Method for synthesizing α-methylene-γ-butyrolactone

Abstract

The present application relates to a process for preparing α-methylene-γ-butyrolactone, comprising the steps of: a) acetylating the C1-hydroxyl group of isoprenol to obtain isoprenyl acetate; b) forming 4-acetoxy-2-methylene-butan-1-ol from said isoprenyl acetate by whole cell biotransformation, i) contacting cells (CB) with a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; and ii) optionally isolating the resulting 4-acetoxy-2-methylene-butan-1-ol, wherein said cells (CB) exhibit at least one alkane monooxygenase enzyme activity that catalyzes the formation of 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; c) oxidizing 4-acetoxy-2-methylene-butan-1-ol to obtain 4-acetoxy-2-methylenebutyric acid; and d) converting the 4-acetoxy-2-methylenebutyric acid to α-methylene-γ-butyrolactone by hydroxylysis to γ-hydroxy-α-methylenebutyric acid and subsequent cyclization of the γ-hydroxy-α-methylenebutyric acid; A method is provided that includes:

Description

[Technical field]

[0001] The present disclosure relates to a method for the synthesis of α-methylene-γ-butyrolactone (MBL) from isoprenol. More specifically, the present disclosure relates to a multi-step method for the synthesis of α-methylene-γ-butyrolactone (MBL) from isoprenol, at least one step of which involves whole cell biotransformation. The method identifies a number of important intermediate compounds that have utility in their own right. [Background technology]

[0002] Significant research is being conducted to develop renewable or sustainable polymeric materials to replace petroleum-based feedstocks for large commodity and specialty chemical markets. Bio-derived polylactic acids and polyhydroxyalkanoates (PHAs), such as poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), and their copolymers, have long attracted attention in this regard. In contrast, unsaturated lactones derived from itaconic or levulinic acids isolated from plant sources or obtained by microbial fermentation of biomass feedstocks have been less widely studied so far.

[0003] The advantage of unsaturated lactones is that they have two different functional groups in one molecule, in particular vinyl and lactone, both of which are polymerizable. These monomers are therefore useful as (co)monomers for replacing (meth)acrylates in vinyl addition polymerizations or for the preparation of degradable polyesters via lactone ring-opening polymerization (ROP). With these unsaturated monomers, in particular the unsaturated monomers from which α-methylene-γ-butyrolactone (MBL), β-hydroxy-α-methylene-γ-butyrolactone (H-MBL), β- and γ-methyl-α-methylene-γ-butyrolactone (β-MBL, γ-MMBL) and angelica lactone (α- and β-AL) are obtained, functional polymers can be derived with pendant double bonds, pendant lactone rings, or other pendant substituents that allow for various post-functionalizations.

[0004] The present disclosure relates to a novel synthetic route for the production of α-methylene-γ-butyrolactone (MBL). In addition to being a platform monomer for the aforementioned homopolymers and copolymers, the MBL structural unit has been recognized to exhibit multiple biological properties, including antibacterial, cytotoxic, anti-inflammatory, antioxidant, allergenic and antimicrobial activity, both in α-methylene-γ-butyrolactone itself and as part of various sesquiterpenoids.

[0005] No. 5,166,357 (Orlek et al.) discloses a synthetic route to α-methylene-γ-butyrolactone that involves a two-step sequence consisting of i) reacting γ-butyrolactone with ethyl formate in the presence of a base, and ii) refluxing the resulting α-formyl-γ-butyrolactone sodium salt with paraformaldehyde in tetrahydrofuran under nitrogen. Distillation affords the desired α-methylene-γ-butyrolactone as a colorless oil.

[0006] U.S. Pat. No. 6,362,346 B1 (Coulson et al.) describes a process for preparing α-methylene-γ-butyrolactone, which comprises heating a mixture of a furoic acid selected from the group consisting of tetrahydro-3-furoic acid and esters of tetrahydro-3-furoic acid and a strong acid catalyst under conditions whereby α-methylene-γ-butyrolactone is formed.

[0007] WO2012 / 116977 (DSM IP Assets BV) describes a process for preparing 3-methylene-Y-butyrolactone (Z), comprising the steps of: i) reacting 1,4-butene-diol (X) or its ester derivative cis-1,4-diacetoxybutene (Y) with H in the presence of a hydroformylation catalyst. 2 and ii) an oxidation step in which said intermediate product, or a hydrolyzed derivative thereof, is oxidized with an oxidizing agent to form Z.

[0008] In an embodiment described in WO2012 / 116977, the intermediate product is a mixture of hydroxy-2-(hydroxymethyl)butanal (X1), 4-hydroxy-2-methylenebutanal (X2), 3-(hydroxymethyl)tetrahydrofuran-2-ol (X3) and 3-methylenetetrahydrofuran-2-ol (X4). The yields of these intermediates are not disclosed, but it is claimed that 1,4-butene-diol (X) cannot be efficiently converted by intermolecular acetalization, and this low efficiency leads to a low yield of Z. Furthermore, the production of Z from said intermediates is not efficient: even under base catalysis, in particular the cyclization and dehydration reactions of X1 and X3; the H reaction of X1 and X2; 2and hydrogenation of X1, X2, X4 and Z to the corresponding alkanes. Such side reactions may be minimized by using a protected starting material, cis-1,4-diacetoxybutene (Y), but this has drawbacks such as additional starting material cost, longer reaction times for hydroformylation and unfavorable molecular economy due to wasted protecting groups.

[0009] Those skilled in the art will further note that the method of WO2012 / 116977 relies on the use of explosive synthesis gas under extreme conditions of temperature and pressure in the presence of transition metal catalysts with toxic phosphorus ligands. It is known that synthesis gas is produced by high temperature processing of fossil carbon sources. Also, natural resources of Co, Rh and Pd-based transition metal catalysts are geopolitically limited.

[0010] Trotta et al., Synthesis of methylene butyrolactone polymers from itaconic acid, J. Polym. Sci., Part A: Polym. Chem. 2017,55, 2730-2737, describes the synthesis of α-methylene-γ,γ-dimethyl-γ-butyrolactone (Me) by a selective addition strategy of β-monomethyl itaconate, which is available as a biorenewable feedstock. 2 The conversion of α-methylene-γ-butyrolactone (MBL, tulipalin A) to α-methylene-γ-butyrolactone (MBL, tulipalin A) has been reported.

