Method for producing an aqueous solution containing ethylene glycol

Abstract

A method for producing an aqueous solution containing ethylene glycol and / or a salt of pyruvic acid, by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase (EC 4.1.2.55) to give an aqueous solution which contains glycol aldehyde and a salt of pyruvic acid, after which the glycol aldehyde is reduced using an NAD(P)H-dependent alcohol dehydrogenase (EC 1.1.1.1) or an NAD(P)H-dependent xylose reductase (EC 1.1.1.307) and subsequently optionally removing either the salt of pyruvic acid or the ethylene glycol from the solution.

Description

[0001] Process for preparing an aqueous solution containing ethylene glycol

[0002] The invention relates to a process for the preparation of an aqueous solution containing ethylene glycol and a salt of pyruvic acid (pyruvate) or ethylene glycol and a salt of lactic acid (lactate).

[0003] Background of the invention

[0004] Ethylene glycol (ethane-l,2-diol), the simplest dihydric alcohol, is an important industrial chemical - it is used, for example, as an antifreeze or as a precursor for the polymers polyethylene terephthalate (PET) and polyethylene furanoate (PEF), a bio-based alternative to PET (Salusjärvi et al., 2019).

[0005] Ethylene glycol is produced by the hydrolysis of ethylene oxide (at 200 °C), which in turn is obtained by the oxidation of ethylene (obtained from fossil fuels). The resulting byproducts (oligoethylene glycols) must be separated by distillation, which is an energy-intensive process. To avoid byproducts, Shell's OMEGA process (Only Mono Ethylene Glycol Advantage) can be used, for example. In this process, ethylene oxide is first reacted with CO2 to form ethylene carbonate, which is then hydrolyzed to ethylene glycol (using K1 and K2MOO4 as catalysts) while releasing CO2. Other processes include the Rh- or Co-catalyzed hydrohydroxymethylation of formaldehyde or the hydroformylation of formaldehyde to glycolaldehyde, which is subsequently reduced to ethylene glycol (Berger, 2016).

[0006] Since the current large-scale processes cannot be described as sustainable due to the high energy consumption (high temperatures) and the fossil origin of the raw materials (ethylene) or the use of expensive, sometimes toxic catalysts, biotechnological processes are being and have been developed that are based on renewable raw materials and are carried out under milder reaction conditions.

[0007] To develop sustainable production processes for chemicals from biomass, less energy-intensive methods are needed that utilize the existing diversity of the biomaterial. In addition to efficient, gentle methods for the depolymerization of renewable raw materials into shorter-chain sugars, efficient technologies are needed to further convert these carbohydrate intermediates into chemical products. A particular challenge lies in efficiently and as completely as possible converting the mixtures of substances resulting from depolymerization into valuable material streams. Straws, wood, and other plant-based raw materials generally contain hemicelluloses and celluloses, from which monomer sugars can be released in varying compositions, primarily the pentoses D-xylose and L-arabinose, the hexoses D-glucose, D-mannose, and D-galactose, as well as acids derived from the sugars (e.g., D-glucuronic acid).Methods for the efficient separation of C6 and C5 sugars allow these stream types to be further processed separately (e.g., US 9970038 B2). For example, corn hulls contain 10% L-arabinose, 16% D-xylose, 64% D-glucose, 4% D-galactose, and 2% D-mannose, based on total sugar (Hromädkovä & Ebringerovä, 1995), whereas wheat straw contains 5% L-arabinose, 30% D-xylose, 56% D-glucose, 1% D-galactose, and 2% D-mannose (Collins et al., 2014). These percentages show that the ratios of L-arabinose and D-xylose in both biomasses differ greatly (maize hulls: 1:1.6 and wheat straw: 1:6).

[0008] Currently, biotechnological efforts primarily focus on processes aimed at converting biomass into chemical products through fermentation, i.e., the conversion of substances during the growth of microbes in a reactor. Despite all technical advances in the field, such as metabolic engineering and heterologous pathway expression, these fermentative whole-cell processes are limited by the physiological limitations of cellular production (tolerance to solvents, temperature, mass transport, and high substrate and product concentrations, as well as by-products from other metabolic pathways in the cell) (Claassens et al., 2019). These intracellular processes, and in particular the energy requirements for the metabolic (often carbon-emitting) steps, lead to long process times and, on the other hand, to the achievable carbon yield being significantly below the theoretically possible.In addition, these processes generally use biosynthetic pathways that release CO2 from the substrate, so that even the theoretical carbon yield in the product is only a fraction of the carbon used: While 1-4 carbons are lost for Cg sugars, depending on the pathway, the figure is 1-3 for C5 sugars. Typical key data of such processes are summarized in the literature (Salusjärvi et al., 2019).

[0009] Alkim et al. describe a process for the production of ethylene glycol from D-xylose via a synthetic pathway in Escherichia coli. D-xylose is first isomerized to D-xylulose using D-xylose isomerase, which is then phosphorylated to D-xylulose-l-phosphate using adenosine triphosphate (ATP) by D-xylulose-l-kinase. The aldol cleavage catalyzed by D-xylulose-l-phosphate aldolase yields dihydroxyacetone phosphate (DHAP) and glycolaldehyde, which is ultimately reduced to ethylene glycol (using glycolaldehyde reductase FucO). In a bioreactor experiment, 55 g / l D-xylose could be converted to 20 g / l ethylene glycol (91% yield and 0.37 g / (lh) productivity) (Alkim et al., 2015).

[0010] The metabolic pathway used by Alkim et al. exists natively in Saccharomyces cerevisiae, as Uranukul et al. demonstrated. The enzymes involved are phosphofructokinase (catalyzes the phosphorylation of D-xylulose) and fructose bisphosphate aldolase (catalyzes the aldol cleavage), the key enzymes of glycolysis. One strain was able to produce 4 g / L of ethylene glycol in 330 h from 50 g / L of D-xylose, with continuous supplementation of D-glucose (Uranukul et al., 2018).

[0011] Pereira et al. used modified E. coli cells to convert D-xylose and L-arabinose to ethylene glycol. Through several steps (isomerization, epimerization, and phosphorylation), D-xylose is converted to D-ribulose-l-phosphate and L-arabinose to L-xylulose-l-phosphate, respectively. These are then cleaved into DHAP and glycolaldehyde by L-fuculose-phosphate aldolase and L-rhamnose-l-phosphate aldolase, respectively. In this way, a mixture of 15 g / l D-xylose and 15 g / l L-arabinose was converted to 10.5 g / l ethylene glycol in 220 h. The data show that D-xylose is converted to ethylene glycol more quickly and that a longer lag phase occurs in the case of L-arabinose and the pentose mixture (Pereira et al., 2016).

