Biocatalytic synthesis of esters

Abstract

The present invention relates to a biocatalytic synthesis of esters that can also be implemented on an industrial scale. In particular, the invention relates to a process for the production of esters, via a hemiacetal intermediate, consisting of the following steps: – a step of bringing into contact, in a reaction medium, • a mixture of a C1– Cn alcohol and a C1– Cn aldehyde with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD(P)+ and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde, or alternatively • a C1– Cn alcohol with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD(P)+ and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal, the starting alcohol, and the formed aldehyde, or alternatively • a C1– Cn aldehyde with an enzymatic catalytic system comprising a reduced cofactor selected from NAD(P)H or NADH and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the starting aldehyde and the formed hemiacetal, where n may be any number, and – a step of isolating the corresponding ester.

Description

[0001] BIOCATALYTIC SYNTHESIS OF ESTERS

[0002] DESCRIPTION FIELD OF THE INVENTION

[0003] The present invention relates to a biocatalytic synthesis of esters that can also be implemented on an industrial scale.

[0004] BACKGROUND OF THE INVENTION

[0005] Esters are among the most important chemical products produced on a large scale (that is, millions of tons ) every year and are used as solvents, fragrances or intermediates in numerous industrial applications worldwide. Esters are generally low-toxicity molecules with a good environmental profile, but their current production is not sustainable, as the processes use energy-intensive procedures and raw materials of fossil origin. Fischer-Speier esterification is the most common of these procedures, involving condensation between a carboxylic acid and an alcohol molecule with the aid of an acid catalyst, high temperature, often high pressure, and stoichiometric release of water. In particular, esterifications are reversible reactions governed by an equilibrium that prevents the complete conversion of reagents into products. Moreover, the water produced is often responsible for catalyst deterioration and, for this reason, energy-intensive procedures are required to remove water from the reaction mixture with the aim of driving the reaction toward the desired product. Other traditional pathways for the production of specific esters are known and widely used in industrial practice, such as the Tishchenko reaction, ethanol dehydrogenation for the synthesis of ethyl acetate, or liquid-phase carbonylation of methanol (MeOH) to produce methyl formate. In any case, the use of corrosive catalysts and high-temperature, high-pressure, and energy-intensive product-recovery procedures are common to all. For these reasons, there is an urgent need for environmentally friendly and low-energy procedures for ester synthesis.

[0006] SUMMARY OF THE INVENTION

[0007] The above-mentioned purpose has been achieved by means of the process of the present invention.

[0008] The invention described below concerns a process for the production of esters occurring under mild temperature and pressure conditions (or close to what can be defined as normal conditions ). The process proceeds with kinetics not previously described, which enables saving energy and raw materials and therefore potentially reducing costs.

[0009] The invention relates to a process for the production of an ester starting from an alcohol and an aldehyde, or alternatively only from an alcohol, or alternatively only from an aldehyde, using one or more polypeptides having hemiacetal-, aldehyde- and alcohol-dehydrogenase activity.

[0010] Enzymatic dehydrogenation of hemiacetal is not new to the scientific community, but so far only a few anecdotal reports and research articles exist on this phenomenon. The maximum titers of esters produced using this strategy are always low or, even when appreciable. They are obtained in vivo after days of cell culture. The vast majority of articles focus on discovering new alcohol dehydrogenases (ADHs ) with hemiacetal-dehydrogenase activity and on the expression of such enzymes in microbial hosts to better understand the metabolism of methylotrophic microorganisms or to find the mechanisms of their resistance to formaldehyde or other aldehydes or, alternatively, to understand the mechanisms of ester formation in wild microorganisms such as yeasts and bacteria. In a general embodiment, an object of the present invention is therefore a process for the production of esters, via a hemiacetal intermediate, consisting of the following steps:

[0011] - a step of contacting, in a reaction medium,

[0012] a mixture of a Ci-Cnalcohol and a Ci- Cnaldehyde with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD (P) + and NAD+ and a polypeptide from the group of oxidoreductases having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde, or alternatively

[0013] • a Ci- Cnalcohol with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD ( P) + and NAD+ and a polypeptide from the group of oxidoreductases having preferential dehydrogenase activity toward the formed hemiacetal, the starting alcohol and the formed aldehyde, or alternatively

[0014] • a Ci- Cnaldehyde with an enzymatic catalytic system comprising a reduced cofactor selected from NAD (P) H or NADH and a polypeptide from the group of oxidoreductases having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde,

[0015] wherein n may be any integer, and

[0016] - a step of isolating the corresponding ester.

[0017] The process in question leads to unexpected ester production yields, thanks to an enzymatic cofactor-regeneration pathway autonomously carried out by the same polypeptide responsible for the oxidation of the hemiacetal starting from the aldehyde, and therefore also without addition of one or more sacrificial substrates.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] Figures 1A, 1B and 1C show three different synthetic pathways for the production of esters according to the process of the invention: Figure 1A - synthesis of an ester starting from an alcohol and an aldehyde, in which a) is the spontaneous formation of the hemiacetal, b) is the oxidation of the hemiacetal to the corresponding ester and c) is the reaction of NAD ( P)+regeneration in which the aldehyde is reduced to the corresponding alcohol

[0020] Figure 1B - synthesis of the ester starting only from an alcohol, in which a) is the oxidation of the alcohol to the corresponding aldehyde, b) is the spontaneous formation of the hemiacetal and c) is the oxidation of the hemiacetal to the corresponding ester

[0021] Figure 1C - synthesis of the ester starting only from an aldehyde, in which a) is the reduction of the aldehyde to the corresponding alcohol and the reaction responsible for the generation and regeneration of NAD ( P)+, b) is the spontaneous formation of the hemiacetal and c) is the oxidation of the hemiacetal to the corresponding ester;

[0022] Figure 2 shows the calibration curve for the quantification of methyl formate.

[0023] Figure 3 shows the kinetics of methyl formate formation through the process of the invention with the enzyme Adhl of S. cerevisiae.

[0024] Figure 4 represents a graph showing the decrease in NADH absorbance at 340 nm during the reaction of formaldehyde reduction catalyzed by Adhl of S. cerevisiae.

[0025] Figure 5 represents a graph showing the results of the experiment of methyl formate synthesis by Adhl of S. cerevisiae with and without the addition of formaldehyde - left columns: without addition; right columns: with addition.

[0026] Figure 6 represents a graph showing the results of the experiment of methyl formate synthesis catalyzed by Adhl of S. cerevisiae starting from methanol or from formaldehyde - left columns: from methanol; right columns: from formaldehyde. Figure 7 represents a graph showing the titer of ethyl acetate over time with (right columns ) and without ( left columns ) the addition of acetaldehyde using the enzyme HS_N (ADH7 of Homo sapiens).

[0027] Figure 8 represents a graph showing the titer of ethyl formate over time with (right columns ) and without ( left columns ) the addition of formaldehyde using the enzyme HS_N (ADH7 of Homo sapiens).

[0028] Figure 9 represents a graph showing the titer of ethyl acetate over time with (right columns ) and without ( left columns ) the addition of acetaldehyde using the enzyme CB_N (a NADP+dependent isopropanol dehydrogenase of C. beijerinckii ).

[0029] Figure 10 represents a graph showing the titer of ethyl formate over time with (right columns ) and without (left columns ) the addition of formaldehyde using the enzyme CB_N (a NADP+dependent isopropanol dehydrogenase of C. beijerinckii ).

[0030] Figure 11 represents the FT-IR spectrum ( in transmittance) of 3 different samples of microfibrillated cellulose: the first curve from the top represents untreated cellulose, the second curve from the top represents cellulose with addition of formaldehyde, the third curve represents cellulose treated with the addition of formaldehyde, the ADH1 enzyme and the NAD+.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] An obj ect of the present invention is a process for the production of esters, via a hemiacetal intermediate, consisting of the following steps:

[0033] - a step of contacting, in a reaction medium,

[0034] a mixture of a Ci - Cnalcohol and a Ci - Cnaldehyde with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD ( P) + and NAD+ and a polypeptide from the group of oxidoreductases having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde, or alternatively

[0035] • a Ci - Cnalcohol with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD (P) + and NAD+ and a polypeptide from the group of oxidoreductases having preferential dehydrogenase activity toward the formed hemiacetal, the starting alcohol and the formed aldehyde, or alternatively

[0036] • a Ci- Cnaldehyde with an enzymatic catalytic system comprising a reduced cofactor selected from NAD (P) H or NADH and a polypeptide from the group of oxidoreductases having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde,

[0037] wherein n may be any integer, and

[0038] - a step of isolating the corresponding ester.

[0039] The term "preferential dehydrogenase activity" refers to the capability of an enzyme to transform one substrate more rapidly and efficiently and therefore preferentially with respect to another. This means that, within a unit of time, more molecules of the preferential substrate will be transformed compared to the molecules of the nonpref erential substrate. The kinetic parameters of an enzyme, such as Kmand Kcat, can be used to quantify the affinity and the rate of transformation of an enzyme toward a given substrate and therefore are useful to establish the preferential nature of the catalytic action of an enzyme toward different substrates. In essence, if two different substrates are present, it is possible to determine which one will be transformed preferentially by analysing or measuring Kmand Kcatof an enzyme for the substrates under study. A substrate can therefore be considered preferential with respect to another for a given enzyme when it has a lower Kmand a higher Kcatcompared to those of the other substrate. The process in question leads to unexpected ester production yields, higher than those calculated stoichiometrically (from 2 to 6 times or, in some examples, even 27 times or more ), thanks to a pathway of enzymatic cofactor regeneration autonomously carried out by the same polypeptide responsible for the oxidation of the hemiacetal starting from the aldehyde, and therefore also without the addition of one or more sacrificial substrates.

[0040] Hemiacetal dehydrogenases, strictly speaking, are enzymes that are not described as such, since the dehydrogenation of the hemiacetal is a collateral activity of certain alcohol dehydrogenases, hereinafter referred to as ADHs. The purpose of the present invention is to provide a process for the production of esters using a polypeptide having preferential dehydrogenase activity toward a hemiacetal and an aldehyde, alternatively in some embodiments also toward an alcohol, to convert:

[0041] • a mixture of a Ci - Cnalcohol and a Ci - Cnaldehyde, or alternatively

[0042] • a Ci-Cn alcohol, or alternatively

[0043] • a Ci-Cn aldehyde,

[0044] into the corresponding ester via a hemiacetal intermediate, with yields suitable for industrial scale. Ci - Cnindicates the number of carbon atoms present in the molecule (alcohols or aldehydes ), where n may be any integer.

[0045] Both alcohols and aldehydes may comprise linear or branched compounds and may be liquids, such as lipids containing short- and long-chain alcoholic or aldehydic functions, or even solids such as waxes, or be contained as functional groups in larger compounds such as the alcoholic groups of cellulose. If the starting raw material is an alcohol, or an aldehyde, the process will comprise, respectively, first the partial oxidation of the alcohol to give the corresponding aldehyde, or the partial reduction of the aldehyde to the corresponding alcohol, then the formation of the hemiacetal intermediate and finally the oxidation of the hemiacetal intermediate to obtain the corresponding ester. In this case, only symmetric esters can be formed.

[0046] If instead the starting raw materials are alcohols and aldehydes in a mixture, the process will first comprise the formation of the hemiacetal intermediate and then the oxidation of that intermediate to give the corresponding ester, using an appropriate polypeptide that simultaneously and preferentially has hemiacetal-dehydrogenase activity and aldehyde-dehydrogenase activity. In the latter case, asymmetric esters may also be produced, depending on the alcohol and aldehyde used as starting raw materials.

[0047] It is known in scientific literature that alcohols and aldehydes may undergo a spontaneous reaction to give the corresponding hemiacetal when mixed together in aqueous or non-aqueous media, whether they are the sole components of the mixture under study. Hemiacetals may then be oxidized to the corresponding esters using specific enzymes with hemiacetal-dehydrogenase activity. Such enzymes usually belong to the alcohol dehydrogenase (ADH) family.

[0048] It is also known that ADHs require a NAD ( P)+or NAD (P) H cofactor to catalyze their reactions; therefore, by selecting one or more specific ADHs, the relevant cofactor and one or more suitable substrates, it is possible to produce an ester of interest through one of the three alternative pathways described above ( see also Figures 1A, IB and 1C) and with yields suitable for industrial scale. It is likewise known that ADHs require the NAD ( P)+cofactor to catalyze the oxidation of a hemiacetal to an ester and that, therefore, the maximum titer of ester obtainable in such a reaction is equal to the molarity of NAD ( P)+used at the start, because for every mole of NAD ( P)+reduced to NAD (P) H only one mole of ester is obtained ( see again Figures 1A, IB and 1C for the reaction stoichiometry).

[0049] The process of the present invention, instead, makes it possible to obtain an amount of ester higher than the molarity of NAD (P)+used at the start, in a short period of time (ranging from one to a few hours ), thanks to a kinetics not previously described: an autonomous pathway for NAD ( P)+regeneration, which in some specific examples leads to an ester yield 2 to 7 times higher (even 27 times higher in one example) than that theoretically predicted stoichiometrically. This excess always occurs, even without further additions of the starting aldehyde (or other sacrificial substrates ), and is caused by the kinetic characteristics of the enzyme ( in particular Kmand Kcat) selected with respect to the substrates used (alcohol and aldehyde ). These kinetic characteristics of the polypeptide, with hemiacetal and aldehyde dehydrogenase activity, allow the formation of an alcohol (the starting alcohol of the reaction) from the starting aldehyde using the NAD ( P) H formed following the initial oxidation of the hemiacetal, and therefore the generation of new NAD (P)+. This NAD ( P)+in turn can be reused to oxidize the hemiacetal again, generating a virtuous cycle that leads to the formation of more ester than is theoretically possible, without the addition of other external systems for the regeneration of the enzymatic cofactor or any sacrificial substrate.

[0050] Nonetheless, in some preferred embodiments of the invention, the reaction may also be carried out by adding an excess of the starting aldehyde, in order to further maximize the amount of ester produced, with respect to the initially used amount, at least up to 10%, or at least up to 50%, or at least up to 100%, or at least up to 200%, or at least up to 300%, more preferably at least up to 400%, still more preferably at least up to 500%, still more preferably at least up to 600% or at least up to 700%. In certain embodiments, it is possible to operate with an excess of aldehyde of at least 10%, or at least 30%, or at least 50%, or at least 100%, without any limitation on the upper quantity. Such additions may be carried out n times (where n is any integer) at individual moments or continuously through an electronically controlled feeding system (preferably for the purpose of maintaining the aldehyde concentration constant in the reaction medium over time).

[0051] It is known that ADHs are capable of reducing aldehydes to alcohols by oxidizing NAD (P) H to NAD (P)+. This pathway is therefore usable according to the invention for the regeneration of NAD ( P)+and allows the formation of a greater quantity of ester than theoretically possible without introducing any external cofactor-regeneration system. Clearly, the same process also regenerates one of the starting raw materials, i. e. the alcohol. In this way, and by selecting the polypeptide with the appropriate kinetic characteristics toward the starting substrates and the desired product, it is possible to increase the ester production titer and simultaneously regenerate two of the starting raw materials (the alcohol and NAD (P)+).

[0052] It is widely known in the literature that cofactor regeneration is one of the main problems to be solved when dealing with optimization and scale-up of certain enzymatic reactions. The stoichiometric addition of complex cofactors to reaction media, the coupling of another enzymatic reaction or the addition of a sacrificial substrate, different from the starting aldehyde, to regenerate the cofactor required for the reaction of interest may enormously increase production costs on a large scale and thus prevent the commercialization of an environmentally friendly process. The invention solves this problem and consequently enables significant savings in energy and raw materials.

[0053] The desired substrates (an alcohol, or alternatively an aldehyde, or alternatively both an alcohol and an aldehyde simultaneously), the selected enzymatic cofactor (NAD (P)+or NAD+or alternatively NAD ( P) H or NADH, depending on the starting substrate or substrates and the enzymes used), and the selected enzyme or enzymes are mixed at an appropriate concentration. It is also possible to prepare beforehand different solutions respectively containing an appropriate concentration of the various aforementioned components and combine said solutions at the time of the reaction.

[0054] The reaction medium may be aqueous or may comprise a solvent that does not interfere with the reaction, in particular one that does not inactivate and / or denature the selected enzymes.

[0055] If water is used as the reaction medium, the process is carried out under acidic or alkaline conditions to improve the formation of the hemiacetal. Preferably, an alkaline condition is used, more preferably one with a pH appropriate to optimize the functionality of the enzymes and the formation of the hemiacetal at the same time. The optimal pH may vary depending on the enzyme or enzymes used and the specific hemiacetal of interest. In general, the optimal pH is between 2 and 10, preferably between 4 and 9, more preferably around 8. For example, in specific embodiments of the present invention the pH is 8.4.

[0056] As mentioned, the solvent may be water or any other organic solvent (es. DMSO-dimethyl sulfoxide ), or alternatively a mixture thereof, in which the selected enzyme or enzymes retain at least partially their activities and in which the formation of the hemiacetal can occur. If the starting compound is only an alcohol, the selected enzyme or enzymes first catalyze the transformation of part of that alcohol, at least 1%, preferably between 1% and 99%, more preferably between 1% and 50%, into the corresponding aldehyde. If the starting compound is only an aldehyde, the selected enzyme or enzymes first catalyze the transformation of part of that aldehyde, at least 1%, preferably between 1% and 99%, more preferably between 1% and 50%, into the corresponding alcohol. Subsequently, in both cases, the second reaction that will occur spontaneously and simultaneously with the first is the formation of the hemiacetal from the alcohol and aldehyde thus formed.

[0057] If instead an alcohol and an aldehyde are used simultaneously as starting substrates, the formation of the hemiacetal will be the first reaction that takes place in the reaction medium. Finally, in every scenario described so far, the oxidation of the hemiacetal into the corresponding ester will be catalyzed by the selected enzyme or enzymes with hemiacetal-dehydrogenase activity, and the regeneration of NAD (P)+will be catalyzed by the enzyme or enzymes having aldehyde-dehydrogenase activity. In specific and preferred embodiments of the present invention, the enzyme responsible for this second reaction is the same.

[0058] All reactions described so far are equilibrium reactions and may occur under standard conditions ( STP: 0 °C and 1 atm), mild conditions ( 25 °C and 1 atm) or even under extreme conditions ( for example high or low temperature and pressure ), but preferably mild conditions will be used, more preferably, in some embodiments of the present invention, at room temperature and atmospheric pressure ( for example 20°C and 1 atm), or even more preferably under conditions close to ambient ( 30 °C and 1 atm), depending on the optimal conditions for the activity of the enzyme or enzymes used. Preferably, the reaction is carried out under stirring to improve Brownian motion and accelerate the overall ester production process. Any means known to the skilled person may be used such as, but not limited to, mechanical stirring, bubbling of a gas, ultrasound irradiation, microwave irradiation, the use of microreactors or the use of flow reactors.

[0059] The process of the invention may be carried out in a single reactor or in sequential reactors for each step.

[0060] Specific concentrations, buffer type used and pH conditions are reported in the examples illustrated below, which are not to be considered as limiting the ways in which the invention may be carried out or the fields of application of the invention, but simply as practical demonstrations of what has been stated so far. The reaction conditions may be adapted by any person skilled in the art according to specific cases, more preferably depending on the esters intended to be produced.

[0061] According to the present disclosure, it has been found that the polypeptides that can be used in the process belong to the group of enzymes known as oxidoreductases. Oxidoreductases are a class of enzymes that catalyze the exchange of electrons between donor and acceptor molecules in reactions involving electron transfer, proton abstraction, hydride transfer, oxygen insertion and others. In general, the catalytic cycle of oxidoreductases includes two half-reactions: an oxidative one and a reductive one.

[0062] Useful for the present invention is a particular class of oxidoreductases, the so-called dehydrogenases. Dehydrogenases are enzymes that catalyze the oxidation of a substrate by transferring hydrogen to an acceptor molecule that is a nicotinamide adenine dinucleotide molecule or a nicotinamide adenine dinucleotide phosphate molecule (NAD+o NADP+). In the classification of the Enzyme Commission, dehydrogenases are classified as EC. 1. X. X. X, where X are integers and indicate, in order, the portions on which the enzyme acts ( for example 1 refers to CH-OH donors ), the type of cofactor used by the enzymes ( for example 1 refers to NAD (P)+and 2 refers to cytochromes as acceptors ) and the last number is a serial number relating to the specific reaction identity. It should be noted that the Enzyme Commission number is a numerical scheme for classifying enzymes based on the chemical reactions they catalyze. Therefore, EC numbers do not specify enzymes, but the reactions catalyzed by enzymes. If different enzymes ( for example from different organisms ) catalyze the same reaction, they receive the same EC number. Preferably, the enzyme is selected from the class of alcohol dehydrogenases of EC 1.1. l. X. Alcohol dehydrogenases (or ADHs ) catalyze the following reactions:

[0063] alcohol <-> aldehyde

[0064] NAD ( P )+- - NAD ( P ) H

[0065] The symbol

[0066]

[0067] indicates that the reactions are reversible. Therefore, ADHs are usually also capable of reducing aldehydes to the corresponding alcohols.

[0068] Preferably, the enzymes useful for the present invention are those ADHs (EC 1.1. l. X) that also have hemiacetal-dehydrogenase activity. It is known that hemiacetal-dehydrogenase activity is a collateral activity of certain ADHs. Typically, the three-dimensional structure of such polypeptides contains two domains in each subunit: one catalytic and one responsible for binding a cofactor (often NAD+o NAD ( P)+and their respective reduced forms ). Such polypeptides often have a conserved amino acid sequence at their active site: GHEX2GX5 (G, A) X2 ( I, V, A, C, S ).

[0069] The selected polypeptide that can be used in the process according to the present invention is chosen from "wild-type" polypeptides such as, but not limited to, Adhl from Saccharomyces cerevisiae (SEQ ID NO: 1 ), ADH7 from Homo sapiens ( SEQ ID NO: 2 ), Adhl from Neurospora crassa ( SEQ ID NO: 3 ) and an NADP+dependent isopropanol dehydrogenase from Clostridium beijerinckii ( SEQ ID NO: 4 ), or from engineered polypeptides designed to enhance their specificity toward a hemiacetal, alcohol or aldehyde of interest or to improve their stability under the reaction conditions. The selected ADH or ADHs may be used in any form known to the skilled person such as, but not limited to, solubilized in the reaction medium or in the form of whole cells expressing or overexpressing the selected ADHs, and both of the above forms may be used immobilized on a solid support to allow their reusability, resulting in increased overall sustainability and economic viability of the process.

[0070] Depending on the specific ester of interest and depending on the selected starting substrate or substrates, one or more different ADHs may be used as catalysts in sequential reactors, or a mixture thereof may be used in a single reactor.

[0071] Due to the degeneracy of the genetic code, there exists a finite but non-unique set of nucleotide sequences that can encode a given amino acid sequence. It is understood that all such equivalent sequences are usable variants of the disclosed sequences, since all give rise to the same protein (that is, the same amino acid sequence ) during translation and are therefore encompassed by the present invention. Of particular interest herein are those nucleotide sequences that encode the enzymes having the amino acid sequences represented respectively by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 reported below:

[0072] SEQ ID NO: 1

[0073] >sp | P00330 | ADH1 YEAST Alcohol dehydrogenase 1 OS=Saccharomyces cerevisiae ( strain ATCC 204508 / S288c) OX=559292 GN=ADH1 PE=1 SV=5 MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVK LPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGY THDGSFQQYATADAVQAAHIPQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAISGAA GGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGEVFIDFTKEKDIVGAVLKATDGGA HGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSYVGNRA DTREALDFFARGLVKSPIKVVGLSTLPEIYEKMEKGQIVGRYVVDTSK SEQ ID NO: 2

[0074] >sp | P40394 | ADH7 HUMAN All-trans-retinol dehydrogenase [NAD (+) ] ADH7 OS=Homo sapiens QX=9606 GN=ADH7 PE=1 SV=2 MFAEIQIQDKDRMGTAGKVIKCKAAVLWEQKQPFSIEEIEVAPPKTKEVRIKILATGICR TDDHVIKGTMVSKFPVIVGHEATGIVESIGEGVTTVKPGDKVIPLFLPQCRECNACRNPD GNLCIRSDITGRGVLADGTTRFTCKGKPVHHFMNTSTFTEYTVVDESSVAKIDDAAPPEK VCLIGCGFSTGYGAAVKTGKVKPGSTCVVFGLGGVGLSVIMGCKSAGASRI IGIDLNKDK FEKAMAVGATECISPKDSTKPISEVLSEMTGNNVGYTFEVIGHLETMIDALASCHMNYGT SVVVGVPPSAKMLTYDPMLLFTGRTWKGCVFGGLKSRDDVPKLVTEFLAKKFDLDQLITH VLPFKKISEGFELLNSGQSIRTVLTF

[0075] SEQ ID NO: 3

[0076] >sp | Q9P6C8 | ADH1 NEUCR Alcohol dehydrogenase 1 OS=Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987 ) OX=367110 GN=adh-l PE=3 SV=1 MPQFEIPEQQWAQVVEKKGGPVVFKQIPVQKPGPDEVLINVKYSGVCHTDLHAMKGDWPL ATKMPLVGGHEGAGVVVAKGELVTEVEVGDHAGIKWLNGSCLACSFCMQADEPLCPHALL SGYTVDGSFQQYAIAKAAHVAKIPKGCDLETTAPVLCAGITVYKGLKESGVRPGQCVAIV GAGGGLGSMAIQYANAMGLHAIAIDGGEEKGKNCRELGAQAYVDFTTTKDLVADVKAATP DGLGPHAVILLAVSEKPFHQAVDYVRSRGTIICIGLPAGAKFQAPVFDTVIRMITIKGSY VGNRQDTQEALDFFARGLIKVPIKTVGLSKLQEVYDLMEEGKIVGRYVVDTSK SEQ ID NO: 4

[0077] >sp | P25984 | ADH GLOBE NADP-dependent isopropanol dehydrogenase OS= Clostridium beij erinckii OX=1520 GN=adh PE=1 SV=2 MKGFAMLGINKLGWIEKERPVAGSYDAIVRPLAVSPCTSDIHTVFEGALGDRKNMILGHE AVGEVVEVGSEVKDFKPGDRVIVPCTTPDWRSLEVQAGFQQHSNGMLAGWKFSNFKDGVF GEYFHVNDADMNLAILPKDMPLENAVMITDMMTTGFHGAELADIQMGSSVVVIGIGAVGL MGIAGAKLRGAGRI IGVGSRPICVEAAKFYGATDILNYKNGHIVDQVMKLTNGKGVDRVI MAGGGSETLSQAVSMVKPGGIISNINYHGSGDALLIPRVEWGCGMAHKTIKGGLCPGGRL RAEMLRDMVVYNRVDLSKLVTHVYHGFDHIEEALLLMKDKPKDLIKAVVIL Two polypeptides are said to be "identical" if the sequence of amino acid residues in the two sequences is the same when aligned for maximal correspondence as described below.

[0078] Sequence comparisons between two (or more) polypeptides are typically carried out by comparing the two sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of the sequences for comparison may be performed using the Smith and Waterman local homology algorithm (Xia Z, Cui Y, Zhang A, Tang T, Peng L, Huang C, et al, A Review of Parallel Implementations for the Smith-Waterman Algorithm. Interdiscip Sci 2022; 14: 1-14, https: / / doi. org / 10.1007 / sl2539-021-00473-0 ), the Needleman and Wunsch homology alignment algorithm (Needleman SB, Wunsch CD, A general method applicable to the search for similarities in the amino acid sequence of two proteins, J Mol Biol 1970; 48: 443-53. https: / / doi. org / 10. 1016 / 0022-2836 ( 70 ) 90057-4 ), the Pearson and Lipman similarity search method ( Pearson WR, Lipman DJ, Improved tools for biological sequence comparison, Proceedings of the National Academy of Sciences 1988; 85: 2444-8, https: / / doi. org / 10. 1073 / pnas.85. 8.2444 ), computerized implementations of these algorithms (BLAST, GAP, BESTFIT, PASTA e TFASTA nel Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin), or by inspection.

[0079] The " BLAST alignment method" is an algorithm provided by the National Center for Biotechnology Information (NCBI ) for comparing polypeptide sequences. In the context of the present disclosure, BLAST is the reference method for comparing polypeptide sequences, using the default parameters of the BLAST software. " Sequence identity" is determined by optimizing the alignment of two sequences in a comparison window, in which the portion of the polypeptide sequence in the comparison window may include additions or deletions (i. e. gaps ) relative to the reference sequence (which does not include additions or deletions ) to obtain the optimal alignment. The percentage is calculated by determining the number of positions at which the same amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0080] The reference sequences in the present document are the coding region defined SEQ ID NO: 1, or alternatively by SEQ ID NO: 2, or alternatively SEQ ID NO: 3, or alternatively SEQ ID NO: 4. A person skilled in the art will recognize that percentage values may be suitably adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0081] Preferably, the enzyme has a sequence identity with respect to SEQ ID NO: 1 of at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, with a sequence identity of at least 98% being most preferred. In another embodiment, preferably the enzyme has a sequence identity with respect to SEQ ID NO: 2 of at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, with a sequence identity of at least 98% being most preferred. In another embodiment, preferably the enzyme has a sequence identity with respect to SEQ ID NO: 3 of at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, with a sequence identity of at least 98% being most preferred. In another embodiment, preferably the enzyme has a sequence identity with respect to SEQ ID NO: 4 of at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, with a sequence identity of at least 98% being most preferred. In another embodiment, the enzyme is simply a functional fragment of SEQ ID NO: 1, or alternatively a functional fragment of SEQ ID NO: 2, or alternatively a functional fragment of SEQ ID NO: 3, or alternatively a functional fragment of SEQ ID NO: 4, or alternatively a mixture thereof.

[0082] It is widely known in the literature that different ADHs have different specificities regarding the chain length of their preferred substrates, which may be branched or cyclic, and a specific enantio-and regio-specificity. Therefore, for example, ADHs active on small alcohols are usually not, or are less, active on longer ones. This means that the choice of the correct ADH is crucial for the success of the ester production reaction of interest according to the invention described herein. As known to the expert in the field of enzymology, Kme Kcatare enzymatic parameters that can be used respectively to quantify the affinity and the turnover number of an enzyme for a specific substrate. Km, or Michaelis-Menten constant, is defined as the substrate concentration at which the reaction rate is half of the maximum rate (Vmax) and represents a measure of the enzyme ' s affinity for a specific substrate. Therefore, if the Kmof an enzyme for a substrate is very low, the affinity of the enzyme for that substrate will be very high. Kcat, on the other hand, or turnover number, is described as the number of substrate molecules converted into products by one molecule of enzyme per unit time when the enzyme is saturated with substrate. If the Kcat of an enzyme for a particular substrate is very high, then the transformation rate of that same substrate into product will also be very high. Kcat and Kmare parameters that can be easily measured and therefore are values widely available in scientific literature for numerous enzymes with respect to many of their known substrates. Public databases also exist, such as the BRENDA Enzyme database, where such data can be found. If data for a specific or relevant enzyme-substrate pair are not available, they may be measured as follows.

[0083] A series of substrate solutions at increasing concentrations ( for example from 0.1× to 10× the estimated Kmconcentration) is prepared and mixed with a fixed concentration of enzyme ( ideally purified). The reaction conditions (such as pH, temperature, concentration of any cofactors and any buffers ) are kept constant and the initial reaction rate (Vo) is recorded for each substrate concentration, obviously by measuring the product concentration over time. Vo will therefore be given by the slope of the product-versus-time curve, under conditions in which the substrate concentration has not yet significantly decreased. The curve of Vo as a function of substrate concentration ( [S] ) is plotted and will show the hyperbolic pattern typical of Michaelis-Menten kinetics. Kmand Vmaxcan then be estimated using two different approaches: the classical graphical method consisting of drawing the Lineweaver-Burk plot or the Eadie-Hofstee plot or the Hanes-Woolf plot, or a more modern method may be used, consisting of applying a direct nonlinear fit of the experimental data to the Michaelis-Menten equation using software such as GraphPad Prism, Origin or even Excel with Solver. Kcatis then calculated using the following equation from Vmaxand the total concentration of active enzyme ( [E] t ).

[0084] Kcat= Vmax / ([E])t

[0085] Unless special conditions apply, it is usually assumed that 100% of the enzyme is catalytically active.

[0086] We have found that the kinetic parameters of the selected polypeptide, relative to the starting substrates (alcohols and aldehydes ), and the steric hindrance of the hemiacetal are crucial in selecting suitable ADHs for the production of an ester of interest with yields higher than those stoichiometrically possible as described in this invention. It is widely known that alcohols and aldehydes are the preferred substrates of ADH enzymes.

[0087] We have discovered that the steric hindrance of the hemiacetal must be as close as possible to that of the preferred alcoholic or aldehydic substrate of the enzyme. Therefore, if an ADH is capable of preferentially oxidizing an alcohol with 2 carbon atoms ( i. e. ethanol ), it will most likely also have sufficient space in its catalytic pocket to accommodate a 2-carbon hemiacetal ( i. e. the one formed between formaldehyde and methanol).

[0088] The selected enzyme must also have suitable kinetic parameters to favor ester production and regeneration of the NAD ( P )+cofactor from the starting aldehyde. Essentially, the polypeptide must be selected so as to possess a higher Kmand a lower Kcatfor the starting alcoholic substrate than for the hemiacetal of interest. This will ensure that the starting alcoholic substrate does not undergo immediate and efficient oxidation to the corresponding aldehyde. If this occurred, concurrent consumption of the cofactor would take place, which would hinder the desired outcome of the process. To support these statements, we attempted to produce ethyl acetate using the Adhl enzyme from Saccharomyces cerevisiae ( SEQ. ID NO. 1 ) starting from ethanol and acetaldehyde. It is widely known that ethanol is the preferred substrate of this ADH and, as expected, virtually no ester was detected in the reaction medium.

[0089] Finally, the enzyme must also have suitable Kcatand Kmvalues for the starting aldehyde substrate; otherwise, cofactor recycling will be impaired.

[0090] The reaction may be optimized using any means known to the skilled person to favor the production of an ester of interest according to the process described in this invention. Depending on requirements, continuous or batch processes may be designed. For example, since all the reactions described are equilibrium reactions, the Le Chatelier principle may be exploited by increasing the concentration of one or more reagents over time and thus increasing the concentration of the desired product in the reaction medium. Alternatively, the product may be separated or extracted from the reaction mixture by any means known to the skilled person (for example, by high-energy-intensity procedures such as distillation, or by low-energy-intensity procedures such as membrane-separation technology), thereby increasing the conversion of reagents toward the desired ester. Ideally, all measures necessary to increase productivity and therefore the cost-effectiveness of the process may be applied with a view to adapting the process to the industrial production of esters. The extraction of small esters ( chain length from C1to C10) from aqueous or low-volatility solvents ( for example DMSO) is theoretically an easier task to perform than the extraction of larger esters (chain length above C10) and water-soluble esters, due to the low volatility and high solubility in water of the latter. In some specific embodiments of the present invention, however, such as those involving the production of methyl formate (see Examples 1, 2 and 3 below), no particular extraction procedures are required, since the reaction may be carried out at a temperature equal to or higher than the boiling point of methyl formate itself (i. e. approximately 31 °C). With suitable equipment designed to recover the gases originating from the reactor or reactors, methyl formate should be easily recoverable in its purest form.

[0091] Finally, other cofactor regeneration strategies may be applied to further improve the yield of the reaction.

[0092] EXPERIMENTAL SECTION

[0093] In the following examples, ester quantification was performed spectrophotometrically using the ferric hydroxamate colorimetric assay (Hill UT. Colorimetric Determination of Fatty Acids and Esters, Industrial & Engineering Chemistry Analytical Edition 1946; 18: 317-9, https: / / doi. org / 10.1021 / i560153a017; Löbs A, Lin J, Cook M, Wheeldon I. High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces niarxianus on C5, C6, and C12 carbon sources, Biotechnol J 2016; 11: 1274-81, https: / / doi. org / 10. 1002 / biot.201600060 ) and with headspace GC-FID (gas chromatography).

[0094] Example 1: synthesis of methyl formate The synthesis of methyl formate was carried out using the process described in the present invention, starting from methanol and formaldehyde in alkaline aqueous solution using a commercial Adh from Saccharomyces cerevisiae (baker' s yeast Adhl; EC 1. 1. 1.1; SEQ ID NO: 1 ). The substrates, the enzymatic cofactor (NAD+) and the enzyme itself were dissolved at appropriate concentration in a phosphate buffer at pH 8.4. A control sample was prepared in which the enzyme was omitted. The final concentration of the reagents in the reaction vials is reported in Table 1.

[0095] Table 1: initial concentration of reagents in the vials

[0096] Methanol Formaldehyde NAD+ En z yme

[0097] 1 M 0, 1 M 3, 1 mM 0, 33 mg / mL

[0098]

[0099] The reactions were carried out at 30 °C, ambient pressure ( 1 atm) and orbital shaking at 160 rpm. Sampling was performed at 0, 15 min, 30 min, 45 min and 60 min.

[0100] Extraction of the ester from the reaction medium was performed in hexane ( 1: 1 ratio with the sample) for 2 minutes with vigorous shaking. The samples were then centrifuged at 9000 rpm, the supernatant was removed and suitably diluted with fresh hexane for the ester quantification assay.

[0101] Determination and quantification of the esters was performed using the ferric hydroxamate assay (see above). The calibration curve was prepared with a commercial standard of methyl formate.

[0102] The results of the time course of the reaction are reported in Table 2 as the average of triplicates, while the graphical representation of these results is shown in Figure 3. The calibration curve for product quantification is shown in Figure 2. No methyl formate was detected in the control samples (without enzyme ).

[0103] Table 2: time course of methyl formate concentration during 1 hour of reaction

[0104] Time (minutes ) Average of triplicates (mg / L)

[0105] 0 0, 00

[0106] 15 1012, 47

[0107] 30 1086, 54

[0108] 45 1255, 00

[0109] 60 1217, 00

[0110]

[0111] As can be seen, methyl formate was efficiently produced using this reaction strategy, with an appreciable titer ( 1.255 g / L as maximum titer after 45 minutes of incubation). Stoichiometrically, the maximum methyl formate titer obtainable is 0.186 g / L (or 3.1 mM), since 3. 1 mM is the concentration of NAD+ used as cofactor for the reaction, and for each mole of NAD+ reduced to NADH only 1 mole of hemiacetal is oxidized to methyl formate. However, the methyl formate titers are higher than this threshold (almost 7-fold higher). This is due to the NAD+ regeneration pathway we discovered, in which S. cerevisiae Adhl is able to regenerate NAD+ by reducing the excess formaldehyde to methanol.

[0112] We also conducted a separate experiment to confirm whether S. cerevisiae Adhl was able to reduce formaldehyde to methanol, consuming NADH and thereby producing NAD+. This can be achieved by monitoring the decrease in absorbance at 340 nm. As shown in Figure 4, the concentration of NADH clearly decreases during the reaction: this means that the enzyme is able to reduce formaldehyde to methanol and thus generate NAD+.

[0113] Example 2: NAD (P)+regeneration pathway

[0114] To verify the cofactor regeneration pathway (NAD+o NAD ( P)+) in ester production with the synthesis described in this invention, another experiment was conducted over a period of 3 hours. Methyl formate production was increased with two repeated additions of formaldehyde at 1 hour and 2 hours of incubation, respectively. The substrates, the enzymatic cofactor, and the enzyme itself (S. cerevisiae Adhl; EC 1. 1. 1. 1; SEQ ID NO: 1 ) were initially prepared as described in Example 1. For the addition steps, a 5. 4 M formaldehyde solution in alkaline buffer at pH 8.4 was also prepared. The sampling and addition schedule is reported in Table 3.

[0115] Table 3: sampling schedule and formaldehyde addition

[0116] Sampling and addition Formaldehyde addition

[0117] schedule (h) ( from a 5.4 M solution)

[0118] 0 - 1 180 pL

[0119] 2 180 pL

[0120] 3 -

[0121]

[0122] Obviously, a blank sample without enzyme was prepared, and a vial was also prepared in which the reaction was carried out without any addition of formaldehyde. To confirm the hypothesis regarding the NAD+regeneration pathway, an increase in the methyl formate titer over time is expected in the vials in which formaldehyde was added, since the excess formaldehyde can be reduced to methanol by the enzyme and thus regenerate the NAD+ necessary for the process.

[0123] In this case, determination and quantification of methyl formate were carried out by gas chromatography. The time-course results are reported in Table 4, while the graphical representation of these results is shown in Figure 5.

[0124] Table 4: methyl formate produced in the 3-hour experiment (without and with formaldehyde addition)

[0125] Time (h) Without addition - With addition - methyl formate methyl formate produced (mg / L) produced (mg / L)

[0126] 0 0, 00 0, 00

[0127] 1 1281, 00 1196, 00

[0128] 2 1222, 00 3625, 00

[0129] 3 1138, 00 5045, 00

[0130]

[0131] Here again, the stoichiometrically obtainable maximum methyl formate titer is 0. 186 g / L (or 3.1 mM). The experiments conducted confirmed that the methyl formate titers obtained following formaldehyde addition are higher than this threshold: almost 7 times higher after 1 hour of incubation (as in Example 1 ), and even 27 times higher after 3 hours of incubation and 2 separate formaldehyde feedings, with a maximum methyl formate titer of 5.045 g / L at 3 hours of incubation.

[0132] No appreciable increase in methyl formate titer was observed after 3 hours in the experiments without formaldehyde addition, and no methyl formate was found in the control vials (without enzyme). These results are consistent with the NAD+regeneration hypothesis. Example 3: synthesis of methyl formate starting only from methanol or only from formaldehyde

[0133] The synthesis of methyl formate starting only from methanol, and in a separate experiment starting only from formaldehyde, was also tested as described in the other two embodiments of the present invention. Five different solutions were prepared in phosphate buffer at pH 8. 4, containing respectively 1.5 M methanol, 1.5 M formaldehyde, 9.3 mM NAD+, 9.3 mM NADH and 1 mg / mL enzyme (a commercially available preparation of S. cerevisiae Adhl; EC 1.1. 1.1; SEQ ID NO: 1 ). The final concentration of reagents in the reaction vials is reported in Table 5. Table 5: concentration of reagents in the vials at the beginning Substrate NAD+ NADH Enzima

[0134] Methanol 0, 5 M 3, 1 mM -- 0, 33 mg / mL

[0135] Formaldehyde 0, 5 -- 3, 1 mM 0, 33 mg / mL

[0136] M

[0137]

[0138] Two different reaction media were assembled: the first containing methanol, NAD+ and the enzyme, and the second containing formaldehyde, NADH and the enzyme. Naturally, in both cases a blank (negative control ) without enzyme was also assembled.

[0139] The reactions were carried out at 30 °C, ambient pressure ( 1 atm), and orbital shaking at 160 rpm for 3 hours. Sampling was performed at 0, 1 hour, 2 hours and 3 hours.

[0140] Extraction and quantification of the esters were conducted in both cases as reported in Example 1. The results of methyl formate production are shown in Table 6, while the graphical representation of these results is shown in Figure 6.

[0141] Table 6: concentration of methyl formate over the 3-hour period Time (h) Methyl formate from Methyl formate from

[0142] methanol (mg / L) formaldehyde (mg / L)

[0143] 0 0, 00 0, 00

[0144] 1 23, 72 45, 92

[0145] 2 33, 16 56, 00

[0146] 3 27, 88 107, 00

[0147]

[0148] The calibration curve for product quantification is again shown in Figure 2 using the ferric hydroxamate colorimetric assay. As can be seen, methyl formate was produced efficiently with this synthesis strategy, with appreciable titers: 107. 00 mg / L as the maximum titer starting only from formaldehyde and 33.16 mg / L starting only from methanol after 3 hours and 2 hours of incubation, respectively. In particular, the rather low titer obtained starting from methanol is due to the fact that most of the cofactor is likely consumed to oxidize methanol to formaldehyde, which is necessary for the subsequent formation of the desired ester.

[0149] Example 4: synthesis of ethyl formate and ethyl acetate with two natural non-commercial enzymes

[0150] Ethyl acetate and ethyl formate were produced with two other natural and non-commercial ADHs (hereafter renamed HS N and CB N) starting respectively from ethanol and acetaldehyde, and from ethanol and formaldehyde. The enzymes were produced using standard molecular biology techniques (followed by recombinant production in bacteria) based on their amino acid sequences reported in the literature ( SEQ ID No: 2 and SEQ ID No: 4 previously reported). Both enzymes, CB N from Clostridium beijerinckii (EC 1. 1. 1. 80; SEQ ID NO: 4 ) and HS N from Homo sapiens (EC 1. 1. 1. 1; SEQ ID NO: 2 ), were chosen because in the literature they have been reported to be more active on higher alcohols, such as propanol, butanol and pentanol, than on ethanol. These enzymes indeed have a high Kmand a low Kcatfor the alcohol used as substrate in this example (ethanol), and they appear to have suitable Km and Kcatvalues also for the aldehydes used, such that cofactor recycling is not impaired.

[0151] For these reasons, according to the present invention, these enzymes should also be active on the hemiacetals formed between ethanol and formaldehyde ( 3 carbon atoms - similar to propanol) and ethanol and acetaldehyde ( 4 carbon atoms - similar to butanol). NADP+ was used as cofactor for CB N and NAD+ as cofactor for HS N, as CB N appears to have higher affinity for NADP+, whereas HS N has higher affinity for NAD+, as reported in the literature ( Ismaiel AA, Zhu CX, Colby GD, Chen JS. Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii. J Bacteriol 1993; 175: 5097-105. https: / / doi.org / 10.1128 / jb.175.16.5097-5105.1993; Moreno A, Pares X. Purification and characterization of a new alcohol dehydrogenase from human stomach. Journal of Biological Chemistry 1991; 266: 1128-33. https: / / doi.org / 10.1016 / S0021-9258(17)35292-4).

[0152] In the same experiment, the production of ethyl formate and ethyl acetate was also tested with three repeated additions of aldehyde as in Example 2 for methyl formate. The addition protocol is shown in Table 7, and samples for ester quantification were taken from the reaction vials after 1, 3, and 4 hours of incubation.

[0153] Table 7: addition schedule

[0154] Time (h) Formaldehyde addition Acetaldehyde addition

[0155] ( from a 5.4 M solution) ( from a 5.4 M solution)

[0156] 0 - - 1 180 pL 180 pL

[0157] 2 180 pL 180 pL

[0158] 3 180 pL 180 pL

[0159] 4 - -

[0160]

[0161] The substrates and enzymatic cofactors were initially prepared as described in Example 1, while the enzymes (CB N and HS N) were used at the concentrations reported in Tables 8 and 9.

[0162] Table 8: concentration of reagents in the reaction vials for the synthesis of ethyl acetate; HS N and CB N were used alternatively and not in the same vials

[0163] Reagents Concentration in the reaction vials

[0164] NAD+o NADP+3, 1 mM

[0165] Ethanol 1 M

[0166] Acetaldehyde 0, 1 M

[0167] HS_N 0, 1 mg / mL

[0168] CB_N 0, 2 mg / mL

[0169]

[0170] Table 9: concentration of reagents in the reaction vials for the synthesis of ethyl formate; HS N and CB N were used alternatively and not in the same vials

[0171] Reagents Concentration in the reaction vials

[0172] NAD+o NADP+3, 1 mM

[0173] Ethanol 1 M

[0174] Formaldehyde 0, 1 M

[0175] HS_N 0, 1 mg / mL

[0176] CB_N 0, 2 mg / mL

[0177]

[0178] A 5. 4 M formaldehyde solution and a separate 5. 4 M acetaldehyde solution were prepared in alkaline phosphate buffer for the addition steps. Naturally, controls without enzyme were always prepared, and for comparison, vials in which the reaction was carried out without any aldehyde addition were also assembled. To confirm the hypothesis regarding the NAD ( P ) + regeneration pathway, also in this case an increase in the titer of ethyl formate or ethyl acetate, depending on the case, is expected over time where formaldehyde or acetaldehyde was added.

[0179] Quantification of ethyl formate and ethyl acetate was performed using the ferric hydroxamate assay as previously described. The results of ester production over time are reported in Tables 10 and 11, while the graphical representation of these results is shown in Figures 7, 8, 9 and 10.

[0180] Table 10: ethyl formate titer in mg / L at various reaction times Time HS N without HS_N with CB N without CB_N with

[0181] ( h ) aldehyde aldehyde aldehyde aldehyde

[0182] addition addition addition addition

[0183] 0 0, 00 0, 00 0, 00 0, 00

[0184] 1 619, 22 613, 86 0, 00 0, 00

[0185] 3 1019, 53 1202, 10 99, 81 100, 08

[0186] 4 1348, 52 1505, 20 160, 43 130, 16

[0187]

[0188] Table 11: ethyl acetate titer in mg / L at various reaction times Time HS N without HS_N with CB N without CB_N with

[0189] ( h ) aldehyde aldehyde aldehyde aldehyde

[0190] addition addition addition addition

[0191] 0 0, 00 0, 00 0, 00 0, 00

[0192] 1 138, 61 158, 03 16, 36 20, 77

[0193] 3 157, 56 312, 88 9, 35 50, 71

[0194] 4 130, 38 648, 47 24, 06 98, 96

[0195]

[0196] In this case, the maximum theoretical titer of ethyl formate obtainable was 230 mg / L (or 3.1 mM), while the maximum theoretical titer of ethyl acetate obtainable was 273 mg / L (or 3.1 mM). The experiments carried out confirmed that titers of both ethyl acetate and ethyl formate above this threshold are obtained with the HS N enzyme (Tables 10 and 11 and Figures 7 and 8 ), whereas they are not with the CB N enzyme (Tables 10 and 11 and Figures 9 and 10 ). The CB N enzyme is scarcely capable of producing ethyl formate and ethyl acetate, and for this reason only a modest increase in ester concentration is observed in the addition experiments. However, in the case of ethyl acetate, a clear increase in the titer over time is observed in the experiment with aldehyde addition compared to that without aldehyde addition. The HS N enzyme, in contrast, is highly active and capable of producing both ethyl acetate and ethyl formate. In both cases, an increase in ester titers is observed in the addition experiments compared to those without aldehyde addition.

[0197] A lower increase in ethyl formate and ethyl acetate titers was clearly observed in the experiments without aldehyde addition over time compared to those with aldehyde addition, and no ester was detected in the control vials (without enzyme ). These results are consistent with the NAD (P) + regeneration hypothesis and therefore support the invention disclosed herein.

[0198] Example 5: synthesis of ethyl formate with Adhl from Neurospora crassa (NC_N; EC 1.1. 1.1)

[0199] The ability of the NC N enzyme (N. crassa Adhl; EC 1.1. 1. 1; SEQ ID NO 3 ) to produce ethyl formate was evaluated in a 4-hour time course. Samples for ester quantification with the ferric hydroxamate colorimetric assay were taken at 2, 3 and 4 hours. The concentration of reagents in the reaction vials was as follows: 1 M ethanol, 0.1 M formaldehyde, 3.1 mM NAD+, and 0. 17 mg / mL NC N. All reagents were dissolved as previously described in a phosphate buffer solution at pH 8.4. The results of ester production over time are reported in Table 12, while the graphical representation of these results is shown in Figure 11.

[0200] Table 12: ethyl formate titer (expressed in mg / L) obtained with the NC N enzyme at the various reaction times

[0201] Time (h) Ethyl formate titer in mg / L

[0202] 0 0, 00

[0203] 2 420, 67

[0204] 3 407, 46

[0205] 4 416, 64

[0206]

[0207] No ester was detected in the blank ( same composition as the vials but without enzyme ). Also in this case, the maximum theoretical titer of ethyl formate obtainable is 230 mg / L (or 3.1 mM), but approximately double that amount is obtained after 4 hours of reaction.

[0208] Example 6: functionalization of cellulose with formaldehyde carried out by Saccharomyces cerevisiae Adhl (EC 1.1. 1.1; SEQ ID NO 1)

[0209] To demonstrate that the technology disclosed in the present invention also allows the formation of ester bonds in larger and branched molecules, we carried out the functionalization of cellulose (which contains 3 alcoholic groups per glucose unit ) with formaldehyde using S. cerevisiae Adhl.

[0210] The reaction mixture was assembled as follows: microfibrillated cellulose (type EXILVA-FO1-L, marketed by Borregaard) was resuspended in phosphate buffer at pH 8.4 at a concentration of 4 mg / L, formaldehyde at a concentration of 0. 1 M, the enzyme at a concentration of 0.33 mg / mL, and the cofactor at a concentration of 3.1 mM. After 24 h of reaction, the vial contents were completely lyophilized and the cellulose was analyzed by FT-IR. Naturally, unmodified cellulose (without formaldehyde and without enzyme) was also analyzed, as well as cellulose with formaldehyde but without enzyme. The obtained spectra are shown in Figure 11.

[0211] The top curve is the spectrum of unmodified cellulose. The second curve from the top represents cellulose with added formaldehyde, in which an additional peak is visible compared to unmodified cellulose at around 1600 cm-1, representing formation of the hemiacetal between formaldehyde and the alcoholic groups of cellulose. The third curve represents the spectrum of cellulose with added formaldehyde and yeast ADH1 enzyme ( SEQ ID NO: 1 ). In the latter curve, a peak is visible at approximately 1750 cm-1, confirming the presence of a C=O double bond, a characteristic signature of an ester bond.

[0212] As can be observed, the cellulose was effectively functionalized, as shown by the characteristic ester C=O peak around 1750 cm-1.

[0213] Example 7: synthesis of methyl acetate with CB_N from Clostridium beijerinckii (EC 1.1.1.80; SEQ ID NO 4)

[0214] The synthesis of methyl acetate starting from methanol and acetaldehyde was carried out in triplicate with the enzyme referred to as CB N. The concentration of reagents in the reaction vials was as follows: 1 M methanol, 0. 1 M acetaldehyde, 3.1 mM NADP+, and 0.33 mg / mL CB N. All reagents were dissolved as previously described in a phosphate buffer solution at pH 8. 4. The results of ester production after 1 hour are reported in Table 13.

[0215] Table 13: methyl acetate titer (expressed in mg / L) obtained with the CB N enzyme at various reaction times Time (h) Methyl acetate titer in mg / L

[0216] 0 0, 00

[0217] 1 725, 86 ± 86, 94

[0218]

[0219] The ester concentration was measured using the ferric hydroxamate colorimetric assay. No ester was detected in the blank (same composition as the vials but without enzyme ). In this case, the maximum theoretical titer of methyl acetate obtainable is 0.230 mg / L (or 3. 1 mM), but more than three times this amount is obtained in just one hour of reaction.

[0220] It is obvious that only some particular embodiments of the present invention have been described, and the skilled person will be able to make all modifications necessary to adapt it to specific applications, without however departing from the scope of protection of the present invention as defined by the appended claims.

Claims

CLAIMS1. A process for producing esters, via a hemiacetal intermediate, consisting of the following steps:- a step of bringing into contact, in a reaction medium,• a mixture of a Ci- Cnalcohol and a Ci- Cnaldehyde with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD (P) + and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde, or alternatively• a Ci- Cnalcohol with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD ( P) + and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal, the starting alcohol, and the formed aldehyde, or alternatively• a Ci- Cnaldehyde with an enzymatic catalytic system comprising a reduced cofactor selected from NAD ( P) H or NADH and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde,where n may be any integer, and- a step of isolating the corresponding ester.

2. The process according to claim 1, wherein, when the starting compound is only an alcohol, or alternatively only an aldehyde, the process comprises, respectively, first i ) the partial oxidation of the alcohol to give the corresponding aldehyde, or in the other alternative ii) the partial reduction of the aldehyde to the corresponding alcohol, then the formation of ahemiacetal intermediate and finally the oxidation of the hemiacetal intermediate to obtain the corresponding ester.

3. The process according to claim 2, wherein said partial oxidation i) of the alcohol to the corresponding aldehyde occurs for at least 1%, preferably between 1% and 99%, more preferably between 1% and 50%, and said partial reduction ii) of the aldehyde to give the alcohol occurs for at least 1%, preferably between 1% and 99%, more preferably between 1% and 50%.

4. The process according to claim 1, wherein, when the starting compound is a mixture of an alcohol and an aldehyde, the process comprises firslyt the formation of a hemiacetal intermediate and then the oxidation of said hemiacetal intermediate to obtain the corresponding ester, using one or more enzymes having preferentially hemiacetal dehydrogenase activity and aldehyde dehydrogenase activity.

5. The process according to claim 4, wherein during the reaction an excess of aldehyde is added, preferably at least up to 10%, or at least up to 50%, or at least up to 100%, or at least up to 200%, or at least up to 300%, more preferably at least up to 400%, still more preferably at least up to 500%, even more preferably at least up to 600% or at least up to 700%.

6. The process according to claim 4 or 5, wherein the excess of aldehyde is at least 10%, or at least 30%, or at least 50%, or at least 100%.

7. The process according to any one of claims 1 to 6, wherein the reaction medium is aqueous, an inert solvent in the reaction environment or a mixture thereof, said inert solvent preferably being DMSO.

8. The process according to any one of claims 1 to 7, wherein the pH is between 2 and 10, preferably between 4 and 9, more preferably about 8, even more preferably about 8.4.

9. Process according to any one of claims 1 to 8, wherein the reactions are carried out under standard conditions (STP: 0 °C and 1 atm), or alternatively under mild conditions (25 °C and 1 atm), or alternatively also under forced conditions (for example high or low temperature and pressure).

10. The process according to any one of claims 2 to 9, wherein the reactions are carried out in a single reactor or in sequential reactors for each reaction or in flow reactors.

11. The process according to any one of claims 1 to 10, wherein the polypeptide is an enzyme belonging to the class of alcohol dehydrogenases with classification EC 1.1.1.X of the Enzyme Commi ssi on, also having hemiacetal dehydrogenase activity, for example in the form of soluble, immobilized enzyme or whole cells expressing and / or over-expressing the selected ADHs.

12. The process according to claim 11, wherein the enzyme is of the wild type and is selected from Adh1 from Saccharomyces cerevisiae (SEQ ID NO: 1), ADH7 from Homo sapiens (SEQ ID NO: 2), Adh1 from Neurospora crassa (SEQ ID NO: 3), and a Clostridium beijerinckii NADP+ dependent isopropanol dehydrogenase (SEQ ID NO: 4).

13. The proces s according to claim 12, wherein the enzyme is a wildtype or engineered polypeptide having:- a sequence identity to SEQ ID NO: 1 of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80 %, even morepreferably at least 90%, even more preferably at least 95%, a sequence identity of at least 98% being most preferred; or- a sequence identity to SEQ ID NO: 2 of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, a sequence identity of at least 98% being most preferred; or- a sequence identity to SEQ ID NO: 3 of at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, a sequence identity of at least 98% being most preferred; - a sequence identity to SEQ ID NO: 4 of at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, a sequence identity of at least 98% being most preferred; or the enzyme is a functional fragment of SEQ ID NO: 1, or alternatively a functional fragment of SEQ ID NO: 2, or alternatively a functional fragment of SEQ ID NO: 3, or alternatively a functional fragment of SEQ ID NO: 4, or alternatively a mixture thereof.

14. The process according to any one of claims 11 to 13, wherein the enzyme is selected so as to have a higher Kmand a lower Kcatfor the starting alcohol substrate compared to the hemiacetal of interest and / or a lower Kmand a higher Kcatfor the starting aldehyde substrate compared to the starting alcohol substrate, in order to ensure cofactor regeneration.

15. Process according to any one of claims 1 to 14, wherein the titer of ester produced is higher than the maximum yield obtainable considering the stoichiometry of the reaction, in some specific examples from at least 2 to at least 27 times the maximum yield obtainable considering the stoichiometry of the reaction or even higher.

16. A nucleotide sequence encoding enzymes having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, or for a polypeptide according to claim 13.

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