Patent Application
20230175029
The present invention relates to a process for the multi-step enzymatic conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), the process comprising the steps of: a) enzyme-catalyzed conversion of adenosine diphosphate in the presence of sucrose and a sucrose synthase to adenosine diphosphate-glucose; and b) enzyme-catalyzed conversion of the adenosine diphosphate-glucose formed in process step a) in the presence of inorganic pyrophosphate and a pyrophosphorylase to adenosine triphosphate and glucose-1-phosphate. Furthermore, the invention relates to the use of the process for the preparation of sugar phosphates, nucleotide sugars, glycans, glycoproteins, glycolipids or glycosaminoglycans.
The efficient and reproducible synthesis of defined macromolecules is still a major challenge for science and industry. Although possible solutions can be given in principle for a large number of syntheses, the successful transfer to the large scale, under the boundary conditions of a cost-effective and high-yield production, is not feasible in all cases. This applies in particular to biological macromolecules whose functionality is based not only on their actual composition but also on a specific, three-dimensional structure. A representative of this substance class is hyaluronic acid (hyaluronan, HA), a non-sulfated glycosaminoglycan (GAG) composed of repeating disaccharide units of β1,4-D-glucuronic acid and β1,3-N-acetyl-D-glucosamine (4GlcAβ1-3GlcNAcβ1-). The HA represents the main component of the extracellular matrix (ECM) and a single molecule can reach a molecular weight (MW) of up to 10 MDa. While a variety of biological manufacturing processes have been successfully brought to market in recent years, these processes still had the disadvantage that HA with only relatively “low” molecular weights, for example up to 500 kDa, can be provided in an economically reasonable way. In particular, higher molecular weights with a defined molecular weight distribution are currently not available in larger quantities at reasonable cost. One of the reasons for the latter is that expensive starting materials, such as adenosine triphosphate (ATP), have to be used for HA, which is usually obtained via fermentation.
In biotechnology, adenosine triphosphate (ATP) is widely used in the synthesis of fine chemicals, pharmaceuticals, or biopolymers by biotransformation and biocatalysis. Since ATP is a major cost factor, regeneration of spent ATP is sought. On the one hand, this can reduce costs and, on the other hand, alleviate the limitations of ATP-dependent reactions.
The most commonly used method for ATP regeneration involves the phosphorylation of ADP with phosphoenolpyruvate (PEP) by a pyruvate kinase (PK). However, this system is subject to limitations because pyruvate is released in this reaction, which has an inhibitory effect on PK. This release affects the overall efficiency of ATP regeneration. Second, PEP is a very expensive substrate and more expensive than ATP, which directly reduces the economics of ATP regeneration. Recent approaches to ATP regeneration utilize polyphosphates (PolyP) and polyphosphate kinases (PPK). Long-chain polyPs are used to regenerate ATP from ADP with high efficiency. However, this system is hampered by undefined chain lengths of polyPs, since polyphosphates cannot be obtained in uniform chain lengths so far. Therefore, the achievable ATP regeneration cycles vary. Furthermore, polyphosphates cannot be completely degraded by PPKs, resulting in contamination of the synthesis approach with polyphosphate residues. These in turn have to be degraded or separated from the product. The possibility of using PPi directly for ATP synthesis has not yet been described for this enzyme class.
The patent literature also contains a wide variety of approaches to enzymatic conversions of ATP derivatives or degradation products such as ADP.
For example, DE 60 2005 026 917 D1 discloses a method for producing recombinant sucrose synthase, use thereof in the production of kits for sucrose determination, as well as methods for producing ADP-glucose and methods for obtaining transgenic plants with leaves and storage organs in which ADP-glucose and starch accumulate in high concentration.
Furthermore, CN 101 294 167 A discloses a method for producing hydrogen by culturing hydrogen-producing microbes in a culture medium containing phosphate oils. In this writing, phosphate is added in a cellular environment to simultaneously promote rapid growth and extraction of ATP, thereby controlling ATP levels and oxidation-reduction potential in cells and accelerating carbon source consumption and production efficiency and hydrogen. In addition, the phosphate can be recycled in the culture system without contaminating the environment.
In another patent document, U.S. Pat. No. 2,007,005 4283 A1, a low-cost DNA sequencing method with high sensitivity is provided. The method comprises the steps of adding a given amount of dATP for stepwise complementary strand synthesis and subtracting the background luminescence intensity caused by dATP from the measured luminescence intensity to obtain the luminescence intensity involved in complementary strand synthesis.
Such solutions known from the prior art may offer further potential for improvement, in particular with regard to the controlled enzymatic provision of ATP within complex conversions by the work-up of ATP degradation products, whereby the conversions can be used to a high degree of flexibility in larger enzyme cascades.
It is therefore the task of the present invention to at least partially overcome the disadvantages known from the prior art. In particular, it is the task of the present invention to provide an improved process and an improved use in which ATP is recovered enzymatically from ATP degradation products, whereby the work-up also takes place in complex reaction environments with further enzymes and reactants without significant interactions.