[0011] WO 2002101013 A2 (DuPont) describes the cloning of genes encoding proteins shown to be involved in the natural biosynthesis of α-methylene-γ-butyrolactone (Tulipalin A) and its intermediates. More specifically, the cited document discloses isolated nucleic acid fragments encoding tuliposide A synthesis proteins selected from the group consisting of: (a) isolated nucleic acid fragments encoding one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:24 of the cited document; (b) isolated nucleic acid fragments hybridizing to the above sequences under the following hybridization conditions: 0.1×SSC, 0.1% SDS, and 2×SSC, 0.1% SDS at 65° C., followed by washing with 0.1×SSC, 0.1% SDS; (c) isolated nucleic acid fragments fully complementary to (a) or (b). The biosynthetic pathway proposed in this reference does not include a hydratase step or other enzymatic reaction leading to the intermediate product γ-methylene glutamate, and for this reason alone, it is believed that the pathway is either not workable or is otherwise incompletely or poorly disclosed in this reference.

[0012] WO2001068803A2 (Maxygen Inc.) describes methods for preparing enzymes, biochemical pathways and whole cell bioprocesses that convert simple carbon sources into polymerizable substrates, such as polyhydroxyalkanoates (PHAs). In particular, an artificial pathway for the synthesis of α-methylene-γ-hydroxybutyrate in the itaconate-producing fungus Aspergillus terreus is described. The reference suggests that itaconate is converted to itaconyl-CoA by endogenous enzymes of the fungus, which is then converted to α-α-methylene succinate semialdehyde and further converted to α-methylene-γ-hydroxybutyrate. These reductive steps are suggested to be catalyzed by succinate-semialdehyde dehydrogenase and γ-hydroxybutyrate dehydrogenase, respectively, from Clostridium kluyveri. Potential enzymes capable of catalyzing these reactions are listed in Table 1 of this reference, but no relevant experimental data are presented. Many of the enzymes from obligate anaerobes are highly oxygen sensitive, which may make their introduction into aerobic organisms problematic.

[0013] The inventors believe that there is a need in the art to provide new synthetic routes to α-methylene-γ-butyrolactone (Tulipalin A) starting from feedstocks that may be biologically renewable. Summary of the Invention

[0014] In a first aspect of the present disclosure, there is provided a method for preparing α-methylene-γ-butyrolactone, comprising the steps of: a) acetylating the C1-hydroxyl group of isoprenol to obtain isoprenyl acetate; b) forming 4-acetoxy-2-methylene-butan-1-ol from said isoprenyl acetate by whole cell biotransformation, i) contacting cells (CB) with a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; and ii) optionally isolating the resulting 4-acetoxy-2-methylene-butan-1-ol; Including, wherein said cells (CB) exhibit at least one alkane monooxygenase enzyme activity that catalyzes the formation of 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; c) oxidizing the 4-acetoxy-2-methylene-butan-1-ol to obtain 4-acetoxy-2-methylenebutyric acid; and d) converting the 4-acetoxy-2-methylenebutyric acid to α-methylene-γ-butyrolactone by hydrolytic decomposition to γ-hydroxy-α-methylenebutyric acid and subsequent cyclization of the γ-hydroxy-α-methylenebutyric acid; A method is provided that includes:

[0015] The starting point for this process step, isoprenol, can be commercially sourced and obtained from the petroleum-derived chemicals isoprenol and formaldehyde, however, it is preferred that the compound be sourced from a synthetic microbial system for producing isoprenol.

[0016] The cell (CB) used in step b) carries a gene for an alkane monooxygenase, optionally as part of the Alk operon. In an important embodiment, said cell (CB) in step b) is genetically modified to exhibit an increased activity of an alkane monooxygenase enzyme encoded by a homologue of the AlkB gene from Pseudomonas putida GP01. Good results have been obtained when said cell (CB) is genetically modified to exhibit an increased activity of an alkane monooxygenase enzyme encoded by the AlkB gene from Pseudomonas putida GP01, selected from the group consisting of the AIkBGT gene cluster; the AlkBGTJH gene cluster; and the AlkBGTJHL gene cluster.

[0017] Step c) of the present disclosure can be carried out chemically, but in important embodiments, step c) is characterized by an enzymatic oxidation process comprising contacting 4-acetoxy-2-methylene-butan-1-ol under aerobic conditions with at least one enzyme exhibiting oxidative activity; and optionally, at least one mediator compound that enhances the oxidative activity of the enzyme.

[0018] Step c) can likewise be carried out by whole cell biotransformation, for which two important embodiments are recognized.

[0019] In a first embodiment, step c) is carried out by whole cell biotransformation, i) contacting the cells (CC) with a culture medium containing 4-acetoxy-2-methylene-butan-1-ol or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-ol under conditions that allow the cells (CC) to form 4-acetoxy-2-methylene-butyric acid from said 4-acetoxy-2-methylene-butan-1-ol; and ii) isolating the resulting 4-acetoxy-2-methylene-butyric acid; Including, wherein said cells (CC) exhibit the activity of at least one enzyme exhibiting oxidative activity, preferably said at least one enzyme exhibiting oxidative activity is selected from the group consisting of alcohol dehydrogenase (ADH); alcohol oxidase (AlcOx); aldehyde dehydrogenase (AlDH); and laccase.

[0020] In a second embodiment, step c) is carried out by whole cell biotransformation, i) contacting a first cell (CC1) with a culture medium containing 4-acetoxy-2-methylene-butan-1-ol, the culture medium being in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-ol, under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-al from said 4-acetoxy-2-methylene-butan-1-ol; ii) optionally isolating the resulting 4-acetoxy-2-methylene-butan-1-al; iii) contacting a second cell (CC2) with a culture medium containing 4-acetoxy-2-methylene-butan-1-al or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-al under conditions that allow the cells to form 4-acetoxy-2-methylenebutyric acid from 4-acetoxy-2-methylene-butan-1-al; and iv) isolating the resulting 4-acetoxy-2-methylenebutyric acid; Including, wherein the first and second cells (CC1, CC2) each exhibit at least one enzyme activity exhibiting oxidative activity. Preferably, the first cell (CC1) is genetically modified to exhibit increased activity of at least one enzyme exhibiting oxidative activity selected from the group consisting of alcohol dehydrogenase (ADH), alcohol oxidase (AlcOx) and laccase; and the second cell (CC2) is genetically modified to exhibit increased activity of at least one enzyme exhibiting oxidative activity selected from the group consisting of aldehyde dehydrogenase (AlDH) and laccase.

[0021] With regard to the enzymes exhibiting oxidizing activity, it is particularly preferred that the alcohol dehydrogenase (ADH) enzyme is encoded by a homologue of the AlkJ gene from Pseudomonas putida GPO1; and / or that the aldehyde dehydrogenase (AlDH) enzyme is encoded by a homologue of the AlkH gene from Pseudomonas putida GPO1.

[0022] The present disclosure also provides the use of α-methylene-γ-butyrolactone (MBL) obtained by the above process as a monomer in an anionic polymerization process to obtain homopolymers or copolymers having a pendant lactone ring in the polymer chain (p-MBL) or in a ring-opening polymerization process to obtain polyesters.

[0023] In a second aspect of the present disclosure, there is provided a method for preparing 4-acetoxy-2-methylene-butan-1-ol, comprising the steps of: a) acetylating the C1-hydroxyl group of isoprenol to obtain isoprenyl acetate; b) forming 4-acetoxy-2-methylene-butan-1-ol from said isoprenyl acetate by whole cell biotransformation, i) contacting cells (CB) with a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; and ii) isolating the resulting 4-acetoxy-2-methylene-butan-1-ol; Including, wherein the cells (CB) exhibit at least one alkane monooxygenase enzyme activity that catalyzes the formation of 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate, A method is provided that includes:

[0024] The cells utilized in step b) (CB) carry a gene for an alkane monooxygenase, optionally as part of the Alk operon.

[0025] The present disclosure provides the use of said 4-acetoxy-2-methylene-butan-1-ol obtained by whole cell biotransformation as defined hereinabove and in the appended claims for the synthesis of α-methylene-γ-butyrolactone (MBL). The present disclosure also provides the synthesis of 2-methylenebutane-1,2-diol by hydrolysis of 4-acetoxy-2-methylene-butan-1-ol, said 4-acetoxy-2-methylene-butan-1-ol obtained by whole cell biotransformation as defined hereinabove and in the appended claims. The hydrolysis may be performed under basic or acidic conditions or enzymatically using a suitable lipase or carboxylesterase enzyme.

[0026] The present disclosure still further provides a method for preparing a compound of formula (BIII) from a compound of formula (BII) by whole cell biotransformation: [ka] [In the formula, n is an integer from 0 to 8; R 1 is C 1 -C 4 is alkyl; R 2 is H or C 1 -C 4 is alkyl; R 3 is H or C 1 -C 4 is alkyl] And, i) contacting cells (CB) with a culture medium containing a compound of formula (BII), or a culture medium in contact with an organic phase containing a compound of formula (BII), under conditions that allow the cells to form a compound of formula (BIII) from said compound of formula (BII); and ii) isolating the resulting compound of formula (BIII), Including, wherein the cell (CB) is a compound of formula (BII) n+3 The present invention provides a method for determining the activity of at least one alkane monooxygenase enzyme that catalyzes -hydroxylation of an alkane monooxygenase. The utilization cell (CB) of this embodiment of the disclosure has a gene for a monooxygenase, optionally as part of the Alk operon.

[0027] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0028] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0029] As used herein, the term "consisting of" excludes any unspecified elements, components, members or method steps. For completeness, the term "comprising" encompasses "consisting of."

[0030] Where amounts, concentrations, dimensions, and other parameters are expressed in the form of ranges, preferred ranges, upper values, lower values, or preferred upper and lower values, it is to be understood that a range obtained by combining any upper value or preferred value with any lower value or preferred value is also specifically disclosed, regardless of whether the resulting range is expressly stated in the context.

[0031] Furthermore, in accordance with standard understanding, weight ranges expressed as "0 to" specifically include 0 wt.%, and the component defined by said range may or may not be present in the composition.

[0032] In this specification, the words "preferred," "preferably," "desirable," and "particularly" are frequently used to refer to embodiments of the present disclosure that may offer certain advantages, under particular circumstances. However, the recitation of one or more preferred, preferred, desirable, or particular embodiments does not imply that other embodiments are not useful, and is not intended to exclude such other embodiments from the scope of the present disclosure.

[0033] As used throughout this application, the term "may" is used in its permissive rather than obligatory sense, i.e., in the sense of the possibility of.

[0034] Room temperature, as used herein, is 23° C.±2° C. As used herein, "ambient conditions" refers to the surrounding temperature and pressure in which the composition is located or in which a sticky or coating resulting from the composition is located.

[0035] The term "isoprenol" refers to a compound of formula C 5 H 10 The IUPAC name for isoprenol is 3-methylbut-3-en-1-ol, and synonyms for isoprenol include isobutenylcarbinol, and methallylcarbinol.

[0036] As used herein, "Tulipalin A" is interchangeably referred to as α-methylene-γ-butyrolactone. The IUPAC name for Tulipalin A is 3-methylideneoxolan-2-one (C 5 H 6 O 2 ).

[0037] The term "acetylate" or "acetylation" as used herein refers to the process of introducing an acetyl group into a molecule of a compound that has a hydroxyl (-OH) group. Acetylation converts the H of the -OH group into CH 3 Replace with a CO-group.

[0038] In this specification, "C 1 -C n An "alkyl" group is a monovalent group containing 1 to n carbon atoms, which is a radical of an alkane, and which includes linear and branched organic groups. 1 -C 4 An "alkyl" group is a monovalent group containing from 1 to 4 carbon atoms, which is a radical of an alkane, and which includes straight-chain and branched organic groups. Examples of alkyl groups include, but are not limited to, methyl; ethyl; propyl; isopropyl; n-butyl; isobutyl; sec-butyl; and tert-butyl. In the present invention, such alkyl groups may be unsubstituted or substituted with one or more halogens. If applicable for a given moiety (R), the permissible range of one or more non-halogen substituents in an alkyl group is described herein.

[0039] The term "water" as used herein is intended to include tap water, spring water, purified water, deionized water, demineralized water and distilled water. Water is included in the composition of the present invention in liquid form. The presence of solid water particles (ice) is undesirable because solid water cannot be mobilized to form hydrates necessary for the strength development of the hardened composition.

[0040] As used herein, "basic" refers to the property of being a base rather than an acid. More specifically, according to the Lewis theory of acids and bases, a base is an electron pair donor. This definition includes, but is not limited to, Bronsted-Lowry bases, which are compounds that act as proton acceptors.

[0041] The terms "monomer" and "comonomer" as used herein refer to a molecule that can be converted into a polymer, synthetic resin, or elastomer by combination with itself or other similar molecules or compounds. The terms are not limited to small molecules, but include oligomers, polymers, and other large molecules that can combine with themselves or other similar molecules or compounds.

[0042] As used herein, "macromonomer" refers to a polymer having at least one functional group capable of undergoing a polymerization reaction. Thus, a macromonomer is a polymeric monomer that can be converted into a homopolymer or copolymer of a defined structure. As used herein, macromonomer does not exclude the inclusion of more than one polymer chain attached to one functional group.

[0043] As used herein, the term "enzyme" refers to a protein that catalyzes a chemical reaction. The catalytic function of an enzyme constitutes its "enzymatic activity" or "activity." As used herein, the term "substrate" refers to a substance on which an enzyme exerts its catalytic activity to produce a product.

[0044] The term "monooxygenase" or "hydroxylase" as used herein means an enzyme as defined in BRENDA:EC (Enzyme Commission) 1.14 that catalyzes the incorporation of a hydroxyl group into a substrate such as an alkane or alkene, which requires an electron donor, such as an electron transfer protein, NADH or NADPH.

[0045] As used herein, "laccase" refers to a multi-copper-containing oxidase as defined in BRENDA:EC (Enzyme Commission) 1.10.3.2 that catalyzes the oxidation of alcohols by single-electron robbery with the reduction of oxygen to water in a four-electron transfer process. Included are zinc enzymes that require NAD+ or NADP+ as an acceptor as defined in BRENDA:EC 1.1.1.1, and metal-independent enzymes that require NAD+ or NADP+ as an acceptor as defined in BRENDA:EC 1.1.1.3. As used herein, "aldehyde dehydrogenase" (AlDH) refers to an enzyme that requires NAD+ or NADP+ as an acceptor as defined in BRENDA:EC 1.2.1.3. As used herein, the term "alcohol oxidase" (AlcOx) refers to an oxidoreductase enzyme that utilizes oxygen as an acceptor as defined in BRENDA:EC 1.1.3.13.

[0046] As used herein, the terms "mediator compound" or "mediator" may be used interchangeably to refer to chemical compounds that function as redox mediators that shuttle electrons between an enzyme exhibiting oxidative activity and a secondary substrate or electron donor. Such chemical mediator compounds are sometimes referred to in the art as "enhancers" and "promoters."

[0047] The term "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide. The term should also be understood to include, as equivalents, either RNA or DNA analogs made from nucleotide analogs, and, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0048] The parameter "nucleotide identity" for the recorded sequence listing (SEQ ID NO:) can be determined herein using established known methods.Smith-Waterman algorithm computer programs implementing this algorithm and equivalent algorithms include, but are not limited to: GCG software, including GAP, as described in Deveroy, J.et al., Nucleic Acid Research 12:387, 1984; and BLASTP, BLASTN and FASTA, as described in Altschul, S.et al., Journal of Molecular Biology 215:403-410, 1990, among others.

[0049] As used herein, the term "expression vector" refers to a DNA construct that contains a DNA coding sequence (e.g., a gene sequence) operably linked to one or more suitable control sequence(s) capable of effecting the expression of the coding sequence in a host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding an appropriate mRNA ribosomal binding site, and sequences that control the termination of transcription and translation. The vector may be a plasmid, a phage particle, or a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself.

[0050] The term "whole cell biotransformation" refers to the conversion of suitable substrates to products by microorganisms.

[0051] The term "wild-type" as used herein with respect to a cell refers to a cell having a genomic configuration that is naturally found in the wild. Thus, the term "wild-type" does not include cells or genes whose gene sequences have been at least partially altered by human mutation.

[0052] The term "mutant" as used herein with respect to a protein means a version of the protein other than the wild type. This change can be affected by methods well known to those skilled in the art, for example, by point mutation, such that the resulting protein can be referred to as a mutant.

[0053] As used herein, the term "non-recombinant laboratory cells" refers to cells derived from a cell line that is a clonal population derived from a progenitor wild-type cell. Clonal populations are often obtained by continuous or long-term growth and division in vitro. During storage or transfer of clonal populations in the laboratory, spontaneous or induced changes in the karyotype may occur. Therefore, laboratory cells derived from a cell line may not be exactly identical to the ancestral cells, and the cell line encompasses variants thereof.

[0054] As used herein, the term "operon" refers to a functional unit of DNA that contains a cluster of genes under the control of a single promoter.

[0055] The optical density at a wavelength of 600 nm (OD 600 " (abbreviated as "cell density") means a standard measure of cell density in a culture obtained by calculating the absorbance of light at 600 nm wavelength at a 1 cm path length of culture minus the absorbance of medium without cell culture at a 1 cm path length.

[0056] The term "medium" as used in reference to cell culture includes the components of the environment surrounding the cells. Medium may be solid, liquid, gaseous, or mixed phase. Medium includes liquid growth media and liquid media that do not support cell growth. Also included are gelatinous media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed. The term "medium" also refers to materials intended for use in cell culture, even if not yet in contact with the cells.

[0057] The term "minimal medium" includes a medium that supports the growth of a species' wild-type but not one or more auxotrophs of that species. A supplemented minimal medium is a minimal medium that contains one or more additional substances to support the growth of an auxotroph. "Defined medium" or "defined minimal medium" refers to a medium that consists of chemically defined (usually purified) components. A "defined medium" does not contain poorly characterized biological extracts, such as yeast extract or beef broth.

[0058] For completeness, a medium suitable for growing high density cultures is one in which the cell culture can grow to an OD of 3 or higher, provided other conditions (such as temperature and oxygen transfer rate) allow such growth. 600 It is a medium that allows the

[0059] The viscosity of the compositions described herein is measured using a Brookfield viscometer at standard conditions of 20° C. and 50% relative humidity (RH), unless otherwise specified. The calibration, spindle type and rotation speed of the Brookfield viscometer are selected according to the manufacturer's instructions appropriate for the composition to be measured.

[0060] As used herein, "polymerization conditions" are conditions that cause the formation of at least one monomer into a polymer, such as temperature, pressure, atmosphere, ratio of starting components used in the polymerization mixture, reaction time, or external stimuli of the polymerization mixture. The polymerization process can be carried out in bulk, solution, or other conventional polymerization modes. The process is operated at any reaction conditions appropriate to the polymerization mechanism.

[0061] The term "anionic polymerization" as used herein refers to an ionic polymerization mechanism in which the kinetic chain carrier is an anion. Thus, an anionic polymerization reaction is a chain reaction in which the growth of a polymer chain proceeds by reaction of a monomer(s) with a reactive site(s) on the polymer chain, with the reactive site(s) being regenerated at the end of each growth step. Anionic polymerization is used herein to produce macromolecules from monomers containing carbon-carbon double bonds. Polymerization is initiated by nucleophilic addition to the double bond of the monomer, and initiators include anions such as hydroxides, alkoxides, cyanides, carbanions, etc.

[0062] The term "ring-opening polymerization" as used herein refers to polymerization in which, in the presence of a suitable catalyst, cyclic compounds (monomers) open to form linear polymers. The reaction system tends toward equilibrium between the desired resulting mixture of polymeric compounds, cyclic compounds and / or linear oligomers, the achievement of which depends heavily on the nature and amount of cyclic monomers, the catalyst used and the reaction temperature. The use of solvents and / or emulsions in the polymerization is not recommended as their removal after the reaction is complete can be complicated.

[0063] In the present specification, the compositions of the present invention may be defined as "substantially free" of a particular compound, element, ion, or other similar component. The term "substantially free" is intended to mean that the compound, element, ion, or other similar component is not intentionally added to the composition and is present at most in trace amounts that do not (adversely) affect the desired properties of the composition. An exemplary trace amount is less than 1000 ppm by weight of the composition. The term "substantially free" encompasses those embodiments in which the particular compound, element, ion, or other similar component is completely absent from the composition or is absent in any amount measurable by techniques commonly used in the art.

[0064] As used herein, the term "anhydrous" is equivalent to the term "substantially free of water." Water is not intentionally added to a given composition and, at most, is present in trace amounts that do not (adversely) affect the desired properties of the composition.

[0065] Detailed Description of the Invention The isoprenol provided in step a) of the present disclosure may be commercially available and, as such, may be derived from petroleum-based isoprenol and formaldehyde. However, it is preferred that the compound is sourced from synthetic microbial systems for the production of isoprenol known in the art.

[0066] In an important embodiment of the present disclosure, said isoprenol of step a) is obtained via a fermentation step, said step comprising: providing a fermentation medium containing a substrate for microbial growth; and introducing into said medium an inoculum comprising a culture of one or more microorganisms selected from the group consisting of bacteria, molds and yeasts; wherein the one or more microorganisms are characterized in that they ferment the carbohydrate to form isoprenol.

[0067] The term "fermentation medium" is used herein to denote a three-phase (solid-liquid-gas) system held within a fermentation vessel. The liquid phase includes water, dissolved nutrients, dissolved substrates for microbial growth, and dissolved metabolic products, with the source of the water being without limitation, particularly process waters, such as backset and / or thin stillage, scrubber water, evaporator condensate or distillate, side stripper water from distillation, or other process waters of fermented plant products. The solid phase includes individual cells, pellets, insoluble substrates for microbial growth, and precipitated metabolic products.

[0068] In the context of microbial growth, the term "substrate" refers to a substance or compound that is or is meant to be converted into another compound by the action of an enzyme. The term "substrate" is intended to encompass not only compounds that provide a suitable carbon source for use as a starting material, such as carbohydrates derived from biomass, but also intermediate metabolites used in metabolic pathways associated with microorganisms. Fermentation media typically may contain one or more fermentable carbohydrates, such as sugars, as substrates.

[0069] The fermentation medium comprising the fermentation substrate and other raw materials used in the fermentation process of the present invention may be processed by comminution, liquefaction, saccharification, etc., prior to or simultaneously with the fermentation process. Thus, fermentation medium can refer to the medium prior to addition of the fermenting microorganism, e.g., the medium in liquefaction and / or saccharification, or the medium resulting from liquefaction and / or saccharification, as well as the medium comprising the fermenting microorganism, e.g., the medium used in a simultaneous saccharification and fermentation (SSF) or one-step fermentation process. For completeness, in the above-mentioned embodiments in which an antimicrobial agent is added to the fermentation medium prior to the inoculating microorganism, it is also included that the agent is added during liquefaction and / or saccharification.

[0070] As used herein, "inoculum" refers to an original source of a complex microbial community that is intended to be added to a fermentation vessel but does not limit the final composition of the microbial community, which is determined by the operating conditions and productivity of the fermentation vessel. The inoculum is generally formed by growing the desired microorganisms in a suitable growth tank, which is much smaller than the fermentation vessel.

[0071] The inoculum typically comprises a culture of one or more producing strains of a microorganism adapted by natural selection or by biotechnological means to produce the desired fermentation product. An exemplary, but non-limiting, inoculum comprises a culture of Escherichia coli as described in Kang et al. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production Metabolic Engineering Vol. 34: 25-25 (2016).

[0072] Further informative literature is known regarding the fermentative production of isoprenol. For example, US 20080092829 (Renninger et al.) describes the production of isoprenol from microorganisms, including bioengineered microorganisms: one of the biosynthetic pathways disclosed involves the mevalonic acid (MVA) pathway, in which mevalonic acid is produced, diphosphorylated, then decarboxylated-dehydrated to isoprenyl-pyrophosphate, and finally dephosphorylated twice to isoprenol. It is also known to form isoprenol from E. coli strains via the 2-methyl-D-erythritol-4-phosphatase (MEP) pathway. Furthermore, EP 2 516 656 A (Global Bioenergies et al.) discloses a method for producing isoprenol, characterized in that it comprises a step of converting mevalonic acid to isoprenol in an enzymatic pathway that includes an enzyme with diphosphomevalonate decarboxylase (EC 4.1.1.33) activity.

[0073] This fermentation embodiment is important in that it allows the coupling of a fermentation process for obtaining isoprenol from a carbohydrate source to steps a) to c) that can be carried out by whole cell biotransformation of the present disclosure. More specifically, as described herein below, specific production strains of microorganisms, such as, but not limited to, Escherichia coli, can be used to obtain 4-acetoxy-2-methylenebutyric acid starting from a carbohydrate source, provided that said production strains exhibit the enzymatic activities by which the cells of step b) (CB) and the cells of step c) (CC) are characterized.

[0074] Process A) Step a) of the present disclosure provides O-protection of the C1-hydroxyl group of isoprenol via acetylation: [ka]

[0075] The acetylation reaction of step a) may simply involve treating isoprenol with an acetylation reagent under effective acetylation conditions. Non-limiting examples of suitable acetylation reagents include acetic acid, acetic anhydride, and acetyl chloride; the use of one or more of said reagents is not excluded. Furthermore, the acetylation conditions may include a temperature of 20°C to 150°C, for example, 20°C to 100°C. The use of a catalyst may be beneficial and, although not intended to limit the disclosure, may include in this regard perchloric acid; magnesium perchlorate; copper perchlorate; sulfuric acid; hydrochloric acid; hydrobromic acid; trichloroacetic acid; copper chloride; copper sulfate; zinc chloride; zinc acetate; zinc sulfate; potassium metaperiodate; dimethyl sulfate; and dimethyl sulfate.

[0076] Regardless of the above, it is not excluded that step a) of the present disclosure is constituted by an enzymatic process carried out either via in vitro or whole cell biotransformation. In the latter respect, US20100180491A1 (Taek-Soon Lee et al.) describes culturing a genetically modified host cell expressing an enzyme capable of catalyzing the esterification of isoprenol with, in particular, acetic acid, such as alcohol acetyltransferase (AAT), wax ester synthase / diacylglycerol acyltransferase (WS / DGAT) or lipase, under conditions such that isoprenyl acetate is produced. Furthermore, Zada ​​et al. Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives, Biotechnology for Biofuels, Volume 11: 210 (2018) also describes the acetylation of the C1-hydroxy group of isoprenol, in which acetyl-coenzyme A (Acetyl-CoA) acts as the acetylating agent under the enzymatic catalysis of acetyltransferases, specifically chloramphenicol acetyltransferase (CAT).

[0077] Regardless of the chemical or biochemical mode of acetylation, the above reactions proceed as follows: 1 The conversion can be monitored by known techniques, including H NMR, Fourier transform infrared spectroscopy, ultra performance liquid chromatography (UPLC), gas chromatography, or thin layer chromatography (TLC). At the appropriate conversion point in the chemical acetylation process, the reaction can be quenched by adding an aqueous base, such as sodium bicarbonate or potassium carbonate.

[0078] The reaction product (II) can be isolated and used in crude form in step b), or alternatively, the isoprenyl acetate (II) can be purified using methods known to those skilled in the art, including, but not limited to, solvent extraction, filtration, evaporation, and chromatography.

[0079] Process B) This process of the present disclosure provides for the formation of 4-acetoxy-2-methylene-butan-1-ol (III) by whole cell biotransformation, which is depicted in the following reaction scheme: [ka]

[0080] The cell (CB) used in this process has a gene for alkane monooxygenase, optionally as part of an Alk operon. In one embodiment, the cell (CB) used can be a wild-type cell or a non-recombinant laboratory cell that has an alkane monooxygenase gene, optionally as part of an Alk operon, in particular as part of an Alk operon that contains gene products AlkB, AlkH and AlkJ. Examples include Pseudomonas putida, whose wild-type genotype contains two Alk operons, the first operon encoding the gene products AlkB, AlkF, AlkG, AlkH, AlkJ, AlkK and AlkL, and the second operon encoding AlkS and AlkT, where AlkS functions to regulate the expression of the first alk operon.

[0081] Alternatively, the cells (CB) used may be genetically modified relative to the wild type so that they are able to produce more 4-acetoxy-2-methylene-butan-1-ol (III) starting from isoprenyl acetate (II) compared to the wild type. This comparison is intended to encompass both: i) when the wild type genetically modified cells produce detectable amounts of 4-acetoxy-2-methylene-butan-1-ol; and ii) when the wild type genetically modified cells are unable to form detectable amounts of 4-acetoxy-2-methylene-butan-1-ol. Thus, for the second category of cells ii), detectable amounts of 4-acetoxy-2-methylene-butan-1-ol are only formed when the wild type is genetically modified to produce the cells used in step b).

[0082] The cells (CB) utilized are preferably genetically engineered to form at least 10 times, for example at least 100 times or at least 1000 times more 4-acetoxy-2-methylene-butan-1-ol (III) than wild-type cells in a defined time interval of 24 hours. The increase in product formation can be determined by culturing the cells used according to step b) of the present disclosure and the wild-type cells separately at the same initial cell density, nutrient medium and culture conditions for a specified time interval, and then measuring the amount of the target product in each nutrient medium.

[0083] The cells (CB) used in step b) of the present disclosure can be prokaryotic or eukaryotic. They can be mammalian cells, including human cells, plant cells, or microorganisms, such as fungi, molds or bacteria, with preferred microorganisms being those deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Yeasts can be important microorganisms (fungi) in this context, but it is preferred to use bacteria, and therefore, reference can be made to http: / / www.dsmz.de / species / bacteria.htm.

[0084] The potential use of wild-type Pseudomonas putida in the present invention has been described above. Without intending to limit the present invention, preferred cells (CB) that can be genetically modified and used in step b) of the present disclosure can be selected from the following genes: Corynebacterium; Brevibacterium; Bacillus; Lactobacillus; Lactococcus; Candida; Pichia; Kluveromyces; Saccharomyces; Escherichia; Zymomonas; Yarrowia; Methylobacterium; Ralstonia; Pseudomonas; Burkholderia; and Clostridium. Particularly preferred is the use of cells (CB) selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida. Good results have been obtained with the Escherichia coli strain BL21.

[0085] Compared to the wild type, the genetically modified cells (CB) according to step b) must show an increase in the activity of at least one of the alkane monooxygenase enzymes that catalyze the C4-hydroxylation of isoprenyl acetate: direct C4-hydroxylation of isoprenyl acetate (II) leading to 4-acetoxy-2-methylene-butan-1-ol (III).

[0086] The enzyme alkane monooxygenase may be encoded by the AlkB gene from Pseudomonas putida GP01, its variants and homologues. For example, the enzyme alkane monooxygenase may be encoded by the AlkB gene from Pseudomonas putida GP01, selected from the group consisting of the AIkBGT gene cluster; the AlkBGTJH gene cluster; and the AlkBGTJHL gene cluster. The isolation of the respective gene sequences is described, for example, in van Beilen et al., "Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria", Journal of Bacteriology, Vol. 184 (6), pages 1733-1742 (2002). Furthermore, DNA sequence information encoding AlkB, AlkF, AlkG and AlkT was obtained from the Pseudomonas putida OCT plasmid alk gene cluster identified as GenBank AJ 245436.1; and the plasmid sequence of the pCom10 vector into which AlkBGT was cloned can be found in GenBank AJ 302087.1.

[0087] Mutants are provided to improve the activity of AlkB on isoprenyl acetate and may be determined by rational design based on predictive models. For example, the structure of AlkB can be predicted by AlphaFold2 prediction software available from the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI); positions are selected from mutations based on docking studies using isoprenyl acetate as a ligand. Single mutants at each selected position can be generated by site-directed mutagenesis of pBT10 constructs. Exemplary mutants include, but are not limited to: AlkB(I233V), a mutant disclosed in Koch et al. [8]; and AlkB(F164L), a single mutant identified from docking studies using isoprenyl acetate as a ligand, which exchanges the amino acid phenylalanine (Phe) of wild-type AlkB with leucine (Leu).

[0088] Enzymes encoded by nucleic acid sequences having at least 40%, preferably at least 50%, more preferably at least 75% identity to the identified sequences are suitable for use in the methods of the invention. For completeness, further exemplary homologs of the AlkB gene include, but are not limited to, AlkB-P1 obtained from Pseudomonas putida P1; Alk1-MO obtained from Marinobacter hydrocarbonoclasticus; and AlkB1 obtained from Alcanivorax borkumensis.

[0089] To achieve increased intracellular activity of the aforementioned enzymes in genetically modified cells (CB), one or more of the following measures can be employed: increasing the copy number of the gene sequence encoding the enzyme; using a stronger promoter for the gene; using a stronger ribosome binding site; using codon optimization; and utilizing genes or alleles encoding the corresponding enzymes with increased activity, as described, for example, in Koch et al., "In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6", Applied and Environmental Microbiology, Vol. 75 (2), pages 337-344 (2009).

[0090] The genetically modified cells used in the methods according to the invention are produced by transformation, transduction and / or conjugation with an expression vector comprising a desired gene, an allele of this gene or a part thereof, and a vector allowing expression of the gene. Heterologous expression is achieved by integrating the gene or an allele thereof into the chromosome of the cell or into an extrachromosomal replicating vector. Without intending to limit the disclosure, WO2009 / 077461 (Evonik Degussa GmbH) provides a useful reference on genetic transformation of cells.

[0091] In the preparation of 4-acetoxy-2-methylene-butan-1-ol the following sub-steps can be identified: b) i) contacting the cells (CB) as defined above with either a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; and Optionally, b) ii) isolating the obtained 4-acetoxy-2-methylene-butan-1-ol.

[0092] The cells (CB) can be contacted with said culture medium in a bioreactor and can thus be cultivated continuously or discontinuously in a batch process, a fed-batch process or a repeated fed-batch process.

[0093] Traditional batch bioreactor systems are often considered "closed" in that the composition of the medium is established at the beginning of each bioproduction event and is not subject to any artificial changes or additions during a period that essentially ends at the end of the bioproduction event. In this situation, at the beginning of the bioproduction event, the medium is inoculated with the desired microorganism(s) and bioproduction is allowed to occur without adding anything to the system. However, in this disclosure, "batch" type bioproduction events encompass systems in which the substrate content of the medium is established at the beginning of each bioproduction event, but control of factors such as pH and oxygen concentration is attempted.

[0094] A variant of the standard batch system is the fed-batch system. A fed-batch bioproduction process includes a typical batch system with the exception that nutrients, including the substrate, are added in small increments as bioproduction proceeds. Fed-batch systems are useful when catabolite repression tends to inhibit cellular metabolism and it is desirable to limit the amount of substrate in the medium. Measurement of actual nutrient concentrations in a fed-batch system may be done directly, for example by sampling, or by measuring pH, dissolved oxygen, CO 2 The estimated value may be based on changes in measurable factors such as waste gas partial pressure.

[0095] Continuous bioproduction is considered an "open" system in which a defined bioproduction medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for further processing. Continuous bioproduction, whether in a chemostat or perfusion culture system, generally maintains the culture within a controlled density range, with the cells primarily in log-phase growth. Continuous bioproduction can limit downtime associated with draining, cleaning, and preparing the equipment for the next bioproduction event. In addition, performing downstream unit operations, such as distillation, continuously tends to be more economical than performing them in batch mode. Continuous bioproduction also allows for the adjustment of one or more factors that affect cell growth and end product concentration.

[0096] The bioproduction system is maintained within a suitable temperature range and a controlled dissolved oxygen concentration range for a sufficient time to obtain the desired conversion (C4-hydroxylation) of the isoprenyl acetate (II) substrate molecule to obtain the chemical product (III) in the bioreactor. Suitable temperature ranges for this step include 20°C to 50°C, for example 20°C to 40°C. The bioreactor used in this step may maintain anaerobic conditions, and aerobic conditions are generally maintained by the addition of oxygen or an oxygen-containing gas such as air. The desired aerobic, microaerobic or anaerobic conditions may be maintained or confirmed by monitoring the dissolved oxygen level of the liquid culture containing the nutrient medium.

[0097] It will be apparent to those skilled in the art that the culture medium used in this step must be suitable for the requirements of the microorganism used to produce the desired end product (III). To form the culture medium, at least one growth factor or its precursor; at least one nitrogen source; at least one phosphorus source; and at least one trace metal source selected from the group consisting of manganese, boron, cobalt, copper, molybdenum, zinc, calcium, magnesium, iron, nickel, and combinations thereof may be appropriately combined. Suitable growth factors include, but are not limited to, amino acids and vitamins, such as biotin, vitamin B12, derivatives of vitamin B12, thiamine, and pantothenate. Nitrogen sources include, but are not limited to, peptone, yeast extract, meat extract, malt extract, corn-steep liquor, soy flour, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, and the like. Phosphorus sources include, but are not limited to, phosphoric acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate. In addition, the medium may contain adjuvants such as pH adjusters including inorganic acids and bases, such as sodium hydroxide, potassium hydroxide, ammonia, aqueous ammonia, etc.; antifoaming agents; and antibiotics for maintaining plasmid stability.

[0098] The medium may be a defined medium. Also, step b) may be carried out using a minimal medium or a supplemented minimal medium. Furthermore, the use of commercially prepared media is not excluded, and may in particular include: Luria-Bertani (LB) broth; M9 minimal medium; Sabouraud dextrose (SD) broth; Yeast medium (YM) broth, and yeast synthetic minimal medium (Ymin).

[0099] In optional step b) ii) above, the obtained 4-acetoxy-2-methylene-butan-1-ol may be isolated from the bioreactor. This substep is considered optional since the present invention encompasses an embodiment in which both steps b) and c) are performed by whole cell biotransformation and the cells (hereafter CB, CC) utilized in each step are the same. That embodiment aside, depending on the cells (CB) utilized, the isolation of 4-acetoxy-2-methylene-butan-1-ol can be either from the culture medium if the host cell secretes it into the medium, or directly from the host cell that produces it if it is not secreted to a great extent. The isolated compound (III) may be optionally purified using methods known in the art, including but not limited to solvent extraction, filtration, evaporation, distillation, crystallization and chromatography.

[0100] Process C) This process of the present disclosure provides for the oxidation of 4-acetoxy-2-methylene-butan-1-ol (III) to 4-acetoxy-2-methylenebutyric acid (V), which can be considered as a two-step process (i), ii) in which 4-acetoxy-2-methylene-butanal (IV) is formed as an intermediate product, as shown in the following reaction scheme: [ka]

[0101] Step c) of the method can be divided into two main embodiments: in the first embodiment, the two oxidation steps (c)i), c)ii)) are separate, with intermediate isolation and optional purification of 2-acetoxy-2-methylene-butanal (IV); and in the second embodiment, the two oxidation steps do not involve this intermediate isolation of 2-acetoxy-2-methylene-butanal (IV), and therefore are carried out as a "one-pot" synthesis.However, the performance of the second embodiment does not exclude changing reaction conditions over time to drive each oxidation step in sequence.

[0102] It is believed that this reaction sequence can be realized by chemical oxidation using an oxidizing reagent, by biocatalytic oxidation, or by whole-cell biotransformation, either in a whole-cell biocatalyst that catalyzes both steps b) and c) or in a separate whole-cell biocatalyst that catalyzes step c) separately from step b). Herein, it is preferred to use either biocatalytic oxidation or whole-cell biotransformation. Conventional chemical oxidation methods often require the use of at least equimolar amounts of oxidizing reagents, and their atom efficiency is relatively low. Furthermore, the practical application of chemical routes is often limited by overoxidation, low selectivity, and the use of organic solvents. Conversely, biocatalytic oxidation and whole-cell biotransformation are characterized by excellent selectivity and mild reaction conditions.

[0103] Step c) Chemical oxidation When step c) is carried out chemically in two different reaction steps, the first step (c)i)) is the oxidation of the primary alcohol (III) to the corresponding aldehyde (IV). There is no intention to limit the oxidizing agents that may find utility in this step. Exemplary reagents include copper, cobalt and chromium oxides; copper, silver and mixtures thereof; platinum; platinum dioxide; cerium ammonium nitrate; sodium bromate; lead tetraacetate; hexavalent chromium; sodium or potassium dichromate; pyridinium dichromate; chromic acid; Collins' reagent; pyridinium chlorochromate; chromyl chloride; di-tert-butylchromate; manganese dioxide; tetrachloro-o-benzoquinone; tetrachloro-p-benzoquinone; 2,3-dichloro-5,6-dicyano-p-benzoquinone; dimethyl sulfide and chlorine; hypervalent iodine compounds; N-chlorosuccinimide (NCS); tert-butyl hydroperoxide (TBHP); and dimethyl sulfoxide (DMSO). US Pat. No. 5,132,465 A provides useful background literature in this regard.

[0104] The second step (c)ii)) requires the oxidation of the aldehyde (IV) to the corresponding carboxylic acid (V). Suitable oxidizing agents for this step include, but are not limited to, Jones' reagent; potassium permanganate; silver oxide; and nitric acid. Such oxidizing agents are preferably used in conjunction with a catalyst such as sulfuric acid, and further, the solvent is preferably either water or a water-miscible solvent such as acetone, tetrahydrofuran, methanol, or ethanol. The oxidation reaction (step c)ii)) is conventionally carried out at a temperature between 20° C. and 50° C. for a period between 0.5 h and 5 h.

[0105] As will be appreciated by those skilled in the art, the direct or "one-pot" oxidation of 4-acetoxy-2-methylene-butan-1-ol (III) to 4-acetoxy-2-methylenebutyric acid (IV) can be carried out under certain circumstances: typically, a stoichiometric excess of oxidizing agent is required and the aldehyde (IV) formed as an intermediate product must be retained in the reaction mixture. 2 CrO 4 );Na 2 CrO 4; K 2 CrO 4 ;K 2 Cr 2 O 7 ;K 2 Cr 2 O 7 When an oxidizing agent selected from; permanganate; and Jones reagent is used in stoichiometric excess, 4-acetoxy-2-methylenebutyric acid (V) is the main product. For completeness, the stoichiometric excess of the Jones reagent means a stoichiometric excess of the chromic acid relative to 4-acetoxy-2-methylenebutan-1-ol (III). Alternative methods that may be suitable for oxidizing 4-acetoxy-2-methylene-butan-1-ol (III) to 4-acetoxy-2-methylenebutanic acid (V) without intermediate isolation of 2-acetoxy-2-methylene-butanal (IV) are disclosed, inter alia, in WO99 / 52849; WO2006 / 001387; and US2009 / 0124806A1.

[0106] The progress of the reaction in step c) is as follows: 1 The activity can be monitored by known techniques such as 1 H NMR, Fourier transform infrared spectroscopy, ultra performance liquid chromatography (UPLC), gas chromatography, or thin layer chromatography (TLC).

[0107] The 4-acetoxy-2-methylenebutyric acid (V) product of step c) can be isolated from the reactor. Prior to use in subsequent synthetic steps, the isolated 4-acetoxy-2-methylenebutyric acid (V) can be optionally purified using methods known in the art, including, but not limited to, solvent extraction, filtration, evaporation, distillation, crystallization and chromatography.

[0108] Step c) Enzymatic oxidation Regarding biocatalytic oxidation, it is not wise to suggest preferential use of either enzymatic oxidation or whole-cell biotransformation based on genetically engineered cells. Indeed, compared to isolated enzymes, the use of whole-cell catalysts simplifies the procedure, reduces costs and improves enzyme stability. However, in certain situations, the active form of the enzymes prevents their use in whole-cell biotransformations.

[0109] The present disclosure provides an enzymatic oxidation process characterized in step c) comprising contacting 4-acetoxy-2-methylene-butan-1-ol (III) under aerobic conditions with at least one enzyme exhibiting oxidative activity and, optionally, with at least one mediator compound enhancing the oxidative activity of the enzyme. The reaction mixture should preferably contain 0.001-10 mg of said at least one enzyme per kg of 4-acetoxy-2-methylene-butan-1-ol (III).

[0110] Said at least one enzyme exhibiting oxidizing activity is usually chosen from alcohol and aldehyde oxidases: such enzymes with a wide substrate spectrum are described in the literature and mention can be made of alcohol dehydrogenases (ADH), e.g. Escherichia coli EcAdhZ3-LND; alcohol oxidases (AlcOx), e.g. Aspergillus fumigatus LCAO_Af; aldehyde dehydrogenases (AlDH); and laccases.

[0111] Without intending to limit the present invention, a useful alcohol dehydrogenase (ADH) enzyme may be encoded by a homologue of the AlkJ gene, and a useful aldehyde dehydrogenase (AlDH) enzyme may be encoded by the AlkH gene from Pseudomonas putida GP01. DNA sequence information for AlkJ alcohol dehydrogenase and AlkH dehydrogenase can be obtained from the Pseudomonas putida OCT plasmid alk gene cluster identified as GenBank AJ 245436.1. And, enzymes encoded by nucleic acids having at least 40%, preferably at least 75%, more preferably at least 90% identity to the recorded sequences are suitable for use in the methods of the present invention.

[0112] As will be appreciated by those of skill in the art, operational activity of alcohol dehydrogenase (ADH) enzymes can only be achieved in the presence of oxidized cofactors as hydride acceptors, particularly oxidized nicotinamide cofactors. Similarly, alcohol oxidase (AlcOx) transfers reducing equivalents to molecular oxygen and produces H as a stoichiometric by-product. 2 O 2 It functions by generating flavin-dependent or Cu 2+It may be dependent on the aldehyde-catalyzed oxidation. Furthermore, it is known that ADH and AlcOx are generally unable to oxidize aldehydes because the aldehyