the production of acetyl phosphate and / or the regeneration of ATP in an industrial scale. The present invention thereby solves the problems of the prior art by providing cell-free ATP generation and / or regeneration systems and methods from low-cost nonphosphorylated carbohydrates (e.g., see for exemplary embodiments Fig. 8-9).

[0038] For example, a phosphoketolase (PKT) enzyme comprising the mutation in H142N:E153D achieves a surprisingly high substrate affinity and good catalytical activity towards nonphosphorylated carbohydrates, compared to respective wild type (wt) PKT enzymes, and even in comparison to the single mutation H142N of PKT. As a further example, the same applies to a phosphoketolase enzyme comprising the single mutation N549D or H548N or the triple mutation H256Y:H260Y:H548Y, which achieve a surprisingly high catalytical efficiency for processing nonphosphorylated carbohydrates compared to respective wild type (wt) PKT enzymes.

[0039] In embodiments, the invention is employed principally in cofactor regeneration, e.g., for synthesis of cell-free protein or phosphorylated fine chemicals. Additionally, in embodiments applications of the present invention may include the synthesis of the platform molecule acetyl-CoA and thus the production of basic chemicals such as 1 -butanol, isoprenoids, polyhydroxyalkanoates and fatty acids.

[0040] As carbohydrates and sugars may be obtained also from renewable raw materials, and ethylene glycol may also be synthesized from CO2 or even PET waste, for example, the present methods may in embodiments also be employed for producing acetyl phosphate from renewable or next generation feedstocks.

[0041] In summary, the PKT enzymes according to the invention comprising one or more mutations (preferably within their active sites) comprise a surprisingly high binding affinity and catalytic activity for processing / converting non-phosphorylated carbohydrates in comparison to respective wild type PKT enzymes.

[0042] In preferred embodiments, a genetically modified (mutated) PKT enzyme facilitates the synthesis of acetyl phosphate, which may, in embodiments, be used by an acetate kinase for ATP (re)generation from ADP or for the synthesis of acetyl-CoA. In embodiments acetyl phosphate and ADP are converted by acetate kinase to ATP and acetate.

[0043] In embodiments, a non-phosphorylated carbohydrate is enzymatically converted into acetyl phosphate using a phosphoketolase, wherein the acetyl phosphate (C2H5O5P) is subsequently used for ATP synthesis from ADP (e.g., using acetate kinase) or acetyl CoA synthesis. In embodiments acetyl phosphate is used to phosphorylate ADP to ATP. In embodiments, acetyl phosphate is used for the synthesis of acetyl-coenzyme-A and phosphate from coenzyme-A (CoA) .

[0044] In general, phosphoketolase (PKT) is a lyase enzyme that catalyzes the reaction of phosphorylated carbohydrates, such as, for example, fructose-6-phosphate to erythrose-4- phosphate, acetyl phosphate and water. However, the wild-type phosphoketolase enzyme is known to possess a low catalytic reactivity for non-phosphorylated substrates, such as nonphosphorylated carbohydrates, e.g., as fructose, erythrulose, and glycolaldehyde.

[0045] A phosphoketolase enzyme according to the invention preferably comprises at least one mutation in its active site, such that it can bind and convert / process non-phosphorylated substrates, such as, e.g., non-phosphorylated carbohydrates, at a significant / high rate. It was entirely surprising that the phosphoketolase enzyme according to the invention could not only bind and convert / process non-phosphorylated substrates, but preferably also binds and converts them with a higher efficiency than wild type PKT enzymes, or even PKT enzymes described in the prior art and lacking the mutations according to the invention.

[0046] In embodiments, the PKT enzymes according to the present invention preferably possess an increased catalytic activity (and / or affinity) for non-phosphorylated substrates, such as, non- phosphorylated carbohydrates. A further beneficial effect of the surprising capability of PKT enzymes according to the present invention to directly process non-phosphorylated substrates is that, e.g., for the conversion of non-phosphorylated carbohydrates no additional enzymes are requited, such as an aldolase enzyme for the conversion of D-erythrulose to glycolaldehyde. As wild type PKT enzymes comprise a very low affinity and enzymatic activity towards non- phosphorylated substrates, it was entirely unexpected that the present PKT enzymes are capable of converting non-phosphorylated substrates, such as D-fructose, D-erythrulose and glycolaldehyde with a high efficiency and significantly increased rate, thus facilitating the industrial or larger scale production of acetyl phosphate and / or regeneration of ATP.

[0047] As is shown in more detail below, the enzymes and methods according to the invention enable a high ATP generation and recycling rate starting from low-cost substrates, without entailing negative effects on the product yield due to the release of large amounts of phosphate or the need for gassing of the reaction environment. In contrast to the prior art enzyme cascades described, e.g., by Marliere

[0014] , the synthesis of acetyl phosphate by the present method using the present PKT enzymes is carried out exclusively by phosphoketolase - the additional use of an aldolase is in the context of the present invention no longer essential.

[0048] In addition to cell-free ATP regeneration, the enzymes and reaction cascades disclosed herein can also be used in embodiments to synthesize acetyl-CoA from inexpensive substrates, which is an important source product for the production of basic chemicals such as 1 -butanol, isoprenoids, polyhydroxyalkanoates and fatty acids.

[0049] In embodiments, the present method for production of acetyl phosphate, comprises: enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product with a phosphoketolase (PKT), preferably wherein the phosphoketolase comprises at least one mutation that preferably enables an increased affinity and / or increased phosphoketolase activity for a non-phosphorylated carbohydrate compared to a wild-type phosphoketolase sequence, and wherein the non-phosphorylated carbohydrate is selected from (i) D-fructose, (ii) D-erythrulose and (iii) glycolaldehyde, and / or the product is selected from (i) D-erythrose, (ii) glycolaldehyde and (iii) water (H2O), respectively, wherein the method comprises one or more of the following reactions, preferably in a consecutive manner: (i) enzymatically converting D-fructose into acetyl phosphate and D-erythrose with a PKT comprising at least one mutation (producing acetyl phosphate and D-erythrose from D- fructose),

[0050] (iia) enzymatically converting D-erythrose, optionally obtained as product in (i), into D- erythrulose, preferably using erythrose isomerase (El),

[0051] (ii) enzymatically converting D-erythrulose, optionally obtained as product in (iia), into acetyl phosphate and glycolaldehyde with a PKT comprising at least one mutation (producing acetyl phosphate and glycolaldehyde from D-erythrulose),

[0052] (iii) enzymatically converting glycolaldehyde into acetyl phosphate and water (producing acetyl phosphate and water (H2O) from glycolaldehyde) with a PKT comprising at least one mutation.

[0053] In embodiments, the method further comprises enzymatically converting acetyl phosphate and adenosine diphosphate (ADP), into adenosine triphosphate (ATP) and acetate with acetate kinase (ACK) and / or enzymatically converting acetyl phosphate and coenzyme-A (CoA) into acetyl-CoA and phosphate with a phosphate acetyl transferase (PTA).

[0054] In embodiments, the present method may thus be employed for producing acetyl phosphate from different non-phosphorylated carbohydrates, preferably selected from D-fructose, D-erythrulose and glycolaldehyde. The inventors advantageously found that it is also possible to perform the reaction according to the invention in a combined and consecutive manner, thereby converting a first non-phosphorylated carbohydrate into a second one and optionally also into a third one, using the same or different PKT mutant(s) according to the invention, thereby creating a reaction cascade that enables producing acetyl phosphate in a highly efficient manner from non- phosphorylated carbohydrates.

[0055] In embodiments, the method according to the invention comprises: a. producing acetyl phosphate and D-erythrose from D-fructose according to a method of the invention, comprising enzymatically converting a non-phosphorylated carbohydrate (D-fructose) into acetyl phosphate and a product (D-erythrose) with a phosphoketolase comprising at least one mutation in its active site, b. enzymatically converting the D-erythrose obtained in a. into D-erythrulose, preferably using erythrose isomerase (El), and c. producing acetyl phosphate and glycolaldehyde from D-erythrulose obtained in b. according to a method of the invention, comprising enzymatically converting a non- phosphorylated carbohydrate (D-erythrulose) into acetyl phosphate and a product (glycolaldehyde) with a phosphoketolase comprising at least one mutation in its active site, d. and optionally producing acetyl phosphate and water (H2O) from glycolaldehyde obtained in c. according to a method of the invention, comprising enzymatically converting a non-phosphorylated carbohydrate (glycolaldehyde) into acetyl phosphate and a product (water (H2O)) with a phosphoketolase comprising at least one mutation in its active site.

[0056] In embodiments, when several reactions (enzymatic conversions) according to the present method are performed consecutively, e.g., as in the afore disclosed embodiment of a reaction cascade, each reaction preferably uses the product (which is a non-phosphorylated carbohydrate) of the afore reaction as input, wherein different or the same PKT enzyme(s), comprising different or thew same mutation(s), may be used for the respective consecutive enzymatic conversions, preferably depending on the respective PKTs advantageously increased affinity and / or increased phosphoketolase activity for the respective non-phosphorylated carbohydrate to be converted. Hence, in some embodiments, by using different or the same PKT enzyme(s), comprising different or thew same mutation(s), the performance of the respective reaction cascade may be advantageously (further) optimized by choosing the ‘best’ performing PKT enzyme (e.g., according to tables 26-28) for each respective enzymatic conversion reaction.

[0057] The inventors surprisingly found that the erythrose intermediate of the cascade formed in step a) (producing acetyl phosphate and D-erythrose from D-fructose by enzymatically converting a D- fructose into acetyl phosphate and D-erythrose with a mutant phosphoketolase) can inactivate the involved enzymes, particularly PKT, thereby, causing incomplete conversion of the substrate and decreased ATP yield of the cascade. The inventors found the same effect for glycolaldehyde. The inventors discovered that it can be of advantage to keep the concentration of inactivating intermediates, such as erythrose and glycolaldehyde, as low as possible within a in vitro and cell free reaction solution to mitigate the inactivating effect of said intermediates on the employed proteins / enzymes. From this unexpected finding the inventors recapitulated that it can be particularly advantageous, when enzymes, e.g., the mutant PKTs disclosed herein, are able process the intermediates, e.g., in steps b.-d. of the above reaction cascade, with an increased efficiency and / or reaction speed, such that intermediates from step a. and / or c. (e.g., erythrose or glycolaldehyde) do not accumulate and inactivate enzyme(s) of the reaction cascade and cannot slow down the synthesis of acetyl phosphate.

[0058] In embodiments, erythrose accumulation may be avoided or reduced by enabling increased or excess erythrose isomerase activity, preferably compared to the fructose-dependent PKT activity. In embodiments, accumulation of erythrose may be avoided or reduced by ensuring adequate amounts of erythrose isomerase (with sufficient enzymatic activity) in the reaction environment, e.g., in addition to sufficient amounts of PKT enzymes.

[0059] Moreover, the inventors found that the use of erythrose as a starting substrate for a PKT cascade in cell-free systems is disadvantageous because erythrose not only inactivates the relevant enzymes, but is also expensive. The same observation they made for glycolaldehyde, which they found to disadvantageously denature proteins. Further, the inventors also found that the use of erythrulose as a starting substrate for the PKT cascade in cell-free systems is not practical, as this substrate is also expensive. Hence, the inventors developed a reaction cascade and suitable PKT mutants, according to embodiments of the invention, that overcome said problems, such that instead of providing the disadvantageous substrates separately, the cascade for producing acetyl phosphate may start from the low-cost substrate fructose. In embodiments, it is particular advantageous that the present PKT enzymes are capable of carrying out one, or several of steps b.-d. (preferably at least step b. and / or d.) with increased efficiency and / or reaction speed, as the reaction product(s) of a reaction as described above in step a. and / or step c. (e.g., the intermediate products of erythrose or glycolaldehyde) is / are inactivating relevant enzyme(s) of the subsequent reaction cascade, thereby avoiding that intermediates from step a. and / or step c. (erythrose or glycolaldehyde) accumulate and inactivate enzymes and thereby inhibit enzymatic reaction(s). In other words, in embodiments of the invention the combination of rate-limiting (first / previous) reactions (e.g., step a) with rapid subsequent reactions (e.g., c-d), is particularly advantageous to avoid the accumulation of erythrose and / or glycolaldehyde, thereby enabling an increased efficiency and speed of the reaction cascades according to the present invention.

[0060] Such increased efficiency and / or reaction speed is particularly enabled by specific PKT mutants capable of processing non-phosphorylated carbohydrates at increased rates and / or reaction speed, such as those PKT mutants disclosed herein. In other words, in preferred embodiments, in any enzymatic reaction disclosed herein, where a reaction product (as final or intermediate product of a reaction (cascade)) is erythrose or glycolaldehyde, it may be advantageous, if said inhibitory reaction products are subsequently processed (‘removed’ / con verted), preferably quickly and efficiently, by one or more mutant PKT enzyme(s) according to the invention and do not accumulate, thereby increasing the efficiency of the overall production of acetyl phosphate.

[0061] In embodiments, the method further comprises enzymatically converting acetyl phosphate and coenzyme-A (CoA) into acetyl-CoA and phosphate with a phosphate acetyl transferase (PTA).

[0062] In embodiments, the method further comprises the regeneration of ATP from fructose and the (subsequent / parallel) production of (sn)-glycerol 3-phosphate (G3P) from acetyl phosphate (e.g., in a one-step enzyme / reaction cascade). In embodiments, the method comprises the coupling / combination of the respective enzymes and / or method according to the invention for producing and / or regenerating acetyl phosphate and / or ATP with the ATP-consuming synthesis of G3P that is catalyzed by glycerol kinase.

[0063] In embodiments, the method further comprises enzymatically converting acetyl phosphate and adenosine diphosphate (ADP), into adenosine triphosphate (ATP) and acetate with acetate kinase (ACK). Such embodiments of ‘regenerating’ ATP are particularly beneficial for the low-cost provision of ATP, e.g., for large or industrial scale in-vitro and / or cell free production approaches.

[0064] In embodiments, the non-phosphorylated carbohydrate is glycolaldehyde, and wherein the method comprises prior to the enzymatic conversion of glycolaldehyde into acetyl phosphate and a product: producing glycolaldehyde and NADH2 from ethylene glycol and nicotinamide adenine dinucleotide (NAD), with an NAD-dependent ethylene glycol dehydrogenase enzyme (NAD- EGDH).

[0065] In embodiments, the non-phosphorylated carbohydrate is glycolaldehyde, and wherein the method comprises prior to the enzymatic conversion of glycolaldehyde into acetyl phosphate and a product: producing glycolaldehyde and reduced pyrroloquinoline quinone (PQQH2) from ethylene glycol and pyrroloquinoline quinone (PQQ), with a PQQ-dependent ethylene glycol dehydrogenase enzyme (PQQ-EGDH). In embodiments, the non-phosphorylated carbohydrate is glycolaldehyde, and wherein the method comprises prior to the enzymatic conversion of glycolaldehyde into acetyl phosphate and a product: producing glycolaldehyde from two molecules of formaldehyde using glycolaldehyde synthase, and wherein the formaldehyde is produced from methanol using a methanol dehydrogenase (which is preferably either PQQ or NAD-dependent).

[0066] In embodiments, the non-phosphorylated carbohydrate is erythrulose, and wherein the method comprises prior to conversion of erythrulose into acetyl phosphate and a product: producing threose from glycolaldehyde using threose aldolase, preferably from two molecules of glycolaldehyde, and / or producing erythrulose from threose using threose isomerase. In embodiments, threose is generated from glycolaldehyde using threose aldolase in a first step, and subsequently erythrulose is generated from (the obtained) threose using threose isomerase.

[0067] In embodiments, the non-phosphorylated carbohydrate is erythrulose, and wherein the method comprises prior to conversion of erythrulose into acetyl phosphate and a product, producing erythrulose from glycolaldehyde using erythrulose aldolase (wherein, for example, 2 mol glycolaldehyde are converted to 1 mol erythrulose). An exemplary scheme of this embodiment is depicted in Figure 9B.

[0068] In embodiments, the erythrulose aldolase is a, preferably genetically modified or mutated (comprising at least one mutation), formolase (FLS) or (variant of a subunit of) alpha- ketoglutarate-dehydrogenase (SucA) enzyme.

[0069] In embodiments, the use of an erythrulose aldolase enables the direct (enzymatic) conversion of glycolaldehyde to erythrulose, e.g., in alternative to a two-step reaction comprising producing threose from glycolaldehyde using threose aldolase, and subsequently producing erythrulose from threose using threose isomerase (e.g., as depicted exemplarily in Figure 9C).

[0070] In embodiments, glycolaldehyde can be obtained from the converting / processing of erythrulose to acetyl phosphate and glycolaldehyde. In embodiments, glycolaldehyde can be obtained from methanol that is first processed / converted to formaldehyde, which is then processed / converted to glycolaldehyde. In embodiments, glycolaldehyde can be obtained from methanol that is first processed / converted from ethylene glycol, which is then processed / converted to glycolaldehyde.

[0071] In embodiments, the method is a cell-free method for producing acetyl phosphate. In embodiments, a cell-free (synthesis) method relies on the in vitro use of cellular machinery (e.g., one or more enzymes) alternative to microbial-based fermentation processes. Preferably a cell- free method according to the invention has the additional advantage of overcoming current limitations of microbial fermentation, such as low conversion yields of substrates and / or required purification of obtained products.

[0072] In embodiments the invention comprises a method for producing acetyl phosphate, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase (PKT), wherein the phosphoketolase comprises at least one mutation, wherein the non-phosphorylated carbohydrate is glycolaldehyde, which is enzymatically converted by said PKT into acetyl phosphate and water (H2O), and wherein the method comprises prior to the enzymatic conversion of glycolaldehyde:

[0073] (a) producing glycolaldehyde and NADH2 from ethylene glycol and nicotinamide adenine dinucleotide (NAD), with an NAD-dependent ethylene glycol dehydrogenase enzyme (NAD-EGDH), or

[0074] (b) producing glycolaldehyde and reduced pyrroloquinoline quinone (PQQH2) from ethylene glycol and pyrroloquinoline quinone (PQQ), with a PQQ-dependent ethylene glycol dehydrogenase enzyme (PQQ-EGDH), or

[0075] (c) producing glycolaldehyde from two molecules of formaldehyde using glycolaldehyde synthase, and wherein the formaldehyde is produced from methanol using a methanol dehydrogenase.

[0076] In embodiments the invention comprises a method for producing acetyl phosphate, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase (PKT), wherein the phosphoketolase comprises at least one mutation, wherein the non-phosphorylated carbohydrate is D-erythrulose, which is converted into acetyl phosphate and glycolaldehyde with a PKT comprising at least one mutation, and wherein the method comprises prior to conversion of erythrulose into acetyl phosphate and a product:

[0077] (a) producing erythrulose from threose using threose isomerase, and

[0078] (b) optionally producing threose from two molecules of glycolaldehyde using threose aldolase.

[0079] In embodiments of the present method, the phosphoketolase enzyme comprises in its active site one or more of the mutations Q321G, Q321 I, Q321 L, Q321 S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q546E, Q321S:H548N, Q546E:N549D, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, H256Y, H256Y:H260Y:H548Y, H142N:H256Y and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14). The invention therefore encompasses any one or more of the mutations described herein, in any given combination.

[0080] In embodiments of the present method, the phosphoketolase enzyme comprises in its active site one or more of the mutations Q321G, Q321 I, Q321 L, Q321 S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321S:H548N, Q546E:N549D, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, H256Y, H256Y:H260Y:H548Y, H142N:H256Y and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14). In embodiments, the present invention encompasses any one or more of the PKT-mutations disclosed herein, in any given combination.

[0081] In embodiments of the present method, the phosphoketolase enzyme comprises in its active site one or more of the mutations Q321G, Q321 I, Q321 L, Q321 S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, H548Y, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321S:H548N, Q321 L:H548N, Q321V:H548C, Q321V:H548L, H548N:K605T, H548K:K605V Q546E:N549D, N549S, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, T2A:I6T:H26OY:N549D, H256Y, H256Y:H260Y, H256Y:H260Y:H548Y, D547E, D547E:H548Y, H142N:H256Y, H142N:H260Y:H548Y, H142N:H256Y:H260Y:H548Y, H142N:H548Y, H142N:H548N, H142Q, H142E, E153D, E153Q, E153P, E153T and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14).

[0082] In embodiments, the present invention encompasses any one or more of the PKT-mutations disclosed herein, in any given combination.

[0083] In preferred embodiments of the present method(s), the phosphoketolase enzyme(s) used in the method of producing acetyl phosphate from a non-phosphorylated carbohydrate by enzymatic conversion comprises a PKT enzyme comprising one or more of the mutations disclosed herein. In embodiments, when several reactions (enzymatic conversions) according to the present method are performed consecutively, each reaction preferably uses the product (which is a nonphosphorylated carbohydrate) of the afore reaction as input, wherein a different or the same PKT enzyme, comprising different or thew same mutation(s), may be used for the respective consecutive steps. As a non-limiting example, if a PKT enzyme according to the invention comprises a (slightly, moderately or significantly) higher reactivity and / or affinity to a certain nonphosphorylated carbohydrate, compared to other PKT enzymes according to the invention, it may be used for converting said non-phosphorylated carbohydrate, while for the preceding or subsequent conversion reaction, if performed, a different PKT enzyme according to the invention may be used. Preferably, for each conversion reaction a PKT is used, e.g., selected from the enzymes disclosed herein and / or in tables 26-28, that has a certain (slightly, moderately or significantly) higher reactivity and / or affinity to the respective non-phosphorylated carbohydrate that is to be converted. Particularly in such embodiments, the present disclosure provides a variety of options for the skilled person to select suitable mutant PKT enzymes for each conversion reaction of a non-phosphorylated carbohydrate that is performed according to the present invention.

[0084] In embodiments, the present method comprises or takes advantage of the (over)expression of one or more enzymes selected from the group comprising phosphoketolase, D-erythrose isomerase, D-threose isomerase, D-threose aldolase, acetate kinase and glycerol kinase in a microbial host, e.g., a bacterium or yeast or a cell-free environment. In embodiments the expressed one or more enzyme(s) comprise an affinity or purification tag enabling purification of the (over)expressed enzyme(s) from a respective production strain, host cell, a culture / growth medium, culture supernatant and / or (cell-free) synthesis medium. In embodiments said one or more (over)expressed enzymes comprise at least one mutation, preferably within their active / catalytic site.

[0085] In embodiments, a respective gene encoding an enzyme disclosed herein, e.g., phosphoketolase, D-erythrose isomerase, D-threose isomerase, D-threose aldolase, acetate kinase or glycerol kinase, is cloned (prior to enzyme-(over)expression), into an expression vector, which preferably carries an antibiotic resistance gene and / or comprises an affinity or enrichment tag, e.g., a His-, FLAG-, GFP- or Strep-tag, either N-terminally or C-terminally from the gene encoding said enzyme. The skilled person is aware of suitable methods for (over)expressing proteins and suitable expression vectors for expressing / producing enzymes in microorganisms or a cell free environment. The skilled person is also familiar with methods of purifying and / or enriching the (over)expressed enzymes therefrom (e.g., using (affinity) chromatography, filtration or equivalent methods). In embodiments enzymes disclosed herein may be used after (over)expression and optional isolation and / or purification from a cell and / or a cell culture supernatant or a cell-free expression system in the methods according to the present invention for producing acetyl phosphate.

[0086] In embodiments a phosphoketolase enzyme is encoded by and / or expressed from the Bifidobacterium adolescentis gene Bad.f6pkt (WP_011743105.1). In embodiments a D-erythrose isomerase and / or D-threose isomerase enzyme are encoded by and / or expressed from the Pseudomonas stutzerii gene Ps.lhrl (AB121136.1).

[0087] In embodiments a D-threose aldolase is encoded by and / or expressed from the Eschericia coli gene Ec.fsaA (AAC73912.2) or its (genetic) variants such as L107Y:A129G. In embodiments, L107Y:A129G is used, due to its high affinity for glycol aldehyde. In addition, the wild-type D- threose aldolase enzyme and other (genetic) variants thereof also possess threose aldolase activity, in some embodiments with a higher Vmax (max. reaction velocity) but lower affinity for glycol aldehyde.

[0088] In embodiments an acetate kinase enzyme is encoded by and / or expressed from the Geobacillus stearothermophilus gene Gs.ackA (CP016552.1), the Eschericia coli gene Ec.ackA (AAC75356.1) or the Thermotoga maritima gene Tm.ack (AAD35363.1). In embodiments a glycerol kinase enzyme is encoded by and / or expressed from the Cellulomonas sp. gene Cs.glpK (AB531502.2), the Eschericia coli gene Ec.glpK (AAB03058.1) or the Streptomyces canus gene Sca.glpK (KUN66102.1).

[0089] In another aspect and in preferred embodiments of the present method the invention relates to a phosphoketolase enzyme comprising in its active site at least one mutation disclosed herein.

[0090] The following and afore disclosure relate to both, the phosphoketolase enzyme according to the invention, as well as to the phosphoketolase enzyme used in the method according to the invention for producing acetyl phosphate.

[0091] In embodiments, the phosphoketolase enzyme according to the invention comprises in its active site one or more of the mutation(s) Q321G, Q321 I, Q321 L, Q321S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q546E, Q321 S:H548N, Q546E:N549D, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, H256Y, H256Y:H260Y:H548Y, H142N:H256Y and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14). In embodiments, the invention therefore encompasses any one or more of the PKT mutations described herein, in any given combination.

[0092] In embodiments, the phosphoketolase enzyme according to the invention comprises in its active site one or more of the mutation(s) Q321G, Q321 I, Q321 L, Q321S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321S:H548N, Q546E:N549D, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, H256Y, H256Y:H260Y:H548Y, H142N:H256Y and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0093] In embodiments, the phosphoketolase enzyme according to the invention comprises in its active site one or more of the mutation(s) Q321G, Q321 I, Q321 L, Q321S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, H548Y, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321S:H548N, Q321 LH548N, Q321V:H548C, Q321V:H548L, H548N:K605T, H548K:K605V Q546E:N549D, N549S, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, T2A:I6T:H26OY:N549D, H256Y, H256Y:H260Y, H256Y:H260Y:H548Y, D547E, D547E:H548Y, H142N:H256Y, H142N:H260Y:H548Y, H142N:H256Y:H260Y:H548Y, H142N:H548Y, H142N:H548N, H142Q, H142E, E153D, E153Q, E153P, E153T and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14). In embodiments, the invention therefore encompasses any one or more of the mutations described herein, in any given combination.

[0094] In embodiments, one or more mutations are introduced into a (wild type, wt) gene of an enzyme described herein, e.g., a phosphoketolase (PKT) enzyme, preferably using site-directed mutagenesis. In embodiments one or more amino acids of the enzyme according to the invention, e.g., PKT, is exchanged / altered, deleted or inserted by exchanging, inserting or deleting one or more nucleic acids in said gene sequence encoding the respective enzyme according to the invention. In exemplary embodiments, for example, a Q (glutamine) may be replaced by an amino acid H, R, Y, W, P, N, S, V, G, or a H (histidine) may be replaced by an amino acid K, C, E, N, L, Y.

[0095] In embodiments, protein modifications of the enzymes of the present invention, which may occur through substitutions within the amino acid sequence, and nucleic acid sequences encoding such enzymes, are also included within the scope of the invention.

[0096] In embodiments, the phosphoketolase enzyme comprises in its active site the mutation(s) H548N, N549D, H256Y, T2A:16T:H260Y:H548N, H548Y, Q321V, S541 N, H260Y:H548Y, H256Y:H260Y:H548Y, H142N:E153D and / or T2A:16T:H260Y:H548Y with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0097] In embodiments, the phosphoketolase enzyme comprises in its active site one or more of the mutation(s) H142, 1219, Q321 , E437, S440, R442, S541 , H548, N549 and / or K605 with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0098] In embodiments, the phosphoketolase enzyme comprises in its active site one or more of the mutation(s) H548N, T2A:I6T:H26OY:H548N, H142, 1219, Q321 , E437, S440, R442, S541 , N549 and / or K605 with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0099] In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation T2A:I6T:H26OY:H548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation N549D. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q546E:N549D. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H260Y:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H256Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H256Y:H260Y:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation T2A:I6T:H26OY:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q321G, Q321 I, Q321 L, Q321 S, or Q321V. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation S541 N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F or H548P. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation K605C, K605E, K605L, K605T or K605V. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation N549D.ln embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q321S:H548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q546E:N549D. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H260Y:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation T2A:I6T:H26OY:H548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation T2A:I6T:H26OY:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142N:H256Y or H142N:E153D. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H260Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation T2A:I6T:H26OY. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q546E.

[0100] In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q321 LH548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q321V:H548C. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation Q321V:H548L. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548N:K605T. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H548K:K605V. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation N549S. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation T2A:I6T:H26OY:N549D. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H256Y:H260Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation D547E. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation D547E:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142N:H260Y:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142N:H256Y:H260Y:H548Y. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142N:H548Y.

[0101] In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142N:H548N. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142Q. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation H142E. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation E153D. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation E153Q. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation E153P. In embodiments, the phosphoketolase enzyme comprises in its active site the mutation E153T. Preferably, each of the afore mutation position(s) is indicated with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0102] In embodiments, the phosphoketolase enzyme comprises in its active site one or more of the mutation(s) Q321G, Q321 I, Q321 L, Q321S, Q321V, S541 N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, H548Y, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321S:H548N, Q546E:N549D, H260Y:H548Y, T2A:I6T:H26OY, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, H256Y, H256Y:H260Y:H548Y, H142N:H256Y, H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0103] In embodiments, the phosphoketolase enzyme comprises (in its active site) one or more mutation(s) at the amino acid position 2, 6, 142, 153, 256, 260, 321 , 546, 547, 548, 549 and / or 605; or at the amino acid position T2, I6, H142, E153, H256, H260, Q321. Q546, D547, H548, N549, and / or K605, preferably with respect to the genomic sequence of Bifidobacterium adolescentis (according to SEQ ID NO 14).

[0104] In embodiments of the phosphoketolase enzyme according to the invention, the at least one mutation enables an increased affinity and / or increased phosphoketolase activity for at least one non-phosphorylated carbohydrate selected from D-fructose, D-erythrulose and glycolaldehyde compared to a wild-type phosphoketolase (sequence) enzyme.

[0105] The afore mentioned PKT mutations / mutants relate to both, the phosphoketolase enzyme according to the invention, as well as to the phosphoketolase enzyme used in the method according to the invention for producing acetyl phosphate.

[0106] The inventors surprisingly found, that the PKT mutant H142N not only shows a significantly higher activity on glycolaldehyde (compared to wt PKT), but that the use of said enzyme in the context of a reaction cascade, e.g., comprising at least two subsequent reactions, according to the present invention, it surprisingly enables the reduction of the intermediate glycolaldehyde in the reaction mixture by processing glycolaldehyde at an increased rate, such that glycolaldehyde does nor accumulate during the reaction cascade and inhibits enzymes of the cascade. In other words, the inventors discovered a surprising new mechanism by unraveling that glycolaldehyde is able to inhibit enzymes of reaction cascades for producing acetyl phosphate from nonphosphorylated carbohydrates according to the invention, which may be mitigated by using a PKT enzyme mutant that is capable of process the intermediate glycolaldehyde quickly to a less inhibiting product, thereby increasing the efficacy of reaction cascades according to the invention. In the context of the present invention the PKT mutant H142N is preferably only employed in combination with further mutations of a PKT enzyme and / or with mutated PKT enzymes comprising other mutations.

[0107] In embodiments, the skilled person would be capable, based on the present disclosure, to combine and use the PKT mutants disclosed herein meaningfully in any of the reaction (steps) described herein. The inventors found that such combination of PKT mutants according to the invention in reaction cascades disclosed herein can be particularly advantageous in certain scenarios. For example, different PKT mutants may be employed for different reaction(s) (steps), wherein respective mutants are used for reactions where they show particularly advantageous enzymatic functionality. As a non-limiting example, the mutant Ba. PKT H548N may show a highly improved activity on fructose, while Ba. PKT H142N shows a significantly higher activity on glycolaldehyde. In such embodiments the skilled person would be capable, based on the present disclosure to combine the PKT mutants Ba. PKT H548N and Ba. PKT H142N in a reaction cascade according to the invention, using each enzyme in a respective reaction step, such that, e.g., the concentration of glycolaldehyde in the reaction mixture could be minimized, as Ba. PKT H142N processes glycolaldehyde at an increased rate, such that glycolaldehyde does nor accumulate during the reaction cascade and inhibits enzymes of the cascade. This stabilizes proteins / enzymes in the reaction mixture, as the inventors surprisingly found, that glycolaldehyde is capable of denaturing proteins, thereby inhibiting enzymes used in the cascade. At the same time, in these embodiments, the productivity on fructose would therefore remain high, such that the entire cascade may run at an increased production efficiency, compared to prior art methods.

[0108] In embodiments, the one or more mutations within the active site of a phosphoketolase disclosed herein may be transferred or applied to other microorganisms that preferably comprised a nucleic acid sequence encoding a phosphoketolase (PKT) that is conserved with respect to, or shares at least 99, 95, 85, 80, 75, or 70 % sequence identity with, the sequence of Bifidobacterium adolescentis according to SEQ ID NO 14.

[0109] In embodiments, nucleic acid sequence encoding a phosphoketolase (PKT) in a microorganism shares at least 60, 65, 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% with, the sequence of Bifidobacterium adolescentis according to SEQ ID NO 14, such that the locations of the one or more mutations of a phosphoketolase disclosed herein may be transferred or applied to other microorganisms. Sequence identity may be determined using methods known to one skilled in the art, such as BLAST or other sequence alignment tools.

[0110] “Sequence variants” or "variants" of proteins or peptides, as defined in the context of the present invention, may be generated that have an amino acid sequence that differs from an original (wildtype) sequence in one or more mutation(s), such as one or more substituted, inserted and / or deleted amino acid(s). In embodiments, the sequence variation described here with respect to amino acid mutations (substituted, inserted and / or deleted amino acid(s)) and / or percentage identity, may apply to any one or more embodiments, described throughout the application as a whole. Preferably the genomic sequence of Bifidobacterium adolescentis refers to NC_008618.1 :861083-863560; ATCC 15703.

[0111] In embodiments a wild-type phosphoketolase enzyme may be derived from one of the following microorganisms: Bifidobacterium adolescentis (ATCC 15703; WP_011743105.1), Bifidobacterium breve (DSM 20213; EFE89336.1), Bifidobacterium breve strain 203 (ADF97524.1), Bifidobacterium animalis subsp. lactis DSM 10140 (ACS47899.1), Bifidobacterium dentium Bd1 (ADB09649.1), Clostridium acetobutylicum ATCC 824 (WP_010964652.1), Lactiplantibacillus pentosus (WP_050339622.1), Saccharomonospora marina (WP_009154258.1), Nitrosospira sp. 56-18 (OJY14508.1), or Achromobactersp. DMS1 (WP_082308260).

[0112] In general, at least the genomic regions and / or amino acid sequences comprising the active site of a PKT enzyme and / or comprising herein disclosed mutations of a PKT enzyme is / are conserved among bacterial species, such that the genomic location / numbering of mutations indicated herein with respect to Bifidobacterium adolescentis may change slightly between bacterial strains. However, from the herein disclosed exemplary alignment of genomic sequences of Bifidobacterium adolescentis with other bacterial strains (as shown in Figure 7) it is evident to a skilled person what the corresponding positions in bacterial species other than Bifidobacterium adolescentis would be. In general, a skilled person knows how to determine and transfer amino acid, or genomic locations disclosed for one non-human organism, e.g., bacterial strain, to another non-human organism, e.g., another bacterial strain.

[0113] In embodiments, the mutations of the phosphoketolase enzyme are with respect to the genomic sequence of Bifidobacterium, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium Bd1, Clostridium, Lactiplantibacillus pentosus, Saccharomonospora marina, Nitrosospira sp. or Achromobacter sp.

[0114] In embodiments, the mutations of the phosphoketolase enzyme are with respect to the genomic sequence of Bifidobacterium (WP_011743105.1), Bifidobacterium adolescentis, Bifidobacterium breve (EFE89336.1 or ADF97524.1), Bifidobacterium animalis subsp. lactis (ACS47899.1), Bifidobacterium dentium Bd1 (ADB09649.1), Clostridium (WP_010964652.1), Lactiplantibacillus pentosus (WP_050339622.1), Saccharomonospora marina (WP_009154258.1), Nitrosospira sp. (OJY14508.1) or Achromobactersp. (WP_082308260.1).

[0115] Table 1 : Genomic sequences of the respective microorganisms. In a further aspect the present invention relates to the use of the phosphoketolase enzyme according to the invention for producing acetyl phosphate from a non-phosphorylated carbohydrate in a method according to the invention.

[0116] For example, in embodiments the present invention facilitates at least 3 mol ATP / mol of nonphosphorylated carbohydrate, e.g., fructose or glucose, or 1 mol ATP / mol ethylene glycol. Thereby preferably reducing the substrate costs to 0.03 or 0.06 € / mol ATP.

[0117] In a further aspect the present invention relates to a non-human organism, preferably a microorganism, comprising a gene encoding and optionally expressing the phosphoketolase enzyme according to the invention disclosed herein.

[0118] In embodiments the non-human organism or microorganism is a bacterium. In embodiments the non-human organism or microorganism is a fungus, such as a yeast (e.g., Saccharomyces).

[0119] The disclosure herein does not relate to genetic modifications in humans, therefore the term ‘organism’ as used herein does not include or relate to human beings such that each genetically modified organism referred to herein is non-human.

[0120] In embodiments the organism or microorganism is selected from the group comprising Escherichia coli (E. coli), Pseudomonas putida Vibrio natriegens, Corynebacterium glutamicum, Geobacillus stearothermophilus, Gluconobacteroxydans, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces marxianus.

[0121] In embodiments the non-human organism or microorganism is the bacterium E. coli, preferably selected from one of the strains E. coli BL21 (DE3), or E. coli Rosetta(DE3) plysS.

[0122] In a further aspect the present invention relates to the use of a non-human organism or microorganism according to the invention for producing acetyl phosphate from a nonphosphorylated carbohydrate, preferably according to a method of the invention disclosed herein.

[0123] In a further aspect the present invention relates to a composition, comprising: i. a non-phosphorylated carbohydrate, preferably selected from D-fructose, D-erythrulose and glycolaldehyde, and ii. a phosphoketolase according to the present invention, and / or a non-human (micro)organism according to the invention, and optionally iii. a phosphate acetyl transferase (PTA), iv. an erythrose isomerase (El), and / or v. an acetate kinase (ACK).

[0124] In a further aspect the present invention relates to a composition, comprising: i. a phosphoketolase according to the present invention, and / or a non-human (micro)organism according to the invention, ii. an ethylene glycol dehydrogenase (EGDH), iii. ethylene glycol, and iv. nicotinamide adenine dinucleotide (NAD), and optionally v. a threose aldolase and / or a threose isomerase and / or vi. an acetate kinase (ACK) and / or a phosphate acetyl transferase (PTA).

[0125] In embodiments, the compositions of the invention, for example the two aspects described immediately above, relate to reaction compositions, comprising or consisting of compositions with reagents, products and / or intermediates that form before, during and / or after the reactions described herein are conducted.

[0126] Each optional or preferred feature of the invention that is disclosed or described in the context of one aspect of the invention, e.g., the method or the PKT enzyme is herewith also disclosed in the context of the other aspects of the invention described herein.

[0127] All features disclosed in the context of the PKT enzyme according to the invention also relate to the methods according to the invention, and are herewith disclosed also in the context of, the methods, the enzymes, the microorganisms and the composition and kits according to the invention, and vice versa. The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding of the improved capabilities of phosphoketolase enzymes, comprising in its active site one or more mutation(s) according to the invention, of binding and processing / converting non-phosphorylated carbohydrates into acetyl phosphate and a product with a high efficiency, particularly in comparison to wild-type PKT enzymes.

[0128] DETAILED DESCRIPTION OF THE INVENTION

[0129] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0130] The present invention is directed to method for production of acetyl phosphate, comprising enzymatically converting a non-phosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase, wherein the phosphoketolase comprises at least one mutation in its active site, wherein preferably the mutation enables an increased affinity and / or increased phosphoketolase activity for a non-phosphorylated carbohydrate compared to a wild-type phosphoketolase sequence.

[0131] Exemplary sequences of genes encoding phosphoketolase from different microorganisms are enlisted in the following and an alignment of the respective PKT amino acid sequences to each other is depicted in Figure 7:

[0132] In general, the ‘active site’ of an enzyme, e.g., of a phosphoketolase (PKT), may also be referred to as the ‘catalytic center’ or ‘catalytic site’ and constitutes the region of the enzyme where substrate molecules are bound and undergo a chemical reaction, conversion or catalytic processing. Commonly, the active site of an enzyme catalyzes a chemical reaction and comprises amino acid residues directly involved in the catalysis of substrates. In embodiments the ‘active site’ of an enzyme, e.g., of a phosphoketolase (PKT), may also refer to the position in an enzyme which is responsible for binding a substrate, such as a non-phosphorylated carbohydrate, and / or to the position where its catalytic / site for processing its substrate, such as said non-phosphorylated carbohydrate, is located.

[0133] In embodiments of the invention the ‘active site’ of a PKT comprising one or more mutations may be located at or within the amino acid sequence position 142 and 605 of a PKT enzyme, preferably with respect to the genomic sequence of PKT of Bifidobacterium adolescentis (SEQ ID NO 14). In other words, in embodiments the one or more mutation in its active site / catalytic center may be located within the amino acid sequence position 142 and 605 or at the amino acid sequence position 142, 153, 256, 260, 321 , 546, 547, 548, 549 and / or 605 of a PKT enzyme.

[0134] Hence, preferably herein, the term ‘active site’ refers to the region of a PKT enzyme comprising one or more of the herein disclosed mutations, which positively influence the affinity and / or phosphoketolase activity of the PKT enzyme. Hence, the ‘active site’ in the context of the invention preferably also includes mutations / mutation sites that have far-reaching / ’long-range’ effects, i.e. , those that, may not necessarily be considered to be located directly in the catalytic center of the enzyme but still significantly influence the PKT-activity and / or affinity of the PKT enzyme towards a non-phosphorylated substrate, for example, by influencing the polarity of amino acid residues in the catalytic / active center.

[0135] In embodiments of the invention a PKT enzyme may comprise one or more mutations within the amino acid sequence position 142 and 605 of a PKT enzyme, preferably with respect to the genomic sequence of PKT of Bifidobacterium adolescentis (SEQ ID NO 14). In certain embodiments of the invention a PKT enzyme may comprise one or more mutations within the amino acid sequence position 2, 6, 142, 153, 256, 260, 321 , 546, 547, 548, 549 and / or 605 of a PKT enzyme, preferably with respect to the genomic sequence of PKT of Bifidobacterium adolescentis (SEQ ID NO 14). In certain embodiments of the invention a PKT enzyme may comprise one or more mutations within the amino acid sequence position T2, I6, H142, E153, H256, H260, Q321 , Q546, D547, H548, N549, and / or K605 of a PKT enzyme, preferably with respect to the genomic sequence of PKT of Bifidobacterium adolescentis (SEQ ID NO 14).

[0136] In embodiments, the one or more mutations of a PKT enzyme include mutations that may be considered having far-reaching / ’long-range’ effects, e.g., those that may not necessarily be considered to be located directly in the catalytic center of the enzyme but still significantly influence the PKT-activity and / or affinity of the PKT enzyme towards a non-phosphorylated substrate, for example, by influencing the polarity of amino acid residues in the catalytic / active center.

[0137] In general, the wild type phosphoketolase (PKT) enzyme is able to effectively generate acetyl phosphate by catalyzing the reaction of D-xylulose 5-phosphate and phosphate to ( ) acetyl phosphate, D-glyceraldehyde 3-phosphate and H2O, by catalyzing the reaction of D-fructose 6- phosphate and phosphate to ( ) acetyl phosphate, D-erythrose 4-phosphate and H2O. Wild type phosphoketolase is only capable of processing sugar phosphates at significant rates.

[0138] In preferred embodiments the non-phosphorylated carbohydrate is selected from (i) D-fructose (CeH^Oe), (ii) D-erythrulose (C4H8O4) and (iii) glycolaldehyde (C2H4O2), and the product is selected from (i) D-erythrose (C4H8O4), (ii) glycolaldehyde (C2H4O2) and (iii) water (H2O), respectively. Further non-phosphorylated sugars are D-ribulose (C5H10O5), D-xylulose (C5H10O5), D-tagatose (CeH^Oe), D-sorbose (CeH^Oe) and D-psicose (CeH^Oe).

[0139] In embodiments, the processing of any C4 - C6 ketose, comprising also those mentioned herein above, by the methods and enzymes according to the invention is envisaged. This is as, for example, the wild-type variants of the enzymes according to the present invention possess catalytic activity for various phosphorylated ketoses, and the modified enzymes according to the present invention were engineered to possess relevant affinity and catalytic activity on non- phosphorylated sugars with 4 (erythrulose) to 6 (fructose) C atoms.

[0140] (D-)fructose is a ketonic sugar with the chemical formula CeH^Oe (IUPAC: (3S,4R,5R)-1 ,3,4,5,6- Pentahydroxyhexan-2-one or D-arabino-Hex-2-ulose).

[0141] Glycolaldehyde has the chemical formula C2H4O2 (IUPAC: Hydroxyethanal or Hydroxyacetaldehyde).

[0142] (D-)erythrose is a saccharide of the aldose family with the chemical formula C4H8O4 (IUPAC: (2R,3R)-2,3,4-Trihydroxybutanal (D); (2S,3S)-2,3,4-Trihydroxybutanal (L)).

[0143] (D-)erythrulose (brief ‘erythrulose’; IUPAC: D-glycero-Tetrulose or (3R)-1 ,3,4-Trihydroxybutan-2- one) is a carbohydrate of the ketose family with the chemical formula C4H8O4.

[0144] An erythrose isomerase (El) preferably refers to an enzyme that catalyzes the isomerization of D- erythrose to D-erythrulose (e.g., Ps.Lrhl).

[0145] Adenosine triphosphate (ATP; Adenosine 5'-(tetrahydrogen triphosphate; C10H16N5O13P3)) is a nucleotide serving as universal energy carrier in biological systems by supplying energy to diverse biological processes. ATP may also be used as phosphate donor, e.g., in chemical synthesis. For example, ATP may be hydrolyzed to adenosine diphosphate (ADP; C10H15N5O10P2) and inorganic phosphate. This reaction may also be inversed using enzymes such as acetate kinase, wherein acetyl phosphate and ADP are converted into acetate and ATP. In the presence of ATP and divalent cations, acetate kinase is also capable of catalyzing the phosphorylation of acetate for the production of acetyl-CoA.

[0146] Threose is monosaccharide with molecular formula C4H8O4 (IUPAC: (2S,3R)-2,3,4- Trihydroxybutanal (D) or (2R,3S)-2,3,4-Trihydroxybutanal (L)).

[0147] Threose isomerase (Tl) preferably refers to an enzyme catalyzing the isomerization of D-threose to D-erythrulose (e.g. Ps.lrhl).

[0148] Threose aldolase preferably refers to an enzyme catalyzing the homo-aldol addition of two molecules glycolaldehyde yielding one molecule of D-threose (e.g. Ec.fsaA).

[0149] Ethylene glycol dehydrogenase (EGDH) is an enzyme capable of converting ethylene glycol and NAD (Nicotinamide adenine dinucleotide, Coenzyme I or diphosphopyridine nucleotide;

[0150] C21H28N7O14P2) to glycolaldehyde and NADH2 (reduced form of NAD; C21H30N7O14P2).

[0151] Ethylene glycol is an organic compound with the formula C2H6O2 (CH2OH)2 (IUPAC: ethane-1 ,2- diol).

[0152] Coenzyme-A (CoA; C21H36N7O16P3S) is a coenzyme capable of assisting the oxidation of fatty acids and pyruvate. The synthesis of CoA requires itself ATP. During glycolysis CoA is acetylated to acetyl-CoA. Phosphate acetyltransferase (PTA) catalyzes the conversion of acetyl-CoA and acetyl phosphate.

[0153] In general, ‘coenzymes’ are organic compounds that facilitate the catalytic activity of enzymes, and which can interact and bind temporarily or permanently to an enzyme. Coenzymes are often also able to catalyze chemical reactions themselves, but to a lower degree than in combination with a partner enzyme.

[0154] Pyrroloquinoline quinone (PQQ; methoxatin; Ci4H6N2O8; IUPAC: 4,5-Dioxo-4,5-dihydro-1 H- pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid) is a redox cofactor.

[0155] Methanol dehydrogenase (MDH) is an enzyme processing methanol that can be either PQQ or NAD-dependent (requires PQQ or NAD as coenzyme).

[0156] In embodiments a non-phosphorylated carbohydrate is selected from (i) D-fructose, (ii) D- erythrulose and (iii) glycolaldehyde, and the product is selected from (i) D-erythrose, (ii) glycolaldehyde and (iii) water (H2O).

[0157] In embodiments, fructose may be obtained from glucose using a glucose isomerase.

[0158] As used herein the addition or deletion of 0 to 10 amino acid means that the polypeptide may have a) 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids or O, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted. “Sequence variants” or "variants" of proteins or peptides, as defined in the context of the present invention, may be generated that have an amino acid sequence that differs from an original (wild type, wt) sequence in one or more mutation(s), such as one or more inserted, substituted and / or deleted amino acid(s).

[0159] "Variants" of proteins or peptides, as defined in the context of the present invention, may comprise one or more amino acid substitution(s), deletions or additions compared to their native (wt), i.e. non-mutated, physiological sequence. These amino acid sequences, as well as their coding nucleotide sequences, more particularly fall within the term "variants" as defined herein. Substitutions in which amino acids originating from the same class are exchanged for each other are called conservative substitutions. These are in particular amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids whose side chains can form hydrogen bonds, e.g., side chains that have a hydroxyl function. This means that, for example, an amino acid with a polar side chain is replaced by another amino acid with a side chain that is also polar, or, for example, an amino acid characterised by a hydrophobic side chain is replaced by another amino acid with a side chain that is also hydrophobic (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Alternatively, insertions and substitutions are possible at such sequence positions that do cause a change in the three-dimensional structure or do affect the binding region and binding capability to substrates, e.g., such as non-phosphorylated sugars. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can be easily determined, e.g., using circular dichroism spectra (CD spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al., (eds.), Elsevier, Amsterdam).

[0160] Furthermore, variants of genes, proteins or peptides as defined herein that may be encoded by a nucleic acid molecule may also comprise such sequences, wherein nucleotides of the encoding nucleic acid sequence are exchanged according to the degeneracy of the genetic code with or without a change in the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least a part thereof may not differ from the original sequence in one or more mutation(s) or may indeed differ from the original (wt) sequence.

[0161] Nucleic acid substitutions as defined herein are modifications made to the nucleic acid sequence of a nucleic acid molecule or a (genomic) gene sequence, whereby one or more nucleic acids are replaced with the same number of (different) nucleic acids, such that the nucleic acid sequence is changed and a protein is encoded, which, in embodiments, contains a different amino acid sequence than the primarily protein encoded by the primary nucleic acid. In embodiments this amendment will either alter or not alter the encoded amino acid sequence of the protein. Like additions, substitutions may be natural or artificial. It is well known in the art that nucleic acid substitutions may be made with or without altering the encoded amino acid sequence of the encoded protein and / or the encoded proteins function.

[0162] Amino acid substitutions as defined herein are modifications made to the amino acid sequence of the protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein which contains a different amino acid sequence than the primary protein. In embodiments this amendment will either (significantly) alter, or not significantly alter, the function of the protein. Like additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made with or without significantly altering the protein's function. A proteins function may be altered, e.g., when the modification relates to a amino acid substitution, which is the substitution of one amino acid for another of different chemical or physical properties and / or structure. Such amino acids can be natural or synthetic amino acids which because of size, charge, polarity and conformation can be substituted with or without affecting the structure and function of the protein.

[0163] In general abbreviations used for amino acids herein are the following: Alanine is Ala or A, Arginine is Arg or R, Asparagine is Asn or N, Aspartic acid is Asp or D, Cysteine is Cys or C, Glutamic acid is Glu or E, Glutamine is Gin or Q, Glycine is Gly or G, Histidine is His or H, Isoleucine is lie or I, Leucine is Leu or L, Lysine is Lys or K, Methionine is Met or M, Phenylalanine is Phe or F, Proline is Pro or P, Serine is Ser or S, Threonine is Thr or T, Tryptophan is Trp or W, Tyrosine is Tyr or Y and Valine is Vai or V. J is Leucine or Isoleucine. In embodiments herein the terms peptide and polypeptide may be used interchangeably. Also, the terms protein and polypeptide may be used herein interchangeably.

[0164] In general, the non-polar amino acids Gly, Ala, Vai, lie and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gin, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu, represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups. A hydrophobic side-chain of an amino acid commonly refers to the chemical side chain of an amino acid which does not like to reside in an aqueous, e.g., water, environment. Hydrophobic side chains are considered to be comprised in the amino acids alanine (Ala), valine (Vai), leucine (Leu), isoleucine (lie), phenylalanine (Phe), methionine (Met), Tyrosine (Tyr) and tryptophan (Trp).

[0165] Functionally analogous sequences refer to the ability to encode a functional gene product. Functionally analogous sequences refer to the ability to encode a functional gene product and to enable the same or similar functional effect as the gene products of the disclosed sequence.

[0166] In general, the function of a protein / enzyme can be determined by suitable tests for the activity of the respective enzyme / protein, which are routine tests for a skilled person. Appropriate assays for determining enzymatic activity are known in the art.

[0167] In embodiments, "nucleic acid" refers to any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids or modified variants thereof. An "exogenous nucleic acid" or "exogenous genetic element" relates to any nucleic acid or nucleic acid sequence introduced into a cell, which is not a component of the cell’s “wild type”, “original” or "natural" genome. Exogenous nucleic acids may be integrated or non-integrated, or relate to stably transfected nucleic acids. Deoxyribonucleic acids (DNAs) are linear molecules composed of four different kinds of ribonucleotide bases, namely Cytosine (C), Adenine (A), Guanine (G) and thymine (T). DNA is in nature rarely present in form of a single strand, but of two strands that form a double helix. The DNA double helix is stabilized mainly by two forces: Hydrogen bonds between the nucleotides and base-stack interactions between the aromatic nucleobases. Generally, a DNA sequence is transcribed by a Polymerase enzyme into an RNA sequence. Ribonucleic acids (RNAs) are linear molecules composed of four different kinds of ribonucleotide bases, namely Cytosine (C), Adenine (A), Guanine (G) and Uracil (U). A ribonucleotide base is composed of three building blocks, namely a ribose sugar, a phosphate group, and a nitrogenous base. In an RNA molecule the ribonucleotide bases are linked through phosphodiester bonds. RNA is generally singlestranded, contrary to DNA, which commonly occurs as single or double-stranded molecule.

[0168] The present invention relates to genetically modified (or genetically engineered) enzymes, such as PKT. Herein, the term “genetically modified” or “genetically engineered" describes enzymes whose genetic material has been modified in comparison to a respective naturally occurring wild type enzyme, for example by inserting / adding, exchanging, or deleting one or more nucleic acids of the wild type gene (DNA) sequence encoding said enzyme.

[0169] For control of expression of the respective enzymes according to the present invention, constitutive and inducible promoters can be used. Examples Positively regulated bacterial expression systems have been reviewed by Brautaset et al (Microbial Biotechnology (2009)2(1), 15-30).

[0170] The skilled person is familiar with suitable assays for testing the activity of an enzyme. Exemplary assays are described herein and in the examples. The relative levels of reaction-products may subsequently be measured via suitable assay, chromatography or mass spectrometry.

[0171] For example, a hydroxamate assay may be used to determine the phosphoketolase activity via the derivatization of acetyl-phosphate to an iron-acetyl-hydroxamate-complex [6], [7] or as discussed herein in the Examples.

[0172] Further, G3P may, for example, be quantified by a coupled, enzymatic assay using sn-glycerol 3- phosphate oxidase (G3Pox) and peroxidase, comprising first the conversion of G3P into dihydroxyacetone phosphate and hydrogen peroxide under oxygen consumption in a reaction catalyzed by G3Pox. In the presence of 4-amino antipyrine (4-AAP) and sodium 3,5-dichloro-2- hydroxybenzenesulfonate (DHBS), the peroxidase-controlled reaction consumes one mole H2O2 to yield one mole of water and quinone imine, which is detected photometrically.

[0173] In a further example, high-performance liquid chromatography may be used for the quantification of sugars (fructose, erythrose, erythrulose, threose), glycolaldehyde, glycerol and acetate.

[0174] The instant disclosure also includes kits, packages and multi-container units containing the herein described enzymes, substrates, cofactors, reagents and / or buffers for performing the method according to the invention.

[0175] Examples of methods and means for carrying out enzymatic methods are generally described in WO 2015 / 181074 A1.

[0176] The methods according to the present invention may be carried out in vitro or in vivo. An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction. Thus, in vitro preferably means in a cell-free system. The term "in vitro" in one embodiment means in the presence of isolated enzymes (or enzyme systems optionally comprising possibly required cofactors). In one embodiment, the enzymes employed in the method are used in isolated and / or purified form.

[0177] For carrying out the method in vitro, the substrates for the reaction and the enzymes are incubated under conditions (buffer, temperature, co-substrates, cofactors etc.) allowing the enzymes to be active and the enzymatic conversion to occur. The reaction is allowed to proceed for a time sufficient to produce the respective product. The production of the respective products can be measured by methods known in the art, such as High-Performance Liquid Chromatography (HPLC) possibly linked to Mass Spectrometry (MS) detection.

[0178] The enzymes may be in any suitable form allowing the enzymatic reaction to take place. They may be purified or isolated or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzymes are immobilized on a suitable carrier.

[0179] The in vitro method according to the invention may be carried out in a “one-pot” reaction, i.e. the substrate is combined in one reaction mixture with the above-described enzyme(s) necessary for the desired conversion and the reaction is allowed to proceed for a time sufficient to produce the respective product. Alternatively, the method may also be carried out by effecting one or more enzymatic steps in a consecutive manner, i.e. by first mixing the substrate with one or more enzymes and allowing the reaction to proceed to an intermediate and then adding one or more further enzymes to convert the intermediate further either into an intermediate or into the final product.

[0180] The in vitro method according to the invention furthermore may comprise the step of collecting the desired product by recovering it employing methods known in the art. Recovering one or more intermediate products may also occur in any given method described herein.

[0181] In another embodiment, the method according to the invention is carried out in culture, in the presence of a non-human organism, preferably a microorganism, producing at least one of the enzymes described above. A method which employs a microorganism for carrying out a method according to the invention may be referred to as "in vivo" method. The respective substrate may either be provided externally or may be produced by the employed microorganism expressing the corresponding enzyme for the production of the respective substrate as described above. Such a microorganism expresses at least one enzyme for one of the above-described enzymatic conversions. Thus, in such embodiments of the invention, an organism, preferably a microorganism, that produces at least one of the enzymes specified in the description, above, is used. It is possible to use a (micro)organism which naturally produces one or more of the required enzymes and to genetically modify such a (micro)organism so that it expresses also those enzymes which it does not naturally express.

[0182] If a (micro)organism is used which naturally expresses one of the required enzyme activities, it is possible to modify such a (micro)organism so that this activity is overexpressed in the (mircro)organism. This can, e.g., be achieved by effecting mutations in the promoter region of the corresponding gene 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 (micro)organisms which express the enzymes which are necessary for achieving the enzymatic conversions as described above, it is possible to carry out the method according to the invention directly in the culture medium, without the need to separate or purify the enzymes.

[0183] In one embodiment the organism employed in the method according to the invention is a nonhuman organism, preferably a microorganism, which has been genetically modified to contain one or more foreign nucleic acid molecules encoding one or more of the enzymes as described above. The term "foreign" in this context means that the nucleic acid molecule does not naturally occur in said organism / microorganism. This means that it does not occur in the same structure or at the same location in the organism / 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. Promoters for driving expression in different types of organisms, in particular in microorganisms, are well known to the person skilled in the art.

[0184] In a further embodiment the nucleic acid molecule is foreign to the organism / microorganism in that the encoded enzyme is not endogenous to the organism / microorganism, i.e. is naturally not expressed by the organism / microorganism when it is not genetically modified. In other words, the encoded enzyme is heterologous with respect to the organism / microorganism. The foreign nucleic acid molecule may be present in the organism / 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.

[0185] The non-human organisms used in the invention can be prokaryotes or eukaryotes, preferably, they are microorganisms such as bacteria, yeasts, fungi or molds, or plant cells or animal cells. In a particular embodiment, the microorganisms are bacteria. In another embodiment, the method according to the invention makes use of a multicellular organism expressing at least one of the enzymes which are necessary for achieving the enzymatic conversions as described above. Examples of such organisms are plants or animals.

[0186] In a particular embodiment, the method according to the invention involves culturing microorganisms in standard culture conditions (30°-37°C at 1 atm, in a fermenter allowing aerobic growth of the bacteria) or non-standard conditions (higher temperature to correspond to the culture conditions of thermophilic organisms, for example). When the method according to the invention is carried out in vivo by using an organism / microorganism providing the respective enzyme activities, the organism, preferably microorganism, is cultivated under suitable culture conditions allowing the occurrence of the enzymatic reaction. The specific culture conditions depend on the specific organism / 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.

[0187] In another embodiment, the method of the invention comprises the step of providing the nonhuman 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 process like the method of the present invention is carried out which involves non-human 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 liters to cubic meters, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in 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.

[0188] As described above, it is possible to use in the method according to the invention a non-human (micro)organism which is genetically modified so as to contain a nucleic acid molecule encoding at least one of the enzymes which are necessary for achieving the enzymatic conversions as described above. Such a nucleic acid molecule encoding an enzyme as described 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.

[0189] The enzyme(s) used in the methods according to the invention can be a naturally occurring enzyme or an enzyme which is derived from a naturally occurring enzyme, e.g., be 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". 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.

[0190] Moreover, mutants possessing a modified substrate or product specificity can be prepared. Preferably, such mutants show an increased 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 optimized.

[0191] In embodiments, an "increased activity" means that the expression and / or the activity of an enzyme 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 enzyme. In even more preferred embodiments, the increase in expression and / or activity may be at least 150%, at least 200% or at least 500%. The term "increased" expression / activity also covers the situation in which the corresponding non-modified enzyme and / or microorganism does not express a corresponding enzyme so that the corresponding expression / activity of the non-modified enzyme and / or in the non-modified microorganism is zero. Methods for measuring the enzymatic activity of the enzymes employed in the methods according to the invention are known in the art and are described herein.

[0192] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of’ and “consisting essentially of’. Thus, the terms “comprising” / “including” / ”having” mean that any further component (or likewise features, integers, steps and the like) can / may be present. The term “consisting of’ typically means that no further component (or likewise features, integers, steps and the like) is present.

[0193] The term “consisting essentially of’ or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method. Thus, the term “consisting essentially of’ means those specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of' (which can be interchangeably used herein with the term "comprising substantially"), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

[0194] The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

[0195] The present project has been funded by BMBF KMU Innovativ, No. 031 B1245A, and the Deutsche Forschungsgemeinschaft, Project No. 450319558.

[0196] FIGURES

[0197] The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0198] Brief description of the figures:

[0199] Figure 1 : Graph depicting glycerol 3-phosphate production over time.

[0200] Figure 2: Cell-free production of G3P using an enzyme cascade with and without D-erythrose isomerase (Ps.LRhl) to regenerate ATP from fructose.

[0201] Figure 3: Cell-free production of G3P by ATP regeneration from erythrose using two different Bad.F6Pkt variants.

[0202] Figure 4 Cell-free production of G3P by ATP regeneration from erythrulose using two different Bad.F6Pkt variants.

[0203] Figure 5: Cell-free production of G3P by ATP regeneration from glycolaldehyde using either Bad.F6Pkt wild type or mutant H256Y:H260Y:H548Y.

[0204] Figure 6: Cell-free production of G3P by ATP regeneration from glycolaldehyde using Bad.F6Pkt mutant H256Y:H260Y:H548Y in presence or absence of threose aldolase (TA) and threose isomerase (Tl).

[0205] Figure 7: Sequence alignment of Phosphoketolase gene sequence in different bacterial species.

[0206] Figure 8: Enzyme cascades for cell-free ATP (re)generation from low-cost sugars.

[0207] Figure 9: Enzyme cascades for cell-free ATP (re)generation from inexpensive ethylene glycol.

[0208] Detailed description of the figures:

[0209] Figure 1 : Cell-free production of sn-glycerol 3-phosphate (G3P) using a one-step enzyme cascade to regenerate ATP from fructose. Either wild type Bad.F6Pkt or mutant H548N were employed. Experiments were carried out at pH 7.0, 37 °C in the presence of 0.8 mM TPP, 4 mM MgCh with starting concentrations of 50 mM glycerol, 100 mM sodium phosphate, 200 mM fructose and 1 mM ADP. Data represent mean and deviation of biological duplicates.

[0210] Figure 2: A. Cell-free production of G3P using an enzyme cascade with and without D-erythrose isomerase (Ps.LRhl) to regenerate ATP from fructose. Experiments were carried out at pH 7.0, 37 °C in the presence of 0.8 mM TPP, 4 mM MgCh with starting concentrations of 1 mM ADP, 100 mM glycerol and sodium phosphate, as well as 45 mM fructose as the limiting substrate. Phosphoketolase Bad.F6Pkt H548N was added at a concentration of 0.8 mg mL-1 . Data represent mean and deviation of biological duplicates. B. Cell-free production of G3P as in A. but with 66 mM glycerol and, 25 mM fructose as the limiting substrate. Phosphoketolase Bad.F6Pkt H548N was added at a concentration of 2.5 mg mL-1. Data represent mean and standard deviation of biological triplicates (without isomerase) or a single experiment (with isomerase).

[0211] Figure 3: Cell-free production of G3P by ATP regeneration from erythrose using two different Bad.F6Pkt variants. Experiments were carried out at pH 7.0, 37 °C in the presence of 0.8 mM TPP, 4 mM MgCh with starting concentrations of 1 mM ADP, 100 mM glycerol and sodium phosphate, as well as 50 mM erythrose as the limiting substrate. Phosphoketolase Bad.F6Pkt variant H548N or H548Y was added at a concentration of 0.8 mg mL-1 . Data represent mean and standard deviation of biological triplicates.

[0212] Figure 4: Cell-free production of G3P by ATP regeneration from erythrulose using two different Bad.F6Pkt variants. Experiments were carried out at pH 7.0, 37 °C in the presence of 0.8 mM TPP, 4 mM MgCh with starting concentrations of 1 mM ADP, 55 mM glycerol and 62.5 mM sodium phosphate, as well as 25 mM erythrulose as the limiting substrate. Phosphoketolase was added at a concentration of 2 mg mL-1. Data represent mean and standard deviation of biological duplicates.

[0213] Figure 5: Cell-free production of G3P by ATP regeneration from glycolaldehyde using either Bad.F6Pkt wild type or mutant H256Y:H260Y:H548Y. Experiments were carried out at pH 7.0, 37 °C in the presence of 0.8 mM TPP, 4 mM MgCh with starting concentrations of 1 mM ADP, 60 mM glycerol and sodium phosphate, as well as 45 mM glycolaldehyde as the limiting substrate. Phosphoketolase Bad.F6Pkt or its variant were added at a concentration of 2.2 mg mL-1 . Data represent mean and deviation of biological duplicates.

[0214] Figure 6: Cell-free production of G3P by ATP regeneration from glycolaldehyde using Bad.F6Pkt mutant H256Y:H260Y:H548Y in presence or absence of threose aldolase (TA) and threose isomerase (Tl). Experiments were carried out at pH 7.0, 37 °C in the presence of 0.8 mM TPP, 4 mM MgCh with starting concentrations of 1 mM ADP, 32 mM glycerol and sodium phosphate, as well as 15 mM glycolaldehyde as the limiting substrate. Phosphoketolase Bad.F6Pkt H256Y:H260Y:H548Y was added at a concentration of 0.2 mg mL-1. Data represent mean and deviation of biological duplicates.

[0215] Figure 7: Sequence alignment of Phosphoketolase gene sequence in different bacterial species showing the high degree of sequence conservation and hence also of potential mutations influencing the binding and / or activity towards non-phosphorylated carbohydrates between the respective bacterial species. Black background indicates amino acids conserved among bacterial strains. WP_011743105.1 : Sequence phosphoketolase Bifidobacterium (SEQ ID NO: 15), EFE89336.1 - Bifidobacterium breve DSM 20213 (SEQ ID NO: 16), ADF97524.1 - Bifidobacterium breve strain 203 (SEQ ID NO: 17), ACS47899.1 - Bifidobacterium animalis subsp. lactis DSM 10140 (SEQ ID NO: 18), ADB09649.1- Bifidobacterium dentium Bd1 (SEQ ID NO: 19), WP_010964652.1 - Clostridium acetobutylicum ATCC 824 (SEQ ID NO: 20), WP_050339622.1 - Lactiplantibacillus pentosus ATCC 824 (SEQ ID NO: 21), WP_009154258.1 - Saccharomonospora marina (SEQ ID NO: 22), OJY14508.1 - Nitrosospira sp. 56-18 (SEQ ID NO: 23), WP_082308260.1- Achromobacter sp. DMS1 (SEQ ID NO: 24). Figure 8: Enzyme cascades for cell-free ATP (re)generation from low-cost sugars. Enzymes that required genetic engineering for the processing of a atypical (non-phosphorylated) substrates are marked with an asterisk. Atypical (non-phosphorylated) substrates are marked with an asterisk. Gl - Glucose isomerase, PKT - phosphoketolase, D-threose isomerase, ACK - acetotkinase

[0216] Figure 9: Enzyme cascades for cell-free ATP (re)generation from inexpensive ethylene glycol. Enzymes that required genetic engineering for the processing of a atypical (non-phosphorylated) substrates are marked with an asterisk. A. ATP production from ethylene glycol using EGDH and a modified PKT enzyme. B. ATP production from ethylene glycol using EGDH, a modified PKT enzyme and optionally FLS. C. ATP production from ethylene glycol using EGDH, a modified PKT enzyme and optionally FsaA and Tl . EGDH - ethylene glycol dehydrogenase, NOX - NADH oxidase, PKT - phosphoketolase, Tl - D-threose isomerase, ACK - acetate kinase, FLS - formolase, FsaA - fructose 6-phosphate aldolase.

[0217] EXAMPLES

[0218] The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0219] Methods

[0220] Plasmid construction

[0221] Target genes listed in Table 2 were cloned into the pET28a(+) expression vector (Novagen™, Merck, Darmstadt, Germany), which carries a kanamycin resistance gene, with the incorporation of a N-terminal hexa-His tag. The plasmid pET28a_Ps. / r / ? / was provided by BioCat GmbH (Heidelberg, Germany) and the plasmid carrying Ec.feaAL107Y:A129Gwas constructed previously via PCR-restriction cloning [1], while homologous recombination was used for all plasmid constructions carried out in this study.

[0222] Table 2: Genes used in this study. Genomic DNAs were purchased from DSMZ (Braunschweig, Germany). In their unavailability, synthetic genes were obtained from BioCat GmbH (Heidelberg, Germany) or Thermo Fisher Scientific Inc. Target genes were amplified with the introduction of backbone-homologues regions by PCR using the primers listed in Table 3. The pET28 plasmid backbone was amplified between the restriction sites Ndel and EcoRI by PCR as well. To digest the remaining template DNA, the PCR products were incubated in the presence of the restriction endonuclease Dpnl (NEB, 20,000 units / ml) for two hours at 37°C, followed by enzyme inactivation at 80°C for 20 minutes. Resulting PCR fragments were then purified using the Monarch PCR Cleanup Kit or the NEB Monarch Gel Extraction Kit (New England Biolabs) after agarose gel electrophoresis.

[0223] The purified target PCR fragment and plasmid backbone were assembled by homologous recombination [2] using the NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturers instructions.

[0224] The resulting reaction mix was transformed into chemically competent E. coli DH5a cells (New England Biolabs) and plasmid DNA was isolated from positive transformants using Monarch Plasmid Miniprep Kit (New England Biolabs). Resulting plasmids were validated by diagnostic PCR and subsequent DNA sanger sequencing carried out by Microsynth AG (Balgach, Switzerland) or Genewiz Germany GmbH (Leipzig, Germany).

[0225] Table 3: Primers used for plasmid construction and verification.

[0226] Production of relevant enzymes in shake flasks

[0227] N-His-tagged enzymes were expressed from E. coli BL21 (DE3) or Rosetta(DE3) plysS bearing pET-28a (+) derived plasmids. E. coli BL21 (DE3) competent cells were obtained from New England Biolabs GmbH (Frankfurt a.M., Germany), while Rosetta(DE3) plysS competent cells were purchased from Merck KGaA, Germany. Media were supplemented with 50 pg mL'1kanamycin and the cultivation of Rosetta(DE3) plysS strains required additional supplementation with 35 pg mL-1chloramphenicol. All cultivations were carried out at 220 rpm in a rotary shaker (Infers HT, Switzerland).

[0228] After transformation of the protein production plasmids into the appropriate host strains (Table 4), pre-cultures of 20 mL LB medium (Carl Roth, Karlsruhe, Germany) in 100 mL shake flasks were inoculated with single colonies from solid LB medium. Following growth for ~16 h at 37 °C, these pre-cultures were used to inoculate the protein expression cultures.

[0229] Optimal expression conditions were established for each protein (Table 4) in an initial screening. Thus, proteins were expressed in 50 mL of either LB or auto-induction medium [3] in 250 mL shake flasks. Cultures in LB medium were inoculated with an initial ODeoo ~ 0.2. After growth at 37 °C to an ODeoo of 0.6 to 0.8, expression was induced by addition of 1 mM IPTG and cultures were further incubated at 25 °C for 20 h. Cultures in auto-induction medium were inoculated with an initial ODeoo ~0.05 and protein was expressed at 25 °C for 24 h.

[0230] Cells were harvested after appropriate incubation times by centrifugation at 3200 g, 4 °C for 10 min and cell pellets were stored at -20 °C until further use.

[0231] Table 4: Expression conditions for his-tagged proteins from pET28a-derived vector in E. coli host strains.

[0232] Protein purification, quantification and storage

[0233] Frozen cell pellets were thawed on ice and suspended in 1 mL lysis buffer (50 mM KH2PO4, 300 mM NaCI, pH 7.5). Cells were disrupted by three successive rounds of ultrasonication (sonication interval: 10 s, power output: 30 %, UDS 751 , Topas GmbH, Germany), followed by centrifugation at 13000 g, 4 °C for 15 min. to separate cellular debris from the cell lysate.

[0234] His-tagged enzymes were then purified from cell lysate by immobilized metal affinity chromatography (IMAC). Therefore, the supernatant was transferred to a fresh 2 mL reaction tube and 100 pL of streptomycin sulfate solution (15 mg mL-1) was added. After centrifugation (13000 g, 4 °C, 10 min), the supernatant was incubated for 1 h at room temperature and 20 rpm rotation with 0.35 mL of Talon™ Cobalt affinity resin (Cytiva, Marlborough, USA). Prior, the resin was subjected to two pre-washes: first with 1 .75 mL deionized water, subsequently with 1 .75 mL lysis buffer. After incubation of the resin with cell-free crude extract and subsequent centrifugation (700 g for 5 min, 4°C), the supernatant was discarded and the resin washed twice with 1 .75 mL wash buffer (10 mM KH2PO4, 300 mM NaCI, pH 7.5). To elute His-tagged enzymes, 0.5 mL elution buffer (10 mM KH2PO4, 300 mM NaCI, 500 mM imidazole, pH 7.0) was applied and the eluted fraction was loaded onto an Amicon Ultra-0.5 centrifugal filter unit (pore size 10 kDa; Merck Millipore, USA) to exchange the buffer. After an initial round of centrifugation (14 000 g, 4 °C, 10 min.) the filtrate was discarded and the retentate was washed twice with 0.45 mL storage buffer (10 mM KH2PO4, 300 mM NaCI, pH 7.0). The protein fraction was recovered (1000 g, 4 °C, 2 min.), 0.3 mL of storage buffer were added and the purified protein solution was stored at 4 °C until further us.

[0235] Protein concentrations were quantified using the Bradford method (Roti©-Quant, Carl Roth, Karlsruhe, Germany), with bovine serum albumin (0 - 100 pg mL'1) serving as the calibration standard.

[0236] Structural analysis of the wild-type Bad. F6pkt enzyme was performed by Molecular Forces (Toulouse, France). The following residues were identified as target positions for single-site saturated mutagenesis: H142, 1219, Q321 , E437, S440, R442, S541 , H548, N549, K605.

[0237] Creation of mutation libraries

[0238] Single-site directed mutagenesis

[0239] Single site-directed mutagenesis of Bad. f6pkt on the plasmid pET28a-Bad. f6pkt was performed using the PCR procedure described by [4], To create the single-site saturated mutation libraries of Bad. f6pkt mentioned above, oligonucleotide primers were designed with the degenerate codon NNK replacing the wild type codon of the respective target position.

[0240] Remaining template DNA was digested by Dpnl-treatment before each of the 9 Bad. f6pkt libraries was transformed individually into E. coli DH5a competent cells (New England Biolabs).

[0241] Subsequent cultivation on LB agar plates supplemented with 50 pg mL'1kanamycin yielded at least 300 colonies per transformation. The plasmid DNA of at least 5 positive transformants per library was isolated individually and sequenced for verification of successful mutation. The remaining colonies of each library were scrapped of the agar plate and used to inoculate 20 mL LB medium supplemented with 50 pg mL'1kanamycin in a 100 mL shake flask. After incubation over night at 37 °C at 220 rpm agitation, plasmid DNA was isolated and transformed into E. coli Rosetta(DE3) plysS competent cells for protein expression in 96-deep-well plates.

[0242] Multi-site directed mutagenesis

[0243] Based on screening results of the site-saturated libraries, mutations in beneficial residue positions should be combined to obtain further improved Bad.F6Pkt variants. Therefore, a focused library comprising 63 Q321 single and Q321 :H548 double mutant variants was created using the QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, US). Mutagenic primers were designed using the web-based QuickChange Primer Design Program by Agilent Technologies to allow the following residues in the respective amino acid position: K, C, E, N, L, Y, H(wild-type) in position 548 and H, R, Y, W, P, N, S, V, G in position Q321 . The PCR for mutant strand synthesis, followed by Dpnl digestion of remaining template DNA was performed according to the manufacturer’s instructions. The resulting PCR product was transformed into competent XL10-Gold ultracompetent cells provided by the commercial kit. Plasmid DNA of 10 clones was isolated and sequenced as described above. The other >300 colonies obtained after transformation were washed of the agar plate with 1 mL liquid LB medium and used to inoculate 20 mL LB medium supplemented with 50 pg mL'1kanamycin in a 100 mL shake flask. After incubation over night at 37 °C at 220 rpm agitation, plasmid DNA was isolated and transformed into E. coli Rosetta(DE3) plysS competent cells for protein expression in 96- deep-well plates. of mutation libraries in 96 i-well

[0244] The method for expression of Bad.F6Pkt in 96 deep-well plates was adapted from [5], All media were supplemented with 35 pg mL'1chloramphenicol and 50 pg mL'1kanamycin. All cultivations were carried out at 25 °C, 850 rpm and 85 % humidity in an incubator shaker (Multitron, Infers AG, Switzerland). For cultivation, all microplates (Falcon®, Corning GmbH, Germany) and deepwell plates (Masterblock®, Greiner Bio-One GmbH, Germany) were sealed with a sterile Breathe Easy® membrane (Merck KGaA, Germany).

[0245] After transformation, single colonies of E. coli Rosetta(DE3) plysS clones were picked from solid LB medium using sterile toothpicks and transferred to 96-well microplates. While each well contained 180 pL LB medium supplemented with 8 % glycerol, only 60 wells per microplate were inoculated as those on the edge of the plate were left out. These cultures were grown for 24 h prior to storage of the microplates at -80 °C.

[0246] Starter culture was inoculated into 96-well microplates containing 180 pL 2xYT medium (16 g L'1tryptone, 10 g L'1yeast extract, 5 g L'1NaCI) per well from thawed storage microplates which were replicated in parallel. After 15 h cultivation, 30 pL of each starter culture were used to inoculated 96-deep-well plates filled with 600 pL auto-induction medium per well. These cultures for gene expression were grown for 24 h and harvested by centrifugation at 2000 rpm and 4 °C for 20 min. After removal of 600 pL supernatant, cell pellets were resuspended in 100 pL lysozyme solution (1 mg mL'1in 10 mM Tris-HCI pH 8.0) and incubated at 30 °C, 250 rpm for 30 min prior to storage at -80 °C for at least 16 h.

[0247] Screening of mutation libraries in terms of activity on non-phosphorylated substrates

[0248] Deep-well plates containing the lysozyme-treated cell pellets were thawed for 1 h at room temperature, followed by addition of 100 pL benzonase solution (15 U mL'1, 1 mM MgCh) to each well and incubation at 30 °C, 250 rpm for 30 min. Storage buffer (10 mM KH2PO4, 300 mM NaCI, pH 7.0) was added in aliquots of 180 pL per well, before centrifugation at 2000 rpm and 4 °C for 20 min. The obtained enzymatic extract was transferred to a microplate and stored on ice until further use.

[0249] The hydroxamate assay was used to determine the phosphoketolase activity via the derivatization of acetyl-phosphate to an iron-acetyl-hydroxamate-complex [6], [7], To screen Bad.F6Pkt libraries in terms of activity on non-phosphorylated substrates, 30 pL of cell-free crude extract were transferred to a fresh microplate prior to the addition of 50 pL assay mixture to initiate the enzymatic reaction. This reaction mixture contained 100 mM MES buffer pH 6.5, 50 mM KH2PO4, 3.8 mM MgCh, 0.8 mM TPP, 1.4 mM L-cysteine, 17 mM NaF, 6 mM iodoacetate, as well as the substrate (200 mM D-fructose or glycolaldehyde, 25 mM fructose 6-phosphate). The enzymatic reaction was performed at 37 °C for 30 min. until 60 pL of hydroxylamine solution (2 M, pH 6.5) were added per well. After incubation for 10 min. at room temperature, 40 pL trichloroacetic acid (15% w / v), 40 pL HCI (4 M) and 40 pL FeCl3*6H2O solution (5% w / v in 0.1 M HCI) were added. Absorbance was measured immediately at 505 nm using the Infinite M200 PRO microplate reader (Tecan AG, Switzerland). Control reactions with the absence of substrate, protein or both were included. Calibration was performed with freshly prepared lithium AcP standard solutions (0-14 mM).

[0250] To ensure a library coverage of > 95%, the number of clones picked for screening should be three-fold higher than the total number of possible variants [8], Therefore, at least 100 individual clones of each single site-saturated mutation library were analysed and 230 clones of the Bad.F6Pkt H548:Q321 -double mutant library (see above). As reference, at least 3 cultures (wells) per microplate were inoculated with different clones of analysed again in terms of activity. Enzyme variants for which at least two-fold higher activity compared to the wild type was observed on D-fructose or glycolaldehyde, were considered a positive hit. Plasmid DNA of these positive hits was isolated and sequenced. For verification of the screening results, non-redundant positive Pkt variants were expressed in shake flasks, purified and analysed again in terms of activity.

[0251] Construction and analysis of individual Bad. f6pkt variants

[0252] Based on the results of the single-site saturated library screenings, specific mutations were proposed by Molecular Forces (Toulouse, France) to further increase performance on nonphosphorylated substrates. For individual construction of these Pkt variants, one or multiple mutations were introduced step-wise into the Bad. f6pkt wild-type gene according to the PCR- based method described by Zheng [4], Template DNA was digested with Dpnl prior to purification of the PCR product using the Monarch PCR Clean Up Kit (New England Biolabs) and transformation into competent E. coli DH5a cells. Plasmid DNA was isolated and sequenced as described above.

[0253] Individually constructed Bad.F6Pkt mutants, as well as those identified as positive hits in the library screenings, were expressed in shake flasks and purified as described above. Phosphoketolase activity of these purified enzyme variants was measured by the hydroxamat assay as described above, with NaF and iodoacetate omitted from the reaction mixture. Standard substrate concentrations were 200 mM D-fructose and glycolaldehyde, as well as 25 mM F6P. For the best performing Bad. F6Pkt variants in terms of fructose and / or glycolaldehyde activity, activity on 25 mM D-erythrulose was measured in addition.

[0254] In order to determine kinetic parameters, phosphoketolase activity was measured at different substrate concentrations ranging from 0 - 100 mM (F6P, D-erythrulose) or 0 - 300 mM (D- fructose, glycol aldehyde). The reactions were started simultaneous by addition of the respective substrate solution using a multichannel pipette. Experimental data were fitted to Michaelis- Menten kinetics using MatLab software to determine vmax and KM values.

[0255] In vitro sn-qlycerol 3-phosphate (G3P) synthesis In order to demonstrate the functionality of the in vitro ATP regeneration systems, the respective enzymes were coupled to the ATP-consuming synthesis of G3P catalysed by glycerol kinase. Solutions of purified enzymes were prepared, quantified and stored as described above prior to their application for in vitro G3P synthesis.

[0256] The enzymatic synthesis was carried out in a 2 mL tube with a total reaction volume of 1 .5 mL. Unless otherwise stated, the reaction mixture comprised 200 mM HEPES buffer pH 7.0, 1 mM ATP, 0.8 mM TPP, 4 mM MgCh, 100 mM sodium phosphate pH 7.0 and glycerol, 5 pg mL'1Gs.AckA and 0.3 mg mL'1Cs.GIpK. Bad. F6Pkt (variants), Ps.Lrhl and Ec.FsaA A129G:L107Y were applied as indicated. The reaction mixture was pre-incubated at 37 °C for 5 min. before the reaction was initiated by addition of the limiting substrate (D-fructose or glycolaldehyde).

[0257] Incubation was carried out at 37 °C for ~ 30 h, while 50 pL samples were taken regularly from the reaction mixture, diluted 1 :4 in 0,1 M HCI and placed on ice to stop the enzymatic reaction. Part of this sample solution was used in-time for offline monitoring of G3P production, while the remaining part was stored at -20 °C for HPLC analysis of substrate, product and intermediate concentrations.

[0258] Quantification of substrates, products and intermediates

[0259] Photometric quantification of G3P

[0260] G3P was quantified by a coupled, enzymatic assay using sn-glycerol 3-phosphate oxidase (G3Pox) and peroxidase. First, G3P is converted into dihydroxyacetone phosphate and hydrogen peroxide under oxygen consumption in a reaction catalysed by G3Pox. In the presence of 4- amino antipyrine (4-AAP) and sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS), the peroxidase-controlled reaction consumes one mole H2O2 to yield one mole of water and quinone imine, which is detected photometrically.

[0261] The G3Pox apoenzyme (Creative Enzymes, USA) was reactivated by incubation for 2 h at 4 °C in 50 mM HEPES buffer pH 7.5 supplemented with 0.25 mM FAD. At these conditions, the enzyme was stored up to 3 weeks.

[0262] HCI-diluted samples from the in vitro G3P synthesis tests were diluted appropriately, but at least two-fold, in 0.1 M HEPES buffer pH 7.5. Subsequently, 50 pL diluted sample were added to the assay mixture containing 50 mM HEPES buffer pH 7.5, 10 mM MgCh, 7 mM DHBS pH 7.5, 2 mM 4-AAP, 2 U mL-1G3Pox and 50 U mL-1horseradish peroxidase (Merck KGaA, Germany). The assay mixture with a total volume of 250 pL was incubated at 37 °C while reading the absorbance at 520 nm every 20 s until a constant signal was observed (=20 min). For every sample, a control reaction without G3Pox was included. The correlation factor between the concentration of H2O2 and the absorption (A) measured at 520 nm was determined here as 0.0167 mM-1. As one mole of G3P yields one mole of H2O2, the G3P concentration in the reaction mix could be determined according to the following equation: crG3->PP rL mMJ= — ^s 0 —ample ^control , .r.0 —167 — mM rr-n1— * dilutionJf actor

[0263] HPLC analysis High-performance liquid chromatography (UltiMate3000, Thermo Fisher Scientific, USA) was used for the quantification of sugars (fructose, erythrose, erythrulose, threose), glycolaldehyde, glycerol and acetate. The system was equipped with a Rezex-ROA-Organic acid H+column, which was operated at 65 °C with 0.5 mM H2SO4 as eluent at a flow rate of 0.5 mL min-1. Erythrulose was measured via DAD-detector (DAD-3000(RS), Thermo Fisher Scientific, USA) at 210 nm, while the other sugars, glycolaldehyde, glycerol and acetate were measured via Rl- detector (RefractoMax 520, ERC, Germany). Prior to injection, frozen samples were thawed, appropriately diluted in ddH2O and processed through a 0.2 pm PTFE filter. The filtered samples were stored at 6 °C in the autosampler until 20 pL were injected into the column.

[0264] Example 1 - Enzyme cascade for ATP regeneration from fructose

[0265] PKT variant H548N improves performance of fructose-based ATP regeneration system

[0266] To test whether the Bad.F6Pkt mutant H548N, which exhibits improved activity on fructose, provides a benefit for ATP regeneration from fructose, the synthesis of G3P by phosphoketolase, acetate kinase and glycerol kinase was investigated with the sugar substrate added in excess. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0267] - 200 mM HEPES buffer pH 7.0,

[0268] - 1 mM ATP,

[0269] - 0.8 mM TPP,

[0270] - 4 mM MgCh,

[0271] - 100 mM sodium phosphate pH 7.0

[0272] - 200 mM D-fructose

[0273] - 50 mM glycerol

[0274] - 0.8 mg mL'1Bad.F6Pkt wild type or mutant H548N

[0275] - 10 pg mL'1Gs.AckA

[0276] - 0.3 mg mL'1Cs.GIpK

[0277] Complete conversion of 50 mM glycerol to sn-glycerol 3-phosphate (G3P) was successfully demonstrated using Bad.F6Pkt mutant H548N to regenerate ATP from fructose. Meanwhile, employing the wild type phosphoketolase instead resulted in lower productivity and incomplete turnover as the reaction stopped after approximately 8 hours (Figure 1 , Table 5-6). These data clearly indicate that the mutant Bad.F6Pkt H548N improves process performance of cell-free ATP regeneration from fructose, compared to the wild type enzyme.

[0278] Table 5: Performance of cell-free G3P synthesis from 50 mM glycerol using either PKT wild type or mutant H548N to regenerate ATP from D-fructose. Yields were calculated as the maximum G3P production in relation to the initial glycerol concentration. G3P was measured by photometrical assay, while glycerol was quantified by HPLC analysis. Data represent mean and deviation of biological duplicates. Table 6: Glycerol consumption and G3P production by cell-free ATP regeneration from 200 mM fructose using either PKT wild type or mutant H548N. G3P was determined by a photometric assay, whereas glycerol was quantified by HPLC. Data represent mean and deviation of biological duplicates.

[0279] Expansion of the reaction system by an erythrose isomerase enables higher yield

[0280] To investigate whether the multi-step, fructose-based enzyme cascade for ATP regeneration is feasible, G3P synthesis by glycerol kinase, acetate kinase and Bad.F6Pkt variant H548N was tested in presence and absence of the erythrose isomerase Ps.LRhl. Here, glycerol was added in excess while fructose was the limiting substrate. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0281] - 200 mM HEPES buffer pH 7.0,

[0282] - 1 mM ATP,

[0283] - 0.8 mM TPP,

[0284] - 4 mM MgCh,

[0285] - 100 mM sodium phosphate pH 7.0

[0286] - 45 mM D-fructose

[0287] - 100 mM glycerol

[0288] - 0.8 mg mL'1Bad.F6Pkt H548N

[0289] - 0.9 mg mL'1Ps.Lrhl (control: without Ps.Lrhl)

[0290] - 5 pg mL'1Gs.AckA

[0291] - 0.3 mg mL'1Cs.GIpK

[0292] In the presence of erythrose isomerase, a higher maximum product concentration and yield could be observed. With the reaction system comprising only a phosphoketolase, acetate kinase and glycerol kinase around one mole ATP was produced per mole of fructose consumed, which could be increased by nearly 30 % through the additional use of erythrose isomerase (Table 7; Figure 2A). A multi-step enzyme cascade comprising erythrose isomerase was observed to result in a 20- fold decrease in erythrose accumulation, as well as the presence of the intermediates erythrulose and glycolaldehyde (Table 9). Threose was detected as well, as it is a by-product of the erythrose isomerase reaction of Ps.LRhl [9], Neither threose, erythrulose nor glycolaldehyde were detected in the absence of the isomerase (Table 8). These data indicate that the proposed multi-step enzyme cascade for ATP regeneration from fructose is functional and that the use of an erythrose isomerase in addition to an improved Bad.F6Pkt variant enables the production of more than one mol ATP per mol of consumed fructose.

[0293] Table 7: Process performance of G3P synthesis with ATP regeneration from fructose by an enzyme cascade with and without D-erythrose isomerase (Ps.LRhl). Yield was calculated as the maximum G3P production in relation to the initial fructose concentration. Selectivity was calculated as the maximum amount of G3P produced per amount of fructose consumed. G3P was measured by photometrical assay, while fructose was quantified by HPLC analysis. Data represent mean and deviation of biological duplicates.

[0294] Table 8: Substrate and (by-) product concentrations of G3P synthesis by A TP regeneration from fructose without erythrose isomerase. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and deviation of biological duplicates, n.a. - not analysed.

[0295] Table 9: Substrate and (by-) product concentrations of G3P synthesis by A TP regeneration from fructose with the use of a erythrose isomerase. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and deviation of biological duplicates, n.a. - not analysed.

[0296] In a further experiment the use of higher enzyme concentrations was tested for the use of erythrose isomerase in a fructose-based reaction system.

[0297] Therein G3P synthesis by glycerol kinase, acetate kinase and Bad.F6Pkt variant H548N was tested in presence and absence of the erythrose isomerase Ps.Lrhl. Here, glycerol was added in excess while fructose was the limiting substrate. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0298] - 200 mM HEPES buffer pH 7.0,

[0299] - 1 mM ATP,

[0300] - 0.8 mM TPP,

[0301] - 4 mM MgCI2,

[0302] - 100 mM sodium phosphate pH 7.0

[0303] - 25 mM D-fructose

[0304] - 66 mM glycerol

[0305] - 2.5 mg mL-1 Bad.F6Pkt H548N

[0306] - 2 mg mL-1 Ps.Lrhl (control: without Ps.Lrhl)

[0307] - 5 pg mL-1 Gs.AckA

[0308] - 0.3 mg mL-1 Cs.GIpK

[0309] In the presence of erythrose isomerase, a higher maximum product concentration and yield could be observed. With the reaction system comprising only a phosphoketolase, acetate kinase and glycerol kinase around one mole ATP was produced per mole of fructose consumed, which could be increased by nearly 250 % through the additional use of erythrose isomerase (Table 0; Figure 2B). The intermediates erythrulose and glycolaldehyde were observed in the presence of erythrose isomerase, with minimal accumulation of erythrose (10). Additionally, threose was detected in trace amounts, as it is a by-product of the erythrose isomerase reaction of Ps.Lrhl [1], In contrast, neither threose, erythrulose nor glycolaldehyde were detected in the absence of the isomerase (Table 7711).

[0310] These data indicate that the proposed multi-step enzyme cascade for ATP regeneration from fructose is functional and that the use of an erythrose isomerase in addition to an improved Bad.F6Pkt variant enables the production of more than two mol ATP per mol of fructose. Table 10: Process performance of G3P synthesis with ATP regeneration from fructose by an enzyme cascade with and without D-erythrose isomerase (Ps.LRhl). Yield was calculated as the maximum G3P production in relation to the initial fructose concentration. G3P was measured by photometrical assay, while fructose was quantified by HPLC analysis. Data represent mean and deviation of biological triplicates (without isomerase) or a single experiment (with isomerase). without with isomerase isomerase max. productivity [mM h'1] 4.93 ± 0.01 5.20 yield [molG3P molfructose, to'1] 1.02 ± 0.01 2.57

[0311] Table 11: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from fructose without erythrose isomerase. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed

[0312] Table 12: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from fructose with the use of a erythrose isomerase. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent results of a single measurement, n.a. - not analysed

[0313] Cell-free ATP regeneration from D-erythrose by different Pkt variants To investigate whether ATP regeneration from the C4-sugars erythrose and erythrulose is feasible by the proposed enzyme cascade, G3P synthesis by glycerol kinase, acetate kinase, phosphoketolase and erythrose isomerase was investigated. Two different Bad.F6Pkt variants were compared in terms of process performance: H548N which exhibits the highest activity on fructose and H548Y with improved erythrulose- and glycolaldehyde affinity. Glycerol was added in excess while D-erythrose was the limiting substrate. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0314] - 200 mM HEPES buffer pH 7.0,

[0315] - 1 mM ATP,

[0316] - 0.8 mM TPP,

[0317] - 4 mM MgCh,

[0318] - 100 mM sodium phosphate pH 7.0

[0319] - 50 mM D-erythrose

[0320] - 100 mM glycerol

[0321] - 0.8 mg mL'1Bad.F6Pkt H548N or H548Y

[0322] - 0.9 mg mL-1Ps.Lrhl

[0323] - 5 pg mL'1Gs.AckA

[0324] - 0.3 mg mL'1Cs.GIpK

[0325] With the use of both phosphoketolase variants synthesis of the target product G3P was observed, as well as the accumulation of the enzyme cascade intermediates and by-products erythrulose, threose, glycolaldehyde and acetate (Table 14-15 and Figure 3). Employing Bad.F6Pkt H548Y instead of H548N lead to a two-fold higher productivity and improved G3P yield by 64 % (Table 13). In addition, lower erythrulose accumulation and higher glycolaldehyde concentration were observed for the reaction involving the BadF6Pkt H548Y variant.

[0326] These data clearly indicate that ATP can be regenerated from C4-sugars by a reaction system comprised of erythrose isomerase, phosphoketolase and acetate kinase. Further, performance of this cell- free ATP regeneration process can be improved by the use of a Bad.F6Pkt variant with improved kinetic parameters for erythrulose and glycolaldehyde.

[0327] Table 13: Process performance of G3P synthesis with ATP regeneration from erythrose using two different Bad.F6Pkt variants. Yield was calculated as the maximum G3P production in relation to the initial erythrose concentration. G3P was measured by photometrical assay, while erythrose was quantified by HPLC analysis. Data represent mean and standard deviation of biological triplicates.

[0328] Table 14: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from erythrose using Bad.F6Pkt variant H548Y. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed

[0329] Table 15: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from erythrose using Bad.F6Pkt variant H548N. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed.

[0330] Cell-free ATP regeneration from D-erythrulose by different Pkt variants

[0331] To demonstrate the feasibility of ATP regeneration from the C4 sugar D-erythrulose by the proposed in vitro reaction system, G3P synthesis by glycerol kinase, acetate kinase and phosphoketolase was investigated. Two different Bad.F6Pkt variants were evaluated in comparison with the wild type enzyme in terms of process performance. Bad.F6Pkt H256Y:H260Y:H548Y has been shown to exhibit enhanced affinity and activity for erythrulose and glycolaldehyde in this study. The other variant, H142N, was previously described by Yang et al.

[0015] as exhibiting advantageous characteristics with respect to these two substrates. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0332] - 200 mM HEPES buffer pH 7.0,

[0333] - 1 mM ATP,

[0334] - 0.8 mM TPP,

[0335] - 4 mM MgCI2,

[0336] - 62.5 mM sodium phosphate pH 7.0

[0337] - 25 mM D-erythrulose

[0338] - 55 mM glycerol

[0339] - 2 mg mL'1Bad.F6Pkt or its variants

[0340] - 5 pg mL'1Gs.AckA - 0.3 mg mL'1Cs.GIpK

[0341] The synthesis of at least 25 mM target product G3P was observed for all three reactions, as well as the accumulation of the enzyme cascade intermediate glycolaldehyde and the by-product acetate (Table 17-19). While the use of Bad.F6Pkt H142N did not result in improved process performance compared to the wild-type enzyme, the use of Bad.F6Pkt H256Y:H260Y:H548Y enabled a two-fold higher productivity and improved G3P yield by 64 % (Table 16; Figure 4).

[0342] These data clearly indicate that ATP can be regenerated from C4 sugar erythrulose by a reaction system comprised of phosphoketolase and acetate kinase. Moreover, the performance of this cell-free ATP regeneration process can be enhanced by the utilisation of a Bad.F6Pkt variant with optimized kinetic parameters for D-erythrulose.

[0343] Table 16: Process performance of G3P synthesis with ATP regeneration from erythrulose using two different Bad. F6Pkt variants. Yield was calculated as the maximum G3P production in relation to the initial erythrulose concentration. G3P was measured by photometrical assay, while erythrulose was quantified by HPLC analysis. Data represent mean and standard deviation of biological duplicates. max. productivity [mM h1] 14.0 ± 0.8 3.8 ± 0.1 27.3 ± 1 .3 yield [molcsp molerytrhoseto-1] 1 .08 ± 0.06 1 .03 ± 0.02 1 .64 ± 0.05

[0344] Table 17: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from erythrulose using Bad.F6Pkt variant H256Y:H260Y:H548Y. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological duplicates, n.a. - not analysed

[0345] Table 18: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from erythrulose using Bad. F6 Pkt variant H142N. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological duplicates, n.a. - not analysed

[0346] Table 19: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from erythrulose using Bad.F6Pkt wild type. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological duplicates, n.a. - not analysed

[0347] Enzyme cascade for ATP regeneration from ethylene glycol (via qlycolaldehyde)

[0348] Process performance of cell-free ATP-reqeneration from qlycolaldehyde improved by Bad. F6Pkt variant

[0349] To prove that a Bad.F6Pkt mutant with improved affinity and activity on glycolaldehyde provides a benefit for ATP regeneration from this C2-substrate, the synthesis of G3P by glycerol kinase, acetate kinase and phosphoketolase was investigated using either the Bad. F6Pkt wild type or variant H256Y:H260Y:H548Y. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0350] - 200 mM HEPES buffer pH 7.0,

[0351] - 1 mM ATP,

[0352] - 0.8 mM TPP,

[0353] - 4 mM MgCh,

[0354] - 60 mM sodium phosphate pH 7.0

[0355] - 45 mM glycolaldehyde

[0356] - 60 mM glycerol

[0357] - 2.2 mg mL’1Bad.F6Pkt (wild type or variant H256Y:H260Y:H548Y)

[0358] - 1 .5 pg mL'1Gs.AckA - 85 pg mL'1Cs.GIpK

[0359] For both reactions, synthesis of the target product G3P was observed, as well as accumulation of the by-product acetate and consumption of the substrates glycolaldehyde and glycerol (Table 21- 22 and Figure 5). Employing Bad.F6Pkt H256Y:H260Y:H548Y instead of the wild type enzyme resulted in a nearly 60 % higher productivity and improved G3P yield by 36 %.

[0360] These data clearly indicate that cell-free ATP regeneration from glycolaldehyde by phosphoketolase and acetate kinase is feasible and that the use of a Bad.F6Pkt variant with improved glycolaldehyde activity and affinity increases process performance significantly.

[0361] Table 20: Process performance of G3P synthesis with ATP regeneration from glycolaldehyde using either Bad. F6Pkt wild type or mutant H256Y:H260Y:H548Y. Yield was calculated as the maximum G3P production in relation to the initial glycolaldehyde concentration. G3P was measured by photometrical assay, while glycolaldehyde was quantified by HPLC analysis. Data represent mean and deviation of biological duplicates.

[0362] Table 21: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from glycolaldehyde using Bad. F6 Pkt wild type. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed.

[0363] Table 22: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from glycolaldehyde using Bad.F6Pkt variant H256Y:H260Y:H548Y. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed.

[0364] Cell-free ATP-reqeneration from qlycolaldehyde by threose aldolase, threose isomerase and phosphoketolase

[0365] Glycolaldehyde accumulation should be prevented in the proposed reaction system for ATP- regeneration from ethylene glycol due to its strong inactivating effect on isolated enzymes and toxicity for living cells. The aldehyde could be degraded directly by a phosphoketolase as described above. A potentially more efficient alternative is the degradation of glycolaldehyde to threose by a threose aldolase, which can subsequently be converted to erythrulose by a threose aldolase. Erythrulose is then used by a phosphoketolase to produce acetyl phosphate for ATP or acetyl CoA synthesis.

[0366] This threose aldolase and isomerase reaction system was tested for in vitro ATP regeneration from glycolaldehyde to investigate whether it provides a benefit compared to the direct synthesis of acetyl phosphate from glycolaldehyde by phosphoketolase. A control reaction without threose aldolase and isomerase was performed in parallel at the same conditions. In both cases Bad. F6Pkt variant H256Y:H260Y:H548Y was used due to its beneficial properties regarding both PKT-substrates erythrulose and glycolaldehyde. The experiment was carried out as described in the Methods section with the reaction mixture comprised as follows:

[0367] - 200 mM HEPES buffer pH 7.0,

[0368] - 1 mM ATP,

[0369] - 0.8 mM TPP,

[0370] - 4 mM MgCh,

[0371] - 32 mM sodium phosphate pH 7.0

[0372] - 15 mM glycolaldehyde

[0373] - 32 mM glycerol

[0374] - 0.33 mg mL'1Ec.FsaA L107Y:A129G (= threose aldolase, TA)

[0375] - 2.5 mg mL'1Ps.Lrhl (= threose isomerase, Tl)

[0376] - 0.2 mg mL'1Bad.F6Pkt H256Y:H260Y:H548Y

[0377] - 1 .5 pg mL'1Gs.AckA

[0378] - 85 pg mL'1Cs.GIpK

[0379] In the presence of threose aldolase and isomerase, glycolaldehyde was consumed completely within 30 minutes and the accumulation of intermediates threose and erythrulose was detected. In contrast, in the absence of this enzyme, approximately half of the initial aldehyde concentration remained as the reaction stopped (Table 24-25, Figure 6). With use of threose aldolase and isomerase higher final product concentration could be observed, resulting in nearly 60 % higher product yield.

[0380] These data indicate, that acetyl-phosphate production from glycolaldehyde for ATP or acetyl CoA synthesis is feasible by a reaction system comprised of threose aldolase, threose isomerase and phosphoketolase. It was demonstrated that this reaction system provides the benefit of fast glycolaldehyde degradation and associated higher product yield compared to direct utilisation of the aldehyde by phosphoketolase.

[0381] Table 23:2 Process performance of G3P synthesis with ATP regeneration from glycolaldehyde by an enzyme cascade with or without threose aldolase (TA) and isomerase (Tl). Yield was calculated as the maximum G3P production in relation to the initial glycolaldehyde concentration. G3P was measured by photometrical assay, while glycolaldehyde was quantified by HPLC analysis. Data represent mean and deviation of biological duplicates.

[0382] Table 24: Substrate and (by-) product concentrations of G3P synthesis by A TP regeneration from glycolaldehyde using Bad.F6Pkt H256Y:H260Y:H548Y. Threose aldolase (TA) and threose isomerase (Tl) were absent from the reaction mixture. G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed.

[0383] Table 25: Substrate and (by-) product concentrations of G3P synthesis by ATP regeneration from glycolaldehyde using Bad.F6Pkt H256Y:H260Y:H548Y in the presence of threose aldolase (TA) and threose isomerase (Tl) . G3P was determined by a photometric assay, all other components were quantified by HPLC. Data represent mean and standard deviation of biological triplicates, n.a. - not analysed.

[0384] The following table summarizes exemplary mutant PKT enzymes according to the invention, without limiting the mutant PKT enzymes according to the invention thereto. From the afore and following disclosure relating to the advantageous PKT mutants identified by the inventors for producing acetyl phosphate from non-phosphorylated carbohydrates, a skilled person will be capable of advantageously selecting and combining (if desired) PKT mutants with improved properties on the respective substrates (non-phosphorylated carbohydrates) without any problems. Advantageous in this case means that the efficacy and / or speed of the production of acetyl phosphate according to the invention is increased and / or the concentration of the inactivating intermediates, such as erythrose and glycolaldehyde, is reduced or kept as low as possible. Accordingly, the following table is intended to provide a skilled person with information for performing the present invention over the whole scope claimed, as PKT mutants according to the invention may be selected therefrom for each reaction according to the method of the invention.

[0385] Table 26: Phosphoketolase (Bad.F6Pkt) variants: kinetic parameters for D-fructose.

[0386] Substrate affinities were estimated by measuring the initial reaction rates at varying substrate concentrations, in the cases where substrate saturation was not observed within the concentration range tested, catalytic efficiency could not be determined and vmax was defined as the specific activity in the presence of 200 mM substrate. Relative activity was determined as the ratio of the specific activities of the mutant and wild-type enzyme at 200 mM D-fructose. Experiments were carried out at pH 6.5, 37 °C in the presence of 50 mM inorganic phosphate. A\7 - average, SD - standard deviation, n.sat. - no saturation observed, n.d. - not determined. All mutations are indicated with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14).

[0387] Table 27: Phosphoketolase (Bad.F6Pkt) variants: kinetic parameters for D-erythrulose.

[0388] Substrate affinities were estimated by measuring the initial reaction rates at varying substrate concentrations. Relative activity was determined as the ratio of the specific activities of the mutant and wild-type enzyme at 25 mM D-erythrulose. Experiments were carried out at pH 6.5, 37 °C in the presence of 50 mM inorganic phosphate. AV - average, SD - standard deviation, n.sat. - no saturation observed, n.d. - not determined. All mutations are indicated with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14).

[0389] Table 28: Phosphoketolase (Bad.F6Pkt) variants: kinetic parameters for glycolaldehyde.

[0390] Relative activity was determined as the ratio of the specific activities of the mutant and wild-type enzyme at 200 mM glycolaldehyde. If substrate saturation was not observed within the concentration range tested, catalytic efficiency could not be determined and vmax was defined as the specific activity in the presence of 200 mM substrate. Experiments were carried out at pH 6.5, 37 °C in the presence of 50 mM inorganic phosphate. AV - average, SD- standard deviation, n.sat.

[0391] - no saturation observed, n.d. - not determined. All mutations are indicated with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14).

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Claims

CLAIMS1 . A method for the production of acetyl phosphate, comprising enzymatically converting a nonphosphorylated carbohydrate into acetyl phosphate and a product, with a phosphoketolase (PKT), wherein the phosphoketolase comprises at least one mutation, wherein the non-phosphorylated carbohydrate is selected from (i) D-fructose, (ii) D- erythrulose and (iii) glycolaldehyde, and the product is selected from (i) D-erythrose, (ii) glycolaldehyde and (iii) water (H2O), respectively, and wherein the method comprises:(i) enzymatically converting D-fructose into acetyl phosphate and D-erythrose with a PKT comprising at least one mutation,(iia) enzymatically converting the D-erythrose obtained in (i) into D-erythrulose, preferably using erythrose isomerase (El),(ii) enzymatically converting D-erythrulose into acetyl phosphate and glycolaldehyde with a PKT comprising at least one mutation, and(iii) optionally enzymatically converting glycolaldehyde into acetyl phosphate and water (H2O) with a PKT comprising at least one mutation.

2. The method according to claim 1 , wherein the at least one mutation enables an increased affinity and / or increased phosphoketolase activity for a non-phosphorylated carbohydrate compared to a wild-type phosphoketolase sequence.

3. The method according to any one of claims 1 - 2, further comprising enzymatically converting acetyl phosphate and coenzyme-A (CoA) into acetyl-CoA and phosphate with a phosphate acetyl transferase (PTA).

4. The method according to any one of claims 1 -3, further comprising enzymatically converting acetyl phosphate and adenosine diphosphate (ADP), into adenosine triphosphate (ATP) and acetate with acetate kinase (ACK).

5. The method according to any one of the preceding claims, wherein the method is a cell-free method for producing acetyl phosphate.

6. The method according to any one of the preceding claims, wherein the fructose converted in (i) of claim 1 has been obtained from glucose using a glucose isomerase.

7. The method according to any one of the preceding claims, wherein the phosphoketolase enzyme in (i), (ii) and / or (iii) comprises in its active site one or more of the mutation(s) Q321 G, Q321 I, Q321 L, Q321S, Q321V, H142Q, H142E, E153D, E153Q, E153P, E153T, S541 N, D547E, H548N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, H548Y, N549S, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321 S:H548N, Q321 LH548N, Q321V:H548C, Q321V:H548L, H548N:K605T, H548K:K605V, Q546E:N549D, D547E:H548Y, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, T2A:I6T:H26OY:N549D, H256Y, H256Y:H260Y:H548Y,H256Y:H260Y, H142N:H256Y, H142N:H260Y:H548Y, H142N:H256Y:H260Y:H548Y, H142N:H548Y, H142N:H548N, and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14).

8. A phosphoketolase enzyme (PKT) comprising in its active site the mutation(s) Q321 G, Q321 I, Q321 L, Q321S, Q321V, H142Q, H142E, E153D, E153Q, E153P, E153T, S541 N, D547E, H548N, H548D, H548C, H548Q, H548E, H548G, H548L, H548M, H548F, H548P, H548Y, N549S, K605C, K605E, K605L, K605T, K605V, H548N, N549D, Q321S:H548N, Q321 LH548N, Q321V:H548C, Q321V:H548L, H548N:K605T, H548K:K605V, Q546E:N549D, D547E:H548Y, H260Y:H548Y, T2A:I6T:H26OY:H548N, T2A:I6T:H26OY:H548Y, T2A:I6T:H26OY:N549D, H256Y, H256Y:H260Y:H548Y, H256Y:H260Y, H142N:H256Y, H142N:H260Y:H548Y, H142N:H256Y:H260Y:H548Y, H142N:H548Y, H142N:H548N, and / or H142N:E153D with respect to the genomic sequence of Bifidobacterium adolescentis (SEQ ID NO 14).

9. The phosphoketolase enzyme according to claim 8, wherein at least one of the mutation(s) enables an increased affinity and / or increased phosphoketolase activity for at least one nonphosphorylated carbohydrate selected from D-fructose, D-erythrulose and glycolaldehyde compared to a wild-type phosphoketolase enzyme.

10. Use of the phosphoketolase enzyme according to claims 8-9 for producing acetyl phosphate from a non-phosphorylated carbohydrate in a method according to any one of claims 1-7.

11. A non-human organism, preferably a microorganism, comprising a gene encoding and optionally expressing the phosphoketolase enzyme according to claim 8-9.

12. Use of a non-human organism or microorganism according claim 11 for producing acetyl phosphate from a non-phosphorylated carbohydrate.

13. The use of a non-human organism or microorganism according to claim 12, wherein acetyl phosphate is produced from a non-phosphorylated carbohydrate according to the method of any one of claims 1-714. A composition, comprising: i. a non-phosphorylated carbohydrate, and ii. a phosphoketolase according to claims 8-9, and / or a non-human (micro)organism according to claim 11 , iii. and optionally iv. a phosphate acetyl transferase (PTA), v. an erythrose isomerase (El), and / or vi. an acetate kinase (ACK).

15. The composition according to claim 14, wherein the non-phosphorylated carbohydrate is selected from D-fructose, D-erythrulose and glycolaldehyde.

16. A composition, comprising:i. a phosphoketolase according to claims 8-9, and / or an microorganism according to claim 11 , ii. an ethylene glycol dehydrogenase (EGDH), iii. ethylene glycol, and iv. nicotinamide adenine dinucleotide (NAD), and17. The composition according to claim 16, wherein the composition further comprises a threose aldolase, and / or a threose isomerase, and / or an acetate kinase (ACK) or a phosphate acetyl transferase (PTA).

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