The task is solved by the features of the independent claims, directed to the method according to the invention and the use of the method according to the invention. Preferred embodiments of the invention are indicated in the dependent claims, in the description or in the figures, whereby further features described or shown in the dependent claims or in the description or in the figures may individually or in any combination constitute an object of the invention, as long as the context does not clearly indicate the contrary.
According to the invention, the problem is solved by a process for the multistage enzymatic conversion of adenosine diphosphate to adenosine triphosphate, the process comprising at least the steps:
(a) enzyme-catalyzed conversion of adenosine diphosphate in the presence of sucrose and a sucrose synthase to adenosine diphosphate-glucose; and
b) enzyme-catalyzed conversion of the adenosine diphosphate-glucose formed in process step
a) in the presence of inorganic pyrophosphate and a pyrophosphorylase to adenosine triphosphate and glucose-1-phosphate;
wherein process steps a) and b) are carried out in aqueous solution and simultaneously or successively.
Surprisingly, it was found that by means of a combination of a sucrose synthase (Sucrose Synthase, SuSy) and an ADP-glucose pyrophosphorylase (AGPase), ATP can be reformed from sucrose (Suc) and inorganic pyrophosphate (PPi) from ADP. The enzyme cascade according to the invention does not generate free phosphate (Pi) and the 2-step cascade is also robust and flexible to a high degree, so that it can be combined with other or further enzyme cascades in which ATP is required (e.g. kinases). In addition, these substeps can be used in combination with enzyme cascades in which PPi is formed, which can then be removed from equilibrium and efficiently used for ATP synthesis. The reactants used are inexpensive compared to the reactants proposed in the prior art and the individual process steps can be run together or independently in wide temperature and concentration parameter ranges. Thus, compared to the prior art, the system is capable of performing ATP regeneration from PPi and sucrose in multiple cycles in a pyrophosphate (PPi) releasing process. The energy required for this is obtained by adding sucrose and PPi, which on the one hand reduces the amount of total ATP to be added and on the other hand keeps the process phosphate-free. Furthermore, the system can also be used for processes that do not release PPi by separately adding PPi to them. In this case, the amount of regenerated ATP is equivalent to the amount of PPi used. This can also significantly reduce the amount of ATP to be used in the reaction. The enzyme cascade can be easily combined with ATP-consuming syntheses and applied to ATP regeneration from the inexpensive and stable substances sucrose and PPi. The enzyme cascade does not release phosphate, which favors large-scale synthesis and product purification. For example, no magnesium phosphate is produced. Due to the favorable market prices of the energy suppliers, this conversion is particularly suitable for large-scale in vitro syntheses, which can also be designed particularly simply due to the possibility of simple process control in only one reaction solution.
The process according to the invention is a process for the multi-step enzymatic conversion of adenosine diphosphate to adenosine triphosphate. The conversion of ADP to ATP thus takes place via the use of at least two, different enzymes, whereby the ATP is not formed within a single step, but via several steps, i.e. via at least one, potentially isolatable, intermediate product. Within the conversion, ADP according to the following structural formula
in converted into ATP with the following structural formula
at the catalytic centers of at least two enzymes.
The process comprises the process step a) of enzyme-catalyzed conversion of adenosine diphosphate in the presence of sucrose and a sucrose synthase (SuSy) to adenosine diphosphate-glucose. The first step is carried out by a bacterial or plant sucrose synthase in an aqueous solution. For the reaction, the presence, addition or formation of sucrose in the solution is mandatory and the activity of the synthase converts the ADP to adenosine diphosphate-glucose (ADP-Glc) with degradation of sucrose (Suc) to fructose (Fru). The reaction in the first substep can be represented by the following partial reaction equation:
ADP+Suc↔ADP-Glc+Fru.
The reaction substep can be carried out at a temperature greater than or equal to 15° C. and less than or equal to 50° C., preferably greater than or equal to 25° C. and less than or equal to 40° C. The reaction solution may be pH buffered via a buffer system. SuSy concentrations may be greater than or equal to 0.001 μg/L and less than or equal to 5 μg/L, preferably from greater than or equal to 0.01 μg/L and less than or equal to 1 μg/L. Preferred concentration of Suc can be greater than or 50 mM and less than or equal to 500 mM, preferably greater than or 100 mM and less than or equal to 350 mM.
The process comprises process step b) of enzyme-catalyzed conversion of the adenosine diphosphate-glucose (ADP-Glc) formed in process step a) in the presence of inorganic pyrophosphate (PPi) and a pyrophosphorylase (AGPase) to adenosine triphosphate (ATP) and glucose-1-phosphate (Glc-1-P). The second step is carried out by an AGPase in an aqueous solution. For the reaction, the presence, addition or formation of PPi in the solution is mandatory and the activity of the AGPase converts the ADP-Glc to Glc-1-P and ATP consuming the PPi. The reaction in the second substep can be represented by the following partial reaction equation:
ADP-Glc+PPi↔Glc-1-P+ATP.
The reaction substep can be carried out at a temperature greater than or equal to 15° C. and less than or equal to 50° C., preferably greater than or equal to 25° C. and less than or equal to 40° C. The reaction solution may be pH buffered via a buffer system. AGPase concentrations may be greater than or equal to 500 μg/L and less than or equal to 5000 μg/L, preferably from greater than or equal to 1000 μg/L and less than or equal to 4000 μg/L. Preferred PPi concentrations can be greater than or 0.1 mM and less than or equal to 100 mM, preferably greater than or 1 mM and less than or equal to 35 mM.
In sum, both enzymatic process steps can be described by the following reaction equation:
ADP+Suc+PPi↔ATP+Fru+Glc-1-P.
Process steps a) and b) are carried out in aqueous solution and can be performed simultaneously or sequentially. The multistage reaction can take place in a single solution or in separate solutions, in which case partial amounts of the liquids are exchanged. The process control can be designed in such a way that the reactants are present in the reaction solution from the beginning of the reaction. However, it is also possible that the reactants and/or the enzymes are added during the course of the reaction. Furthermore, it is possible that the substances necessary for the synthesis performance of the enzymes are only formed by other reactions in the course of the reaction. For this purpose, these further reactions can take place at other reaction sites or in one and the same solution of process steps a) and/or b). The two enzymes can move “freely” in the reaction solution or one or both enzyme systems can be bound to or on carriers in the solution.
In a preferred embodiment of the process, process steps a) and b) can be carried out in a common aqueous reaction solution. For an efficient process flow, process steps a) and b) can take place in a “one-pot” reaction in one and the same reaction solution. Surprisingly, it has been shown that the proposed process steps can be carried out with high efficiency under the same reaction conditions. By coupling the two substeps in one solution, the procedural effort can be kept low. Significant reaction rates and high space-time yields result. This is particularly astonishing since competitive inhibition or other negative effects in enzymatic performance had to be expected with the partial enzymatic conversions proposed here. Surprisingly, however, these were not observed, or only to a small extent, and a synergistic increase in performance can also result from the coupling of both systems in solution.
In a preferred process design, process steps a) and b) can be carried out simultaneously. It has also been found to be particularly advantageous that both reaction steps are carried out simultaneously. Both steps are carried out simultaneously in that at least partial or total amounts of both enzymes are present in a common reaction solution at the start of the reaction and the intermediate product formed is always in equilibrium with both enzyme systems. The simultaneous process design explicitly does not include that both partial steps are carried out at the same rate or that the same amount of intermediate/final product is always obtained in terms of time.
Within another preferred aspect of the process, the pH in process step a) and/or b) may be greater than or equal to 5.0 and less than or equal to 8.5. Surprisingly, it has been shown that both process step a) and process step b) can be carried out with high conversions within a relatively limited range of pH values. The reaction rates of the substeps of the enzyme cascade presented here thus lie in the same subranges of the pH spectrum and thus allow the preferred simultaneous execution of the subreactions in only one aqueous process solution without a significant reduction of the reaction rates in the individual substeps.
In a preferred embodiment of the process, process step a) and/or b) can be carried out in the presence of fructose-1,6-bisphosphate at a concentration of greater than or equal to 5 μM and less than or equal to 200 μM. To increase the reaction rate and to improve ATP regeneration, the addition of fructose-1,6-bisphosphate in the concentration indicated above has proven to be particularly suitable. The addition accelerates not only one but both partial steps of the reaction, which was not to be expected in this way. In this respect, the addition can in particular improve the performance of the reaction in only one reaction solution and increase the ATP regeneration performance.
Within a preferred embodiment of the process, the inorganic pyrophosphate can be formed in process step b) by an enzyme-catalyzed reaction in the aqueous solution. To control the reaction within the cascade, it has been found to be particularly advantageous that the inorganic pyrophosphate is formed in situ and not added separately from the outside. This has the advantage that the local PPi quantities can be kept small, which in turn improves the catalytic performance of the enzymes by reducing inhibitory substances.
According to a preferred embodiment of the process, the glucose-1-phosphate formed in process step b) can be removed from the aqueous solution by a further enzymatic reaction. In order to increase ATP synthesis, it has been found to be particularly advantageous that parts of the products are removed from the equilibrium by a further enzymatic reaction. This removal can be achieved, for example, by a further enzyme-catalyzed step with an enzyme selected from the group consisting of phosphoglucomutase, glucose 1-phosphatase, nucleoside triphosphate monosaccharide 1-phosphate nucleotidylyltransferases, uridine triphosphate monosaccharide 1-phosphate uridylyltransferase, disaccharide phosphorylases such as sucrose phosphorylase or trehalose phosphorylase, or mixtures of at least two of these enzymes. These enzymes have proved to be particularly suitable for this task and interact only to a small extent with the other cascade steps, so that a one-pot reaction can also be carried out here without reducing the basic yield.
Furthermore, according to the invention, the use of the process according to the invention for the in-situ provision of adenosine triphosphate in multistage, adenosine triphosphate-consuming enzyme cascades in the production of compounds selected from the group consisting of sugar phosphates, nucleotide sugars, glycans, glycoproteins, glycolipids, glycosaminoglycans, phospho-adenosine phosphosulfate, nucleotide-activated compounds or mixtures of at least two compounds from this group. The present provision of ATP by the regeneration of ATP degradation products may be particularly suitable for the production of the above substances in larger enzyme cascades. The two-step conversion is robust and tolerant to the presence of further enzymes and compounds and, in this respect, a long-lasting, controlled ADP work-up can be carried out without the need to add new enzyme or ATP to the system. Moreover, the proposed cascade is applicable under different environmental conditions, so that ATP regeneration can be used as a flexible building block in the context of substance synthesis. Moreover, the required regeneration rate can be precisely adjusted via the choice of the individual enzyme concentrations. In this respect, an additional increase in synthesis performance can be achieved via a simple addition of the cascade according to the invention to the synthesis cascades. Overall, the regeneration cascade can contribute to an extension of the service life of the synthesis cascades and to an optimization of the space-time yields.
Within a preferred embodiment of the use, the adenosine triphosphate-consuming enzyme reaction may comprise the conversion of N-acetylglucosamine (GlcNAc) and adenosine triphosphate (ATP) to N-acetylglucosamine 1-phosphate (GlcNAc-1-P) and adenosine diphosphate (ADP) by means of an N-acetylhexosamine 1-kinase (BlNahK). The proposed ATP regeneration can enable particularly efficient syntheses, especially with partial or total cascades in the context of enzymatic hyaluronic acid production. For example, the above-described conversion of N-acetylglucosamine according to the following equation can precede or parallel the equilibrium of the proposed two-step cascade:
GlcNAc+ATP↔GlcNAc-1-P+ADP.
The reaction is enzyme-catalyzed by means of BlNahK with consumption of ATP to ADP. The consumed ATP can be returned to the system via conversion or by using PPi. This regeneration can extend the run times of the synthesis without addition of further ATP. The overall reaction in the context of this use, including the described ATP regeneration, can be represented as follows:
GlcNAc+PPi+Suc↔GlcNAc-1-P+Glc-1-P+Fru.
In a preferred embodiment of use, the N-acetylglucosamine-1-phosphate can be converted in a further enzymatic reaction using uridine triphosphate by means of a uridine diphosphate-N-acetylglucosamine diphosphorylase to uridine diphosphate-N-acetylglucosamine and inorganic pyrophosphate. In order to shift the equilibrium of the individual enzymatic steps, it has been found to be particularly advantageous that, for example, for hyaluronic acid synthesis, the formed N-acetylglucosamine-1-phosphate is removed from the system in the course of a further reaction. In addition to shifting the equilibrium, the type of removal described here also achieves that PPi is specifically generated in the process. This PPi can be used as reactant in the subsequent steps, so that at best the addition of further PPi can be omitted. This sub-step can be represented by the following equation:
GlcNAc-1-P+UTP↔UDP-GlcNAc+PPi.
In the context of an overall consideration of the 4-step system including the two steps of ATP regeneration, the following overall reaction equation can be obtained:
GlcNAc+Suc+UTP↔UDP-GlcNAc+Fru+Glc-1-P.
In a further preferred aspect of the use, the glucose-1-phosphate formed in process step b) can be converted to uridine 5′-diphosphoGlucose and inorganic pyrophosphate by further enzymatic conversion using uridine triphosphate with a uridine triphosphate monosaccharide-1-phosphate uridylyltransferase. In general, regardless of the upstream cascade steps, it may be useful for the Glc-1-P formed in the final step of regeneration to be removed from equilibrium. It has proven to be particularly efficient to also perform this step enzymatically on the basis of a uridylyltransferase. This step not only removes product from the equilibrium and shifts the equilibrium towards products, it also simultaneously provides PPi for further reaction. This can reduce overall production costs and contribute to on-demand PPi supply. A possible reaction equation for this single step results in:
Glc-1-P+UTP↔UDP-Glc+PPi.
Within an overall consideration with the upstream cascade steps for the production of hyaluronic acid or derivatives, the following equation can result:
GlcNAc+Suc+2UTP↔UDP-GlcNAc+Fru+UDP-Glc+PPi.
In a preferred aspect of the use, the glucose-1-phosphate formed in process step b) can be converted to glucose-6-phosphate by further enzymatic reaction with a phosphoglucomutase. Another way to convert the Glc-1-P in the reaction solution by an enzymatic step is via the use of a phosphoglucomutase. This enzyme can be used in the same reaction solution of the previous steps and in this respect it results in a flexible and efficient ATP regeneration system. This conversion can be represented as:
Glc-1-P↔Glc-6-P.
In the context of the overall consideration of the use, the following overall equation results:
GlcNAc+Suc+UTP↔UDP-GlcNAc+Fru+Glc-6-P.
Further advantages and advantageous embodiments of the objects according to the invention are illustrated by the examples and drawings and explained in the following description. It should be noted that the drawings are descriptive only and are not intended to limit the invention.
The figures show:
In the following examples, the following enzymes were used:
E. coli
E. coli
Corynebacterium
glutamicum
Nitrosomonas
europea
Arabidopsis
thaliana
Streptococcus equi
zooepidemicus
Bifido-
bacterium
longum
Pasteurella
multocida
Cells were transformed with the appropriate vector via heat shock and proteins were expressed in “terrific broth” (TB) medium overnight using isopropyl-β-D-thiogalactopyranoside (IPTG) induction. Cell disruption was performed by sonication and the expressed enzymes were purified by Ni2+-immobilized metal ion affinity chromatography (IMAC) on HisTrap™ HP columns (GE Healthcare, Chicago, USA) on an AKTApurifier™ (GE Healthcare, Chicago, USA) system. Subsequently, the eluate was dialyzed using dialysis tubing (C. Roth, Karlsruhe, Germany) overnight in the respective storage buffer of the enzyme. EcAGPase and CgAGPase were stored in 100 mM HEPES pH 8; NeSuSy in 100 mM Tris-HCl pH 7 and the remaining enzymes in 100 mM HEPES pH 7.5. Protein concentrations of the eluates were performed after dialysis by Bradford assay using RotiQuant solution (C. Roth, Karlsruhe, Germany).
The reaction scheme of ATP synthesis reaction from sucrose (Suc) and inorganic pyrophosphate (PPi) by coupling EcAGPase and CgAGPase results in substeps a) and b) and in the overall summary to:
ADP+Suc ↔ADP-Glc+Fru a)
ADP-Glc+PPi↔Glc-1-P+ATP b)
ADP+Suc+PPi↔ATP+Fru+Glc-1-P Σ)
The interaction of the individual enzymatic cascade elements is shown in
EcAGPase (2.9 mg/mL) and NeSuSy (0.1 μg/mL) are combined in a one-pot synthesis to synthesize ATP from sucrose and PPi. For this purpose, ADP and PPi are present in the experimental series in a concentration ratio of 1:1 at different concentrations (2 mM to 15 mM), respectively. In addition, the synthesis batch contains a MgCl2 concentration corresponding to the sum of the concentration of ADP and PPi in the respective experiment (4 mM to 30 mM). In addition, 1 mM fructose bisphosphate (Fru-1,6-P2) is added to the reaction. The batch is buffered with 100 mM MOPS-NaOH buffer at pH 8 and ATP synthesis is performed at 37° C. The synthesis is stopped at the respective measurement points with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, AMP, ADP, ATP) is performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid). The analytes are detected at 254 nm.
The analytical results of this conversion are shown in
The reaction shown in
The combination of NeSuSy with EcAGPase in an enzyme cascade for ATP synthesis from sucrose and PPi shows that after only a few minutes, the concentration of ADP in the reaction decreases and ATP is formed. However, the synthesis of ATP reaches an ATP synthesis limit between 1.96 mM and 2.14 mM after 30 min of reaction time at ADP and PPi starting concentrations of 5 mM and 10 mM, respectively, corresponding to ATP yields of 39% (5 mM ADP/PPi) and 21% (10 mM ADP/PPi), respectively. The experiments with EcAGPase show that the synthesis of ATP from PPi and ADP-glucose is subject to the reaction equilibrium of EcAGPase (
This reaction sequence involves the in-situ generation of PPi through the use of a complex cascade involving the use of an AtUSP.
This ATP synthesis reaction can be represented as follows:
Glc-1-P+UTP ↔UDP-Glc+PPi
ADP-Glc+PPi↔Glc-1-P+ATP
ADP-Glc+UTP ↔UDP-Glc+ATP
The interaction of the individual enzymatic cascade elements is shown in
AtUSP and EcAGPase are combined in a one-pot synthesis to use PPi formed in the UDP-Glc synthesis for the synthesis of ATP in a subsequent reaction. The resulting Glc-1-P is again used by AtUSP for the synthesis of UDP-Glc. This attenuates the back reaction of EcAGPase towards ADP-Glc synthesis. This allows a more accurate description of the effect of Glc-1-P on ATP synthesis.
The experimental setup is as follows: 2.9 mg/mL EcAGPase and 0.5 mg/mL AtUSP are combined in a reaction with 3 mM ADP-Glc, 3 mM UTP, and 1 mM F-1,6-P2. UDP-Glc is synthesized with starting concentrations ranging from 0.5 mM to 10 mM Glc-1-P over a 10 min period at 37° C. in 100 mM HEPES buffer (pH 8). In addition, the reaction contains MgCl2 whose concentration was adjusted according to the sum of the concentrations of Glc-1-P and UTP. The synthesis is stopped at the respective measurement points with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, AMP, ADP, ATP, UTP, UDP, UMP, UDP-Glc) is performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and the analytes are detected at 254 nm.
The combination of AtUSP and EcAGPase shows that ATP is formed from the PPi of the UDP-Glc synthesis reaction and ADP-Glc (
Through this experiment, it is demonstrated that EcAGPase is able to synthesize ATP from in situ nascent PPi, in a coupled enzyme reaction. ATP can in turn be converted as a substrate by ATP-utilizing enzymes.
This reaction sequence involves the BlNahK/NeSuSy/EcAGPase 3-enzyme cascade for the synthesis of GlcNAc-1-P using the new ATP regeneration system.
This ATP synthesis reaction can be represented as follows:
GlcNAc+ATP ↔GlcNAc-1-P+ADP
ADP+Suc ↔ADP-Glc+Fru
ADP-Glc+PPi↔Glc-1-P+ATP
GlcNAc+PPi+Suc ↔GlcNAc-1-P+Fru+Glc-1-P
The interaction of the individual enzymatic cascade elements is shown in
Reactions were based on 1.6 mg/mL BlNahK, 1.3 mg/mL EcAGPase, 25 μg/mL NeSuSy, 5 mM GlcNAc, 200 mM sucrose, 0.5 mM to 5 mM ATP, 5 mM PPi, 10 mM MgCl2, and 0.5 mM Fru-1,6-P2. The experiment was performed in 100 mM MOPS buffer, pH 7 at 37° C. for 24 h. GlcNAc-1-P and Glc-1-P were reacted separately to give UDP-GlcNAc and UDP-Glc, respectively, and measured by MP-CE. Here, 250 μg/mL AtUSP; 2.5 mg/mL SzGlmU and 2.6 mg/mL PmPpA with 10 mM UTP and 10 mM MgCl2 in 100 mM MOPS buffer, pH 7 at RT for 2 h were added to the reaction after removing the synthesis enzymes. ATP regeneration was calculated using the following formula:
The new ATP regeneration system NeSuSy/EcAGPase is combined with an ATP-consuming enzyme, such as a sugar-1-phosphate kinase (using BlNaHK as an example), for the synthesis of GlcNAc-1-P. ATP is consumed by the kinase BlNahK to form GlcNAc-1-P. The resulting ADP is converted by NeSuSy with sucrose to ADP-Glc and fructose. ADP-Glc is subsequently converted to Glc-1-P and ATP by EcAGPase with the addition of PPi. In this way, ATP is made available again (regenerated) from ADP for the sugar kinase reaction.
The experimental setup is as follows: The reaction contains 1.6 mg/mL BlNahK, 1.3 mg/mL EcAGPase and 25 μg/mL NeSuSy, 5 mM GlcNAc, 200 mM sucrose, 0.5 mM to 5 mM ATP, 5 mM PPi and 10 mM MgCl2 and 0.5 mM Fru-1,6-P2. Enzyme reactions are performed in 100 mM MOPS buffer, pH 7 at 37° C. for 24 h in a 96-microtiter plate in a volume of 200 μL. After each measurement time point, 150 μL is removed from each of the reaction mixtures and the enzymes are separated from the reaction by ultrafiltration (30 K filter, cut-off 30 kDa, AcroPrep™ Advance filters; Pall) for 15 min. Then, to 100 μL of sample, 50 μL of a solution of AtUSP, SzGlmU, PmPpA, 10 mM UTP and 10 mM MgCl2 are added to synthesize the nucleotide sugars UDP-GlcNAc and UDP-Glc. These are then analyzed by capillary electrophoresis. The reaction of the enzymes from the follow-up reaction for nucleotide sugar synthesis are stopped with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, ADP, ATP, UTP, UDP, UDP-Glc and UDP-GlcNAc) is performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and the analytes detected at 254 nm. The UDP-GlcNAc concentration is proportional to the GlcNAc-1-P formed. The UDP-Glc concentration is proportional to the ATP formed.
Reduction of the ATP concentration (2 mM and 0.5 mM) below the substrate concentration (5 mM GlcNAc) shows that GlcNAc is phosphorylated with 41%-68% yield (
In combination with a sugar kinase (ATP-consuming enzyme), the new ATP regeneration system NeSuSy/AGPase is capable of regenerating ATP from ADP with sucrose and PPi.
This ATP synthesis reaction using a 4-enzyme cascade BINahK/SzGlmU/NeSuSy/EcAGPase to synthesize UDP-GlcNAc with new ATP regeneration system can be shown as follows:
GlcNAc+ATP ↔GlcNAc-1-P+ADP
GlcNAc-1-P+UTP ↔UDP-GlcNAc+PPi
ADP+Suc ↔ADP-Glc+Fru
ADP-Glc+PPi↔Glc-1-P+ATP
GlcNAc+Suc+UTP ↔UDP-GlcNAc-1-P+Fru+Glc-1-P
The interaction of the individual enzymatic cascade elements is shown in
The ATP regeneration system NeSuSy/EcAGPase is combined with the enzyme cascade BlNahK/SzGlmU for the synthesis of UDP-GlcNAc. GlcNAc is converted to GlcNAc-1-P and ADP with BlNahK consuming ATP. GlcNAc-1-P is then converted with SzGlmU to UDP-GlcNAc with release of PPi. NeSuSy converts ADP and sucrose to ADP-Glc and fructose. EcAGPase uses the released PPi and ADP-Glc to form Glc-1-P and ATP, which is thus regenerated.
The experimental setup is as follows: The synthesis reaction contains 57.5 μg/mL EcAGPase, 58 μg/mL NeSuSy, 84 μg/mL BlNahK, 94 μg/mL SzGlmU, 5 mM UTP, 200 mM sucrose, 0.5 mM Fru-1,6-P2, 10 mM MgCl2 and the ATP concentration is 0.5 mM to 5 mM. Reactions are performed on a 200 μL scale in a 96-microtiter plate at 37° C. for 24 h in 100 mM HEPES buffer pH 7. With each measurement time point, 150 μL of a reaction is removed and the enzymes are separated from the reaction by ultrafiltration (30 K filter, cut-off 30 kDa, AcroPrep™ Advance filters; Pall) for 15 min. To determine the resulting Glc-1-P concentration, 50 μL of a solution of AtUSP, PmPpA, 10 mM UTP, and 10 mM MgCl2 are then added to 100 μL of sample. The resulting nucleotide sugar UDP-Glc is analyzed by capillary electrophoresis. The UDP-Glc concentration is proportional to the ATP formed. The reaction of the enzymes from the follow-up reaction to the nucleotide sugar synthesis are stopped with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, ADP, ATP, UTP, UDP, UDP-Glc and UDP-GlcNAc) is performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and analytes are detected at 254 nm.
Reduction of ATP concentration (2 mM and 0.5 mM) below the substrate concentration (5 mM GlcNAc) shows that UDP-GlcNAc is synthesized with 25%-63% yield (
In combination with a sugar kinase (ATP-consuming enzyme) and a pyrophosphorylase (PPi generating enzyme), the new ATP regeneration system NeSuSy/AGPase is capable of regenerating ATP from ADP with sucrose and PPi.
This ATP synthesis reaction using a 5-enzyme cascade BINahK/SzGlmU/NeSuSy/EcAGPase/AtUSP to synthesize UDP-GlcNAc with the ATP regeneration system of the invention can be described as follows:
GlcNAc+ATP ↔GlcNAc-1-P+ADP
GlcNAc-1-P+UTP ↔UDP-GlcNAc+PPi
ADP+Suc ↔ADP-Glc+Fru
ADP-Glc+PPi↔Glc-1-P+ATP
Glc-1-P+UTP ↔UDP-Glc+PPi
GlcNAc+Suc+2 UTP ↔UDP-GlcNAc+Fru+UDP-Glc+PPi
The interaction of the individual enzymatic cascade elements is shown in
The enzyme cascade for UDP-GlcNAc synthesis is completed with the enzyme AtUSP. Glc-1-P is converted to UDP-Glc with AtUSP and thus removed from the reaction of EcAGPase to suppress the back reaction of EcAGPase and provide more ATP for the enzyme cascade BINahK/SzGlmU. This results in a higher product yield for UDP-GlcNAc synthesis.
The experimental setup is as follows: The synthesis is performed in a one-pot procedure with five enzymes. 41.5 μg/mL NeSuSy, 834 μg/mL BlNahK, 960 μg/mL EcAGPase, 1.2 mg/mL SzGlmU and 75.5 μg/mL AtUSP are used. Synthesis was performed using 5 mM GlcNAc, 10 mM UTP, 0.5 mM F-1,6-P2, 10 mM MgCl2, and 0.25 mM to 2.5 mM ATP. The reaction was carried out in 200 μL in 100 mM HEPES pH 7 at 37° C. for 24 h in a 96-microtiter plate. The synthesis was stopped with 28 mM SDS (final concentration 7 mM) and the analysis of nucleotides (ADP-Glc, AMP, ADP, ATP, UTP, UDP, UMP, UDP-Glc, and UDP-GlcNAc) was performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and the analytes were detected at 254 nm.
Removal of Glc-1-P from the reaction equilibrium of EcAGPase by reaction with AtUSP significantly increases the yield for UDP-GlcNAc and regeneration of ATP. After 24 h, UDP-GlcNAc yields ranging from 61% (0.25 mM ATP) to 85% (2.5 mM ATP) are achieved (
In combination with the UDP-sugar pyrophosphorylase AtUSP (PPi generating enzyme), a sugar kinase (ATP-consuming enzyme) and another pyrophosphorylase (PPi generating enzyme), the new ATP regeneration system NeSuSy/AGPase is capable of regenerating ATP from ADP with sucrose and PPi very efficiently and achieving high product yields. A key function is assigned to the nascent Glc-1-P in the reaction of EcAGPase. Glc-1-P should be removed from the reaction equilibrium. This can be achieved, for example, with the enzyme AtUSP, which converts Glc-1-P with UTP to UDP-Glc and PPi. AtUSP thus additionally forms PPi, which drives ATP synthesis and thus ATP regeneration.
Other enzymes such as sugar P mutases (phosphoglucomutase, formation of Glc-6-P) and sugar phosphate isomerases (Fru-6-P isomerase, formation of Fru-6-P) would also be suitable to remove Glc-1-P from the equilibrium of EcAGPAse.
The ATP synthesis reaction can proceed in the context of one of the phosphate-free UDP-GlcNAc synthesis with PGM to remove Glc-1-P according to the following equations:
GlcNAc+ATP ↔GlcNAc-1-P+ADP
ADP+Suc ↔ADP-Glc+Fru
GlcNAc-1-P+UTP ↔UDP-GlcNAc+PPi
ADP-Glc+PPi↔Glc-1-P+ATP
Glc-1-P ↔Glc-6-P
GlcNAc+Suc+UTP ↔UDP-GlcNAc-1-P+Fru+Glc-6-P
The interaction of the individual enzymatic cascade elements is shown in
Using AtUSP to reduce Glc-1-P in the reaction resulted in an additional unit of PPi, which could be used by EcAGPase to regenerate ATP. Therefore, this experiment tests how the system behaves when only one unit of PPi is provided during UDP-GlcNAc synthesis.
The experimental setup is as follows: The synthesis was performed in a one-pot procedure on a 96-well plate with a volume of 200 μL. Each reaction batch contained ATP at different concentrations (0.25 mM-2.5 mM), UTP (7 mM), GlcNAc (5 mM), sucrose (200 mM), F-1,6-P2 (0.5 mM), and MgCl2 (10 mM). The reactions were additionally carried out at 37° C. in 100 mM MOPS-buffer pH 7 for 24 h. BlNahK and SzGlmU enzymes were added at concentrations of 0.5 mg/mL and 5 μg/mL, respectively. The enzymes of the ATP regeneration cascade were used at the concentrations of 23.5 μg/mL (NeSuSy) and 175 μg/mL. The enzyme phosphoglucomutase (PGM) from hare muscle (Sigma Aldrich, USA) was added to the cascade at a concentration of 600 μg/mL. The reactions were stopped at the respective measurement points using a stop solution (28 mM SDS, 5 mM PAPA, 1 mM PABA) and analyzed by MP-CE. The nucleotides (ATP, ADP, AMP, UTP, UDP and UMP) and nucleotide sugars (UDP-GlcNAc and ADP-Glc) were detected on MP-CE using UV at 254 nm.
By replacing the AtUSP with PGM, UDP-GlcNAc yields of up to 73% could be achieved from 5 mM GlcNAc and 2.5 mM ATP after 24 h (
The use of PGM for the reduction of Glc-1-P shows that coupling of the system according to the invention with further enzymes for Glc-1-P reduction is possible.
The influence of PPi concentrations on the ATP synthesis activity of the EcAGPase used was investigated.
The experiment included 4.35 μg/mL EcAGPase, 2.5 mM ADP-Glc, 10 mM MgCl2, 1 mM Fru-1,6-P2 and PPi, at concentrations ranging from 0.75 mM to 10 mM, and a control reaction without PPi. ATP synthesis was performed in 600 μL of 100 mM HEPES buffer, pH 8 at 37° C. for 12 min. The synthesis was stopped at the respective measurement points with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, AMP, ADP, ATP) was performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and the analytes were detected at 254 nm.
EcAGPase already shows a reduction in specific activity at PPi concentrations above 3 mM (
EcAGPase is subject to substrate inhibition by PPi in the ATP synthesis direction. Therefore, ATP regeneration by this enzyme may be less efficient in reactions that involve a lot of PPi or release a lot of PPi very rapidly. This disadvantage can be compensated by increasing the EcAGPase concentration.
Fru-1,6-P2 was used as an activator for EcAGPase. It is Fru-1,6-P2 a relatively expensive compound and its use in the EcAGPase reaction should be reduced as much as possible. Therefore, the activity of EcAGPase was investigated for different Fru-1,6-P2 concentrations to determine a minimum concentration of the activator.
The experiment included 3.9 μg/mL EcAGPase, 3 mM ADP-Glc, 3 mM PPi, 10 mM MgCl2 and Fru-1,6-P2 at concentrations ranging from 0.1 mM to 1 mM, as well as a control reaction without Fru-1,6-P2. The synthesis was performed in 600 μL of 100 mM HEPES buffer, pH 8 at 37° C. for 12 min. The synthesis was stopped at the respective measurement points with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, AMP, ADP, ATP) was performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and the analytes were detected at 254 nm.
With 0.5 mM Fru-1,6-P2, a high EcAGPase activity (60 U/mg, 12-fold higher than without activator) is still achieved (
The activator Fru-1,6-P2 significantly increases the activity of EcAGPase. The concentration of the activator can be significantly reduced. Therefore, it is also possible to perform ATP regeneration with EcAGPase efficiently with very small Fru-1,6-P2 concentrations and even without activator.
The EcAGPase prefers the ADP-Glc synthesis. Thus, the accumulation of Glc-1-P negatively affects the reaction equilibrium for ATP synthesis. Therefore, the effect of Glc-1-P on the ATP synthesis activity of EcAGPase was investigated.
The experiment included 8.5 μg/mL EcAGPase, 3 mM ADP-Glc, 3 mM PPi, 0.5 mM Fru-1,6-P2, 10 mM MgCl2 and Glc-1-P at concentrations ranging from 1 mM to 10 mM, and a control reaction without Glc-1-P. The synthesis was performed in 600 μL of 100 mM HEPES buffer, pH 8 at 37° C. for 12 min. The synthesis was stopped at the respective measurement points with 28 mM SDS (final concentration 7 mM) and the analysis of the nucleotides (ADP-Glc, AMP, ADP, ATP) was performed by multiplex capillary electrophoresis (internal standards (final concentrations): 1 mM para-aminobenzoic acid and 1 mM 4-aminophthalic acid) and the analytes were detected at 254 nm.
EcAGPase shows a strongly reduced ATP synthesis activity at increasing Glc-1-P concentrations (
The invention underlying this patent application was developed in a project funded by the BMBF under the grant number 031B0104B.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2020 111 560.1 | Apr 2020 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/060401 | 4/21/2021 | WO |