[0012] Glycolaldehyde, the precursor to ethylene glycol, also occurs as a product in another metabolic pathway starting from D-xylose or L-arabinose, known as the Dahms pathway (Dahms & Anderson, 1969; Dahms, 1974). D-xylose / L-arabinose is first oxidized to 1,4-D-xylonolactone / 1,4-L-arabinolactone, which is then opened to D-xylonate / L-arabonate under the action of a lactonase. A dehydratase converts D-xylonate / L-arabonate to 2-keto-3-deoxy-D-xylonate (KDX) / 2-keto-3-deoxy-L-arabonate (KDA), with both 2-keto-3-deoxy intermediates being cleaved by an aldolase into pyruvate and glycolaldehyde. The use of promiscuous enzymes, for example, in Sulfolobus solfataricus, allows the simultaneous conversion of D-xylose and L-arabinose (Kopp et al., 2020).

[0013] Salusjärvi et al. expressed enzymes of the Dahms pathway (D-xylose dehydrogenase and D-xylonate dehydratase from Caulobacter crescentus, as well as an aldolase from E. coli) in Saccharomyces cerevisiae. In addition to glycolic acid (through oxidation by an aldehyde dehydrogenase from E. coli), they also found small amounts of ethylene glycol (through reduction by an endogenous aldo-keto reductase). Some of the intermediates were converted by S. cerevisiae into byproducts such as 3-deoxypentanoic acid or 3,4-dihydroxybutanoic acid (Salusjärvi et al., 2017).

[0014] Chae et al. constructed the Dahms pathway in E. coli strains, using the glycolaldehyde reductase yqhD to reduce glycolaldehyde to ethylene glycol. Under fed-batch conditions, 108.2 g / L of ethylene glycol could be produced in a D-xylose minimal medium (yield 0.36 g / g D-xylose and productivity 2.25 g / (Lh)) (Chae et al., 2018). Wang et al. used a similar approach, using the NADH-dependent aldehyde reductase FucO instead of the NADPH-dependent aldehyde reductase yqhD for reduction to glycolaldehyde. This enabled the production of 72 g / L of ethylene glycol from D-xylose under fed-batch conditions (52 h runtime) (Wang et al., 2018).

[0015] Zhang et al. also identified the Dahms pathway in the microorganism Enterobacter cloacae S1. In a fed-batch culture of E. cloacae S1, 34 g / L ethylene glycol and 13 g / L glycolic acid could be simultaneously produced in 46 h from D-xylonic acid (0.99 mol products / mol D-xylonic acid) (Zhang et al., 2020).

[0016] WO 2014162063 A1 describes modified S. cerevisiae strains capable of producing up to 1.25 g / l ethylene glycol from 20 g / l D-xylose in 120 h (with the addition of 10 g / l D-glucose and 10 g / l CaCO3) via the Dahms pathway.

[0017] US 10774348 B2 describes recombinant microorganisms (E. coli, S. cerevisiae) for the biosynthesis of ethylene glycol in combination with a C3 compound such as acetone, 2-propanol, or propene (starting from pyruvate as a product of the Dahms pathway or from DHAP as a cleavage product of D-ribulose-l-phosphate or D-xylulose-l-phosphate). For example, an E. coli strain converts 15 g / l of D-xylose into 5 g / l of ethylene glycol, 0.17 g / l of acetone, and 0.42 g / l of 2-propanol via the Dahms pathway within 48 hours.

[0018] Pyruvate, the second product of the Dahms pathway, is a key intermediate in cellular metabolism and serves, for example, as a precursor to the amino acid alanine or acetyl-CoA, which is involved in the citric acid cycle. Reduction of pyruvate yields lactic acid (lactate), which has a wide range of applications. Lactic acid is used, for example, as an acidifier in the food industry, as a descaling agent, pH regulator, or cleaning agent in the chemical industry, as an additive in anti-acne creams or moisturizers in the cosmetics industry, and as a precursor for acrylic acid or ethyl lactate (Wee et al., 2006). Lactic acid can also be used for skin exfoliation (chemical peeling) (Smith, 1996).The bifunctional lactic acid can also be polymerized to produce polylactide (PLA), a biodegradable and biocompatible plastic that has applications in the packaging industry, textile industry, electronics and medicine (Balla et al., 2021).

[0019] Jia et al. developed an enzymatic cascade for the production of (R)-acetoin ((R)-3-hydroxy-2-butanone) and ethylene glycol from D-xylose via the Dahms pathway. The enzymatic reduction of glycolaldehyde and ethylene glycol drives the oxidation of D-xylose to D-xylonolactone (with regeneration of the cofactor NAD). Two pyruvate molecules are finally converted to (R)-acetoin by α-acetolactate synthase and α-acetolactate decarboxylase, with double CO2 elimination. In this way, 10 mM (1.5 g / L) D-xylose can be converted to 5.5 mM ethylene glycol and 3.2 M (R)-acetoin after 24 h (Jia et al., 2018).

[0020] Boer et al. presented an in vitro variant of the Dahms pathway using purified enzyme isolates, in which D-xylose or D-xylonolactone (1 mM substrate concentration) is converted to ethylene glycol (reduction of glycolaldehyde), glycolic acid (oxidation of glycolaldehyde), or lactate (reduction of pyruvate). The main focus of the study was the application of a spectrophotometric assay to evaluate the efficiency of the cascade, which is based on the formation or consumption of NADH. For this purpose, 2 mM NAD was added as an external cofactor. Furthermore, the study also investigated the role of lactonase (from Caulobacter crescentus) in the Dahms pathway. The data suggest that the spontaneous ring opening of D-xylonolactone is rate-limiting, especially at pH 7. Only with the addition of lactonase is the overall kinetics of the cascade noticeably accelerated, which can also be achieved by increasing the pH value (Boer et al., 2019). Overall, Boer et al.There is no process that allows the chemicals mentioned to be produced on a preparative scale. For example, the concentration of the reactants is very small, approximately 0.3 mM in the case of ethylene glycol.

[0021] This is where the present invention comes in and aims to provide a process for producing an aqueous solution containing ethylene glycol and a salt of lactic acid (lactate) or ethylene glycol and a salt of pyruvic acid (pyruvate), which starts from 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA), is technically feasible at higher concentrations and also enables high yields.

[0022] Detailed description of the invention

[0023] The object of the invention for producing an aqueous solution containing ethylene glycol and a salt of pyruvic acid is achieved by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase, whereby an aqueous solution is obtained which contains glycolaldehyde and a salt of pyruvic acid, after which the glycolaldehyde is reduced to ethylene glycol by treatment with an NAD(P)H-dependent alcohol dehydrogenase or with an NAD(P)H-dependent xylose reductase in vitro, wherein the oxidized cofactor NAD(P) formed by the reduction with the alcohol dehydrogenase or together with the xylose reductase +is reduced by means of a secondary alcohol to form a ketone and then, if necessary, either the pyruvic acid salt or the ethylene glycol is removed from the solution. The object of the invention for producing an aqueous solution containing ethylene glycol and a salt of lactic acid is achieved by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase, whereby an aqueous solution is obtained which contains glycolaldehyde and a salt of pyruvic acid, after which the glycolaldehyde is reduced in vitro by treatment with an NAD(P)H-dependent alcohol dehydrogenase or with an NAD(P)H-dependent xylose reductase to ethylene glycol and the salt of pyruvic acid is reduced in vitro by means of a lactate dehydrogenase to a salt of lactic acid, wherein the oxidized cofactor NAD(P) formed by the reduction with the alcohol dehydrogenase or together with the xylose reductase+ is reduced by means of a secondary alcohol to form a ketone and then, if necessary, either the lactic acid salt or the ethylene glycol is removed from the solution.

[0024] The invention further relates to a process for the preparation of an aqueous solution containing ethylene glycol, in which glycolaldehyde, which is dissolved in an aqueous solution, is reduced to ethylene glycol by treatment with an NAD(P)H-dependent alcohol dehydrogenase or together with an NAD(P)H-dependent xylose reductase in vitro and the oxidized cofactor NAD(P) formed by the reduction + is reduced by means of a secondary alcohol to form a ketone.

[0025] In the processes according to the invention, 2-propanol is preferably used as the secondary alcohol.

[0026] Surprisingly, it has been shown that the objectives set by the invention can be achieved if the oxidized cofactor NAD(P) produced by the reduction with alcohol dehydrogenase or together with xylose reductase + by means of a secondary alcohol, preferably 2-propanol, to form a ketone.

[0027] The processes according to the invention are schematically illustrated in the accompanying Figures 1 and 2. The designation A stands for 2-keto-3-deoxy-D-xylonate (KDX), B for 2-keto-3-deoxy-L-arabonate (KDA), C for glycolaldehyde, D for pyruvate, E for ethylene glycol, F for 2-propanol, G for acetone, H for lactate, 1 for aldolase, 2 for xylose reductase, 3 for NAD-dependent alcohol dehydrogenase, 4 for NADP-dependent alcohol dehydrogenase, 5 for NAD(P)-dependent alcohol dehydrogenase, and 6 for lactate dehydrogenase. The dashes between the reference numbers (e.g., 2 / 3 in Figure 1) stand for "or." When using a xylose reductase 2 for the reduction of glycolaldehyde C to ethylene glycol E, an NADP-dependent alcohol dehydrogenase 4 (Figure 1) or an NAD(P)-dependent alcohol dehydrogenase 5 (Figure 2) must be used for the oxidation of 2-propanol F to acetone G.Furthermore, it is crucial that the reaction is not carried out fermentatively, but that the enzymes are contained as such in the aqueous solution, i.e. that the reaction is carried out in vitro.

[0028] The regeneration of the cofactor by alcohol dehydrogenase is previously known, for example, from EP 2812439 Bl or described by Xu et al. (2021).

[0029] A further preferred variant of the process according to the invention is characterized in that it is carried out as a one-pot reaction without isolation of intermediate products.

[0030] In further preferred embodiments of the process according to the invention, the concentration of KDA and / or KDX in the aqueous solution is 20 - 300 g / l.

[0031] The particularly preferred temperature range is between 25 and 40 °C.

[0032] The most preferred pH range of the reaction is between 7.0 and 8.5.

[0033] It has further been shown that the enzymes in the process according to the invention are best present as lysate of the corresponding cells producing them.

[0034] In another variant, the enzymes can be present as a suspension, in a homogenate or immobilized on a solid carrier material.

[0035] In this context, suspension means a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and suspended in a suitable buffer system. In contrast to fermentative processes, which also work with whole cells, the resting cells can no longer grow due to the lack of carbon sources and nutrients; instead, they only serve to convert substrates (Lin & Tao, 2017). Homogenate in this context refers to a physically and / or chemically treated suspension (e.g., treated by pressure, lysozyme, or ultrasound), whereby the cellular components are released from the cells. A lysate is obtained when the insoluble cellular components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0036] The aldolase used in the process for the cleavage of KDX and KDA into glycolaldehyde and pyruvate can be selected from one of the groups EC 4.1.2.18 (2-dehydro-3-deoxy-L-pentonate aldolase), 4.1.2.20 (2-dehydro-3-deoxyglucarate aldolase), 4.1.2.21 (2-dehydro-3-deoxy-6-phosphogalactonate aldolase), 4.1.2.28 (2-dehydro-3-deoxy-D-pentonate aldolase), 4.1.2.29 (5-dehydro-2-deoxyphosphogluconate aldolase), 4.1.2.51 (2-dehydro-3-deoxy-D-gluconate aldolase), 4.1.2.52 (4-hydroxy-2-oxoheptandioate aldolase), 4.1.2.53 (2-keto-3-deoxy-L-rhamnonate aldolase), 4.1.2.54 (L-threo-3-deoxy-hexylosonate aldolase), 4.1.2.55 (2-dehydro-3-deoxy-phosphogluconate / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase) and 4.1.3.39 (4-hydroxy-2-oxovalerate aldolase), with group 4.1.2.55 (2-dehydro-3-deoxy-phosphogluconate / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase) being particularly preferred.

[0037] The oxidoreductase used to reduce pyruvate to lactate preferably comes from group EC 1.1.1.27 (L-lactate dehydrogenase).

[0038] The oxidoreductase used to reduce glycolaldehyde to ethylene glycol is preferably an alcohol dehydrogenase or a xylose reductase.

[0039] The NAD(P)H-dependent alcohol dehydrogenase for the reduction of glycolaldehyde to ethylene glycol preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0040] The NAD(P)H-dependent xylose reductase for the reduction of glycolaldehyde to ethylene glycol preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 7 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0041] SEQ ID NO. 1: M KAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGVIEEVGPGV

[0042] THLKVGDRVGIPWLYSACGHCDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYVVKIPDNLSFEEAAPIFC

[0043] AGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYAKAMGLNVVAVDLGDEKLELAKQLGADLVVNPKHDD

[0044] AAQWIKEKVGGVHATWTAVSKAAFESAYKSIRRGGACVLVGLPPEEIPIPIFDTVLNGVKIIGSIVGTRKDLQEAL

[0045] QFAAEGKVKTIVEVQPLENINDVFDRMLKGQINGRVVLKVD

[0046] SEQ ID No. 2:

[0047] ATGAAAGCTGCAGTTGTGGAACAATTTAAAAAGCCGTTACAAGTGAAAGAAGTGGAAAAAACCTAAGATCT

[0048] CATACGGGGAAGTATTAGTGCGCATCAAAGCGTGTGGGGTATGCCATACAGACTTGCATGCCGCACATGG

[0049] CGACTGGCCTGTAAAGCCTAAACTGCCTCTCATTCCTGGCCATGAAGGCGTCGGTGTAATTGAAGAAGTAG

[0050] GTCCTGGGGTAACACATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTTATTCGGCGTGCGGTCAT

[0051] TGTGACTATTGCTTAAGCGGACAAGAAACATTATGCGAACGTCAACAAAACGCTGGCTATTCCGTCGATGG

[0052] TGGTTATGCTGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAATTCCTGATAACTTATCGTTTGAAGA

[0053] AGCCGCTCCAATCTTTTGCGCTGGTGTAACAACATATAAAGCGCTCAAAGTAACAGGCGCAAAACCAGGTG

[0054] AATGGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTCGCAGTCCAATACGCAAAGGCGATGGGGTT

[0055] AAACGTCGTTGCTGTCGATTTAGGTGATGAAAAACTTGAGCTTGCTAAACAACTTGGTGCAGATTCTTGTCG

[0056] TCAATCCGAAACATGATGATGCAGCACAATGGATAAAAGAAAAAGTGGGCGGTGTGCATGCGACTGTCGT

[0057] CACAGCTGTTTCAAGCCGCGTTCGAATCAGCCTACAAATCCATTCGTCGCGGTGGTGCTTGCGTACTCGT

[0058] CGGATTACCGCCGGAAGAAATACCTATTCCAATTTTCGATACAGTATTAAATGGAGTAAAAATTATTGGTTC

[0059] TATCGTTGGTACGCGCAAAGACTTACAAGAGGCACTTCAATTTGCAGCAGAAGGAAGGTAAAAACAATT

[0060] GTCGAAGTGCAACCGCTTGAAAACATTAACGACGTATTCGATCGTATTGTTAAAAGGGCAAATTAACGGCCG

[0061] CGTCGTGTTAAAAGTAGATTAA

[0062] SEQ ID No. 3:

[0063] MSGKM KAAVVHEFGKPLTIEELDIPTIKPTQILVKM IACGVCHTDLHAASGDWPKKPHLPFIPGHEGVGTVVQV

[0064] GSEVDWVKEGDVVGVPWLYSACGHCEHCLAGWETLCAKQEETGYSVNGCFAEYVVADPNYIAHLPKGVDPV

[0065] KVAPVLCAGLTVYKGLKMTDTRAGNWVAISGVGGLGQMAVQYAVAMGLNWAVDIDDEKLATAKKLGATYT

[0066] VNARNTDPAAFMQEKVGGVHGGLITAVSTKAFSQAMGYVRAGGTLVLNGLPPGDFPISIFDVVM NAITIRGSI

[0067] VGTRLDM IEALSFFAEGKVTSVTTTDRIDNINAIFDALKNGRVEGRVVLDFRN

[0068] SE ID Nr. 4:

[0069] ATGTCCGGAAAAATGAAAGCCGCTGTGGTTCATGAATTTGGCAAACCACTGACCATTGAAGAACTGGACAT

[0070] TCCCACCATTAAACCCACCCAGATTTTGGTTAAAATGATTGCCTGCGGCGTGTGCCATACAGATTTGCATGC

[0071] TGCCAGCGGGGATTGGCCCAAAACCACATCTGCCGTTTATTCCCGGCCACGAAGGCGTTGGCACGGTTG TGCAGGTTGGCAGCGAAGTAGACTGGGTAAAGGAAGGCGACGTTGTTGGCGTGCCTTGGCTCTATTCTGC

[0072] CTGCGGCCATTGCGAGCATTGTCTGGCTGGGTGGGAAACCCTTTGTGCGAAGCAAGAAAACCGGCTAT

[0073] TCCGTAAACGGCTGCTTTGCCGAATACGTGGTAGCAGACCCTAACTACATTGCTCATCTTCCCAAAGGCGTA

[0074] GACCCTGTAAAAGTTGCCCCCGTACTGTGCGCAGGCCTGACCGTTTATAAGGGCCTGAAAATGACAGACAC

[0075] GCGGGCAGGGAACTGGGTTGCCATTTCCGGCGTTGGGGGGTTAGGCCAGATGGCTGTGCAGTACGCCGT

[0076] AGCAATGGGGCTGAATGTTGTGGCGGTAGATATTGATGAAAAACTGGCAACCGCCAAAAAGCTTGGT

[0077] GCAACGTACACTGTTAACGCCAGAAATACAGATCCGGCAGCATTTATGCAGGAAAAAGTTGGTGGCGTAC

[0078] ACGGTGGATTGATCACCGCTGTTTCCACCAAAGCATTTTCTCAGGCTATGGGTTATGTGCGGGCAGGGGGC

[0079] ACTTTGGTGCTAAATGGGCTGCCGCCGGGTGATTTCCCAATTTCTATCTTTGACGTGGTCATGAATGCCATT

[0080] ACCATTCGCGGCTCCATAGTAGGCACACGGCTGGACATGATTGAAGCTCTTTCCTTCTTTGCGGAAGGGAA

[0081] GGTAACATCTGTCACCACAACGGATCGGATTGATAACATCAACGCAATTTTTGATGCGCTCAAGAACGGTC

[0082] GGGTGGAAGGCCGCGTGGTTCTGGACTTCCGCAACTGA

[0083] SE ID Nr. 5:

[0084] MNK™AVARAFGKPLEIEVEVPRPRAGELLVKIEACGVCHTDLHAVEGDWPVKPNPPFIPGHEGVGHVV

[0085] AVGEGVTHVKEGDRVGIPWLYSACGHCEHCLGGWETLCEQQNAGYSVNGGFAEYALAAADYVGLLPKNVG

[0086] FVDIAPVLCAGVTVYKGLKMTDTRPGNWVVVSGIGGLGHMAVQYARAMGLNVAAVDIDDDKLDFAKRLGAE

[0087] VVVNAKATDPAAYLKKEIGGAHGALITAVSPKAFEQALGMVRRGGTVALNGLPPGDFPLSIFDMVLNGVTVRG

[0088] SIVGSRLDLQESLQFAEEGKVRATVATEKLENINSVFDRMRRGQIEGRIVLDMAA

[0089] SEQ ID Nr. 6:

[0090] ATGAACAAGACCATGAAGGCCGCGGTGGCCCGCGCGTTCGGCAAACCCCTGGAAATCGAAGAAGTCGAG

[0091] GTGCCGCGCCCGCGCGCGGGCGAACTGTTGGTGAAGATCGAGGCCTGCGGCGTCTGCCACACCGATCTGC

[0092] ACGCGGTCGAGGGCGACTGGCCGGTCAAGCCCAATCCCCCTTTCATCCCGGGCCACGAAGGCGTGGGCCA

[0093] CGTGGTGGCGGTGGGCGAAGGCGTGACGCACGTCAAGGAAGGCGACCGCGTCGGCATCCCCTGGCTGTA

[0094] TTCCGCCTGTGGCCATTGCGAGCATTGCCTGGGCGGCTGGGAGACGCTGTGCGAACAGCAGCAGAACGCC

[0095] GGCTACTCCGTCAACGGCGGCTTCGCCGAGTACGCGCTGGCCGCGGCCGACTACGTGGGCCTGCTGCCGA

[0096] AGAATGTCGGCTTCGTCGACATCGCCCCGGTACTGTGCGCCGGCGTCACGGTCTACAAAGGCCTGAAGAT

[0097] GACGGACACACGGCCAGGCAACTGGGTCGTGGTCTCCGGCATCGGCGGCCTGGGCCACATGGCGGTGCA

[0098] GTATGCGCGCGCCATGGGCTTGAACGTGGCGGCCGTGGACATCGACGACGACAAACTGGATTTCGCCAAG

[0099] CGTCTCGGCGCCGAGGTGGTCGTGAATGCCAAGGCCACCGATCCAGCGGCTTACCTGAAGAAGGAAATCG

[0100] GCGGCGCCCACGGCGCGCTGATCACGGCGGTTTCGCCCAAGGCCTTCGAGCAGGCGCTGGGCATGGTGC

[0101] GTCGCGGCGGCACGGTGGCGCTCAACGGCCTGCCGCCGGGCGACTTCCCGCTGTCGATCTTCGACATGGT

[0102] GCTCAACGGCGTGACGGTGCGCGGTTCGATCGTGGGTTCGCGCCTGGACTTGCAGGAATCGCTGCAGTTC GCCGAGGAAGGCAAGGTGCGCGCGACCGTGGCGACCGAGAAGCTGGAGAACATCAACAGCGTGTTCGAC CGCATGCGCCGCGGCCAGATCGAAGGCCGCATCGTGCTGGACATGGCGGCGTAA

[0103] SEQ ID Nr. 7:

[0104] MSTTVNTPIKLNSGYEMPLVGFGCWKVTNATAADQIYNAIKTGYRLFDGAEDYGNEKEVGEGINRAIKDGLVK REELFITSKLWNNFHDPKNVETALNKTLSDLNLDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGDNFHYEDVPLLD TWKALEKLVEAGKIKSIGISNFTGALIYDLIRGATIKPAVLQIEHHPYLQQPKLIEYVQKAGIAITGYSSFGPQSFLEL ESKRALNTPTLFEHETIKSIADKHGKSPAQVLLRWATQRNIAVIPKSNNPERLAQNLSVVDFDLTKDDLDNIAKLD IGLRFNDPWDWDNIPIFV

[0105] SEQ ID No. 8:

[0106] ATGAGCACGACCGTTAATACTCCGACGATCAAACTGAACTCCGGTTACGAGATGCCGCTGGTTGGCTTTGG CTGTTGGAAAGTTACCAATGCGACGGCGGCGGACCAGATTTATAACGCTATTAAAACCGGTTACCGTCTGT TCGATGGCGCAGAGGACTACGGTAACGAGAAAGAAGTGGGTGAAGGTATTAATCGCGCAATTAAAGACG GTCTGGTCAAACGTGAAGAGCTGTTTATTACCTCGAAACTGTGGAATAACTTCCATGATCCTAAGAACGTG GAGACTGCGCTGAATAAGACCCTGAGCGATCTGAACCTGGACTATGTGGATCTGTTTCTGATTCACTTCCC GATTGCTTTCAAATTTGTCCCGATCGAAGAAAAGTACCCACCGGGCTTCTACTGCGGTGACGGCGACAACT TCCATTATGAGGATGTTCCGCTGCTGGACACGTGGAAGGCGCTGGAGAAATTGGTGGAAGCCGGTAAGAT CAAGTCCATTGGCATTAGCAACTTCACCGGTGCCTTGATTTACGATTTGATCCGTGGTGCGACCATTAAACC GGCGGTTCTGCAGATCGAGCATCACCCGTATCTGCAACAGCCGAAACTGATCGAATACGTTCAGAAAGCA GGTATCGCCATCACTGGCTATAGCAGCTTCGGTCCACAGAGCTTCCTGGAGCTGGAGAGCAAGCGTGCCCT GAATACCCCGACGTTGTTTGAACACGAAACCATCAAGTCTATCGCTGACAAACACGGTAAGAGCCCTGCAC AAGTCCTGCTGCGCTGGGCAACGCAACGTAATATTGCGGTTATTCCGAAGAGCAATAACCCGGAGCGTCTG GCGCAGAATCTGTCTGTCGTCGACTTTGATTTGACCAAGGATGACCTGGATAATATCGCGAAGCTGGACAT CGGCCTGCGCTTCAACGATCCGTGGGATTGGGACAACATCCCGATCTTTGTGTA

[0107] The NAD(P)H-dependent alcohol dehydrogenase for the reduction of glycolaldehyde to ethylene glycol preferably comprises or consists of an amino acid sequence which has an identity to SEQ ID No. 1, 3 or 5 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the NAD(P)H-dependent alcohol dehydrogenase according to the invention for the reduction of glycolaldehyde to ethylene glycol comprises or consists of the amino acid sequence SEQ ID No. 1, 3 or 5. Alternatively, the NAD(P)H-dependent alcohol dehydrogenase for the reduction of glycolaldehyde to ethylene glycol preferably comprises or consists of an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No.2, 4 or 6 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the NAD(P)H-dependent alcohol dehydrogenase according to the invention for reducing glycolaldehyde to ethylene glycol comprises or consists of the nucleic acid sequence SEQ ID No. 2, 4 or 6.

[0108] The NAD(P)H-dependent xylose reductase for reducing glycolaldehyde to ethylene glycol preferably comprises or consists of an amino acid sequence having an identity to SEQ ID No. 7 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the NAD(P)H-dependent xylose reductase according to the invention for reducing glycolaldehyde to ethylene glycol comprises or consists of the amino acid sequence SEQ ID No. 7.

[0109] Alternatively, the NAD(P)H-dependent xylose reductase for reducing glycolaldehyde to ethylene glycol preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the NAD(P)H-dependent xylose reductase according to the invention for reducing glycolaldehyde to ethylene glycol comprises or consists of the nucleic acid sequence SEQ ID No. 8.

[0110] The term "identity," as used herein, refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps into the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.

[0111] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein. According to the invention, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI) is used to determine identity. The BLAST software suite includes various programs, including a tool called "BLAST 2 Sequences," which is used for the direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used interactively via the NCBI World Wide Web. The blastn program (for nucleotide sequences) uses a word length (W) of 28, an expectation (E) of 0.05, M = 1, N = -2, and a comparison of both strands.For amino acid sequences, the blastp program uses as defaults a word length of 3 and an expectation (E) of 0.05 and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 0.05, M = 1, N = - 2.

[0112] Alternatively, the NAD(P)H-dependent alcohol dehydrogenase for reducing glycolaldehyde to ethylene glycol preferably comprises or consists of an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 2, 4, or 6. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed.

[0113] For example, hybridization can be performed by conventionally known methods, such as those described by J. Sambrook et al. in "Molecular Cloning, A Laboratory Manual," 2nd Ed., Cold Spring Harbor Laboratory (1989). Stringent conditions refer to washing at 65 °C and a salt concentration of 0.1x to 2x SSC (where 1xSSC is a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0114] The enzymes used according to the invention for reducing glycolaldehyde to ethylene glycol are preferably NAD(P)H-dependent alcohol dehydrogenases and NAD(P)H-dependent xylose reductases. Surprisingly, it has been found that these enzymes are capable of converting glycolaldehyde to ethylene glycol in the presence of the cofactor NAD(P)H. Accordingly, a further aspect of the present invention relates to the use of these enzymes for reducing glycolaldehyde to ethylene glycol in the presence of the cofactor NAD(P)H.

[0115] Preferably, one aspect of the present invention relates to the use of an NAD(P)H-dependent alcohol dehydrogenase and / or an NAD(P)H-dependent xylose reductase for the reduction of glycolaldehyde to ethylene glycol, wherein the NAD(P)H-dependent alcohol dehydrogenase preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which ligates under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No.2, 4 or 6, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC, and the NAD(P)H-dependent xylose reductase preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 7 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0116] The following examples describe preferred embodiments of the invention in more detail.

[0117] Materials

[0118] Sodium pyruvate, sodium L-lactate and glycolaldehyde dimer, ethylene glycol and IPTG (isopropyl-ß-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, magnesium chloride hexahydrate, potassium dihydrogen phosphate, di-potassium hydrogen phosphate and sodium dodecyl sulfate (SDS) were purchased from Carl Roth, NAD + , NADH disodium salt, NADP + -Disodium salt, NADPH tetrasodium salt, and methanol were purchased from PanReac AppliChem (ITW Reagents) and triethanolamine was purchased from Chem-Lab NV.

[0119] Solutions of 2-keto-3-deoxy-L-arabonate (KDA) and 2-keto-3-deoxy-D-xylonate (KDX) were prepared enzymatically using L-arabonate dehydratase (Azotobacter vinelandii DJ; NCBI Protein Database Number: ACQ81022.1) from potassium L-arabonate and potassium D-xylonate, respectively (Weimberg, 1959), which were obtained by chemical oxidation of L-arabinose and D-xylose, respectively (Moore & Link, 1940).

[0120] Production of enzymes & preparation of lysates

[0121] General information on the expression of recombinant enzymes in E. coli

[0122] For recombinant enzyme production in an Escherichia coli strain, the gene to be expressed was first amplified by PCR using genomic DNA or its synthetic equivalent, synthetically adapted to the codon usage of E. coli, as a template, together with specific oligonucleotides additionally carrying recognition sequences for restriction endonucleases. The gene fragment encoding the target enzyme was isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the SphI and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. coli cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis.

[0123] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0124] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into the competent expression cells RB791. After 24 h of incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays.

[0125] The next day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37°C until an OD550 of 0.3 was reached. The temperature was then lowered to 25°C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and an activity determination (use in a USE test or optical enzymatic assay).

[0126] Preparation of cell lysates using sonifier disruption

[0127] To prepare a cell suspension, the cell pellet prepared according to the above procedure was weighed into a suitable container and mixed with buffer (e.g., triethanolamine HCl) and lysozyme (final concentration 0.5 mg / ml) and dissolved with stirring. The mass fraction of biomass is typically 20%, with the remainder being buffer.

[0128] A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3 - 5).

[0129] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0130] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples.

[0131] Analytical methods

[0132] High Performance Anion Exchange Chromatography (HPAEC)

[0133] Substrate conversions and product concentrations were determined by HPAEC (High Performance Anion Exchange Chromatography). A Dionex ICS6000 system with an AS-AP autosampler was used for this purpose. The organic acids and their anions (2-keto-3-deoxy-D-xylonate, 2-keto-3-deoxy-L-arabonate, lactate, and pyruvate) were measured using conductivity detection (CD) coupled to a Dionex AERS 500 electrolytically regenerated suppressor in external water mode. A Dionex IonPac AS11-HC-4pm column with a corresponding precolumn and a NaOH gradient was used to separate the analytes. The mobile phase was additionally pretreated with a Dionex ATC Anion Trap Column.

[0134] High Performance Liquid Chromatography (HPLC)

[0135] HPLC (high-performance liquid chromatography) was used to quantify glycolaldehyde and ethylene glycol. Detection is performed using a refractive index detector. A Phenomenex Rezex ROA-Organic Acid H+ (8%) column with an appropriate precolumn was used for the measurement and eluted isocratically with 1 mM sulfuric acid.

[0136] Determination of enzyme activities (optical-enzymatic assay)

[0137] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation and consumption of NAD(P)H were monitored at a wavelength of 340 nm via the change in absorbance. The measurements were performed with 0.2 mM cofactor (NAD(P) +or NAD(P)H). For this purpose, 20 μl of a 10 mM stock solution of the cofactor was placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH was adjusted with 100 mM TEA-HCl buffer (870 μl). 10 μl of lysate (diluted or undiluted) and 100 μl of substrate solution were added to the cuvette, and the measurement was started immediately. The measurements were carried out at 25 °C as standard. The extinction coefficient of NADH or NADPH at 340 nm (E = 6220 L mol 1 cm 1 ), the enzyme activity of the lysate can be determined in U / ml (based on the volume of the lysate) or U / g (based on the biomass used for production). 1 U represents 1 pmol of substrate turnover per minute (1 U = 1 pmol / min = 1.67-10' 8kat). The following examples describe preferred variants of the method according to the invention in more detail. The lysates used in these examples were prepared according to the methods described above.

[0138] Example 1

[0139] Reduction of glycolaldehyde to ethylene glycol

[0140] The following components were mixed in two 2 ml glass vials (batches 1 and 2): 100 μl of a 500 mM TEA-HCl buffer (pH 7), 230 μl of deionized water, 100 μl of a 50 g / l glycolaldehyde solution (final concentration 10 g / l, 167 mM), 10 μl of alcohol dehydrogenase lysate (batch 1: ADH I; batch 2: ADH II), 10 μl of a 10 mM NAD + -solution (0.2 mM final concentration) and 50 pl 2-propanol.

[0141] In three additional 2 ml glass vials (batches 3 and 4), the following components were mixed: 100 μl of a 500 mM TEA-HCl buffer (pH 7), 123 μl of deionized water, 100 μl of a 50 g / l glycolaldehyde solution (final concentration 10 g / l, 167 mM), 10 μl of alcohol dehydrogenase lysate (batch 3: ADH I; batch 4: ADH II) and 167 μl of a 500 mM NADH solution (167 mM final concentration).

[0142] The following components were mixed in a 2 ml glass vial (preparation 5): 100 μl of a 500 mM TEA-HCl buffer (pH 7), 225 μl of deionized water, 100 μl of a 50 g / l glycolaldehyde solution (final concentration 10 g / l, 167 mM), 10 μl of xylose reductase lysate, 10 μl of alcohol dehydrogenase IV lysate and 5 μl of a 5 mM NADP + -solution (0.05 mM final concentration).

[0143] The following components were mixed in a 2 ml glass vial (preparation 6): 100 μl of a 500 mM TEA-HCl buffer (pH 7), 123 μl of deionized water, 100 μl of a 50 g / l glycolaldehyde solution (final concentration 10 g / l, 167 mM), 10 μl of xylose reductase lysate and 167 μl of a 500 mM NADPH solution (167 mM final concentration).

[0144] The mixtures were incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer).

[0145] For processing, 200 μl of a sample was mixed with 200 μl MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at max. 1 g for 10 min. 150 μl of the clear supernatant was transferred to HPLC vials and analyzed (RI detection). The results are shown in Table 2 below.

[0146] Table 2

[0147] The table shows that cofactor regeneration leads to higher yields of ethylene glycol compared to approaches with equimolar cofactor concentration.

[0148] Example 2

[0149] Conversion of a 9 / 1 mixture of 2-keto-3-deoxy-D-xylonate (KDX) and 2-keto-3-deoxy-L-arabonate (KDA) to ethylene glycol and lactate

[0150] The following components were mixed in a 2 ml glass vial (preparation 1): 50 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 96 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 2 U of alcohol dehydrogenase I lysate, 2 U of lactate dehydrogenase lysate, 5 μl of a 10 mM NAD + -solution and 50 pl 2-propanol.

[0151] The following components were mixed in a 2 ml glass vial (preparation 2): 50 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 111 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 2 U of alcohol dehydrogenase II lysate, 2 U of lactate dehydrogenase lysate, 5 μl of a 10 mM NAD + -solution and 50 pl 2-propanol.

[0152] The following components were mixed in a 2 ml glass vial (preparation 3): 50 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 111 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 2 U of alcohol dehydrogenase III lysate, 2 U of lactate dehydrogenase lysate, 5 μl of a 10 mM NAD + -solution and 50 pl 2-propanol.

[0153] The mixtures were incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). For processing, 100 μl of a mixture was mixed with 100 μl of ultrapure water and 200 μl of MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged for 10 min at max. 1 g. The clear supernatant was diluted 1:250 and analyzed by HPAEC (CD). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial and analyzed (RI detection). The results are shown in Table 3 below.

[0154] Table 3

[0155] In all approaches, the substrate (KDX & KDA) was fully converted and similar concentrations of ethylene glycol and lactate were achieved.

[0156] Example 3

[0157] Conversion of a 9 / 1 mixture of 2-keto-3-deoxy-D-xylonate (KDX) and 2-keto-3-deoxy-L-arabonate (KDA) to ethylene glycol and pyruvate

[0158] The following components were mixed in three 2 ml glass vials (batches 1 to 3): 50 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 120 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 20 μl of alcohol dehydrogenase lysate (batch 1: ADH I; batch 2: ADH II; batch 3: ADH III) and 50 μl of 2-propanol.

[0159] The mixtures were incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer).

[0160] For processing, 100 μl of a sample was mixed with 100 μl of ultrapure water and 200 μl of MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:250 and analyzed using HPAEC (CD). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial and analyzed (RI detection). The results are presented in Table 4 below.

[0161] Table 4

[0162] In all approaches 1 to 3, the substrate (KDX & KDA) was fully converted and similar concentrations of ethylene glycol and pyruvate were achieved.

[0163] Example 4

[0164] Conversion of a 9 / 1 mixture of 2-keto-3-deoxy-D-xylonate (KDX) and 2-keto-3-deoxy-L-arabonate (KDA) to ethylene glycol and pyruvate

[0165] The following components were mixed in a 2 ml glass vial (preparation 1): 50 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 175 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 10 μl of alcohol dehydrogenase IV lysate, 5 μl of a 5 mM NADP + -solution and 50 pl 2-propanol.

[0166] The following components were mixed in a 2 ml glass vial (preparation 2): 50 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 165 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 10 μl of xylose reductase lysate, 10 μl of alcohol dehydrogenase IV lysate, 5 μl of a 5 mM NADP + -solution and 50 pl 2-propanol.

[0167] The mixtures were incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer).

[0168] For processing, 100 μl of a sample was mixed with 100 μl of ultrapure water and 200 μl of MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:250 and analyzed using HPAEC (CD). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial and analyzed (RI detection). The results are presented in Table 5 below. Table 5

[0169] Alcohol dehydrogenase IV also catalyzes the reduction of glycolaldehyde to ethylene glycol to a certain extent, but only 6 mM ethylene glycol can be detected in batch 1. Addition of xylose reductase (batch 2) improves the product concentration to 70 mM. Due to the shift in the equilibrium of the retroaldol reaction toward the cleavage products, more pyruvate is also produced (67 mM vs. 8 mM).

[0170] Example 5

[0171] Conversion of a 9 / 1 mixture of 2-keto-3-deoxy-D-xylonate (KDX) and 2-keto-3-deoxy-L-arabonate (KDA) to ethylene glycol and pyruvate or lactate - using an NAD(P)-dependent ADH

[0172] The following components were mixed in a 2 ml glass vial (preparation 1): 100 μl of a 1000 mM TEA-HCl buffer (pH 8.5), 115 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 10 μl of xylose reductase lysate, 10 μl of alcohol dehydrogenase V lysate, 5 μl of a 5 mM NAD(P) + -solution and 50 pl 2-propanol.

[0173] The following components were mixed in a 2 ml glass vial (preparation 2): 100 μl of a 1000 mM TEA-HCl buffer (pH 8.5), 100 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 10 μl of xylose reductase lysate, 10 μl of alcohol dehydrogenase V lysate, 2 U of lactate dehydrogenase lysate, 5 μl of a 5 mM NADP + -solution, 5 μl of 10 mM NAD +-solution and 50 pl 2-propanol.

[0174] The following components were mixed in a 2 ml glass vial (preparation 3): 100 μl of a 1000 mM TEA-HCl buffer (pH 8.5), 125 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 10 μl of alcohol dehydrogenase V lysate, 5 μl of a 5 mM NADP + -solution and 50 pl 2-propanol.

[0175] The following components were mixed in a 2 ml glass vial (preparation 4): 100 μl of a 1000 mM TEA-HCl buffer (pH 8.5), 110 μl of deionized water, 200 μl of a solution of KDX / KDA (9 / 1 m / m), 10 μl of aldolase lysate, 10 μl of alcohol dehydrogenase V lysate, 2 U of lactate dehydrogenase lysate, 5 μl of a 5 mM NADP + -solution, 5 μl of 10 mM NAD + -solution and 50 pl 2-propanol.

[0176] The samples were incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). For processing, 100 μl of a sample was mixed with 100 μl of ultrapure water and 200 μl of MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged for 10 min at max. 1 g. The clear supernatant was diluted 1:250 and analyzed by HPAEC (CD). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial and analyzed (RI detection). The results are shown in Table 6 below.

[0177] Table 6

[0178] Table 6 shows that NAD(P)-dependent alcohol dehydrogenase V regenerates both NADPH for ethylene glycol reduction by xylose reductase and NADH for pyruvate reduction by lactate dehydrogenase. The highest product concentrations were achieved in approaches 1 (ADH V + XR) and 2 (ADH V + XR + LacDH), whereas the use of ADH V alone and in combination with LacDH resulted in only 6 mM ethylene glycol and 14 mM pyruvate or lactate, respectively.

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing ethylene glycol and a salt of pyruvic acid by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase, whereby an aqueous solution is obtained which contains glycolaldehyde and a salt of pyruvic acid, after which the glycolaldehyde is reduced to ethylene glycol by treatment with an NAD(P)H-dependent alcohol dehydrogenase or with an NAD(P)H-dependent xylose reductase in vitro, wherein the oxidized cofactor NAD(P) formed by the reduction with the alcohol dehydrogenase or together with the xylose reductase + is reduced by means of a secondary alcohol to form a ketone and then, if necessary, either the pyruvic acid salt or the ethylene glycol is removed from the solution.

2. A process for the preparation of an aqueous solution containing ethylene glycol and a salt of lactic acid by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase, whereby an aqueous solution is obtained which contains glycolaldehyde and a salt of pyruvic acid, after which the glycolaldehyde is reduced in vitro by treatment with an NAD(P)H-dependent alcohol dehydrogenase or with an NAD(P)H-dependent xylose reductase to ethylene glycol and the salt of pyruvic acid is reduced in vitro by means of a lactate dehydrogenase to a salt of lactic acid, wherein the oxidized cofactor NAD(P) formed by the reduction with the alcohol dehydrogenase or together with the xylose reductase + is reduced by means of a secondary alcohol to form a ketone and then, if necessary, either the lactic acid salt or the ethylene glycol is removed from the solution.

3. A process for the preparation of an aqueous solution containing ethylene glycol, in which glycolaldehyde, which is dissolved in an aqueous solution, is reduced to ethylene glycol by treatment with an NAD(P)H-dependent alcohol dehydrogenase or together with an NAD(P)H-dependent xylose reductase in vitro and the oxidized cofactor NAD(P) formed by the reduction + is reduced by means of a secondary alcohol to form a ketone.

4. Process according to one of claims 1 to 3, characterized in that the secondary alcohol is 2-propanol.

5. Method according to one of claims 1 to 4, characterized in that the enzymes are present as a lysate of the corresponding cells producing them.

6. The method according to any one of claims 1 to 5, characterized in that the NAD(P)H-dependent alcohol dehydrogenase for the reduction of glycolaldehyde to ethylene glycol comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6.

7. The method according to any one of claims 1 to 6, characterized in that the NAD(P)H-dependent xylose reductase for the reduction of glycolaldehyde to ethylene glycol comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 7 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

8.

8. Use of an NAD(P)H-dependent alcohol dehydrogenase and / or an NAD(P)H-dependent xylose reductase for the reduction of glycolaldehyde to ethylene glycol, wherein the NAD(P)H-dependent alcohol dehydrogenase preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6, and the NAD(P)H-dependent xylose reductase preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 7 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid that has an identity to SEQ ID No. 8 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8

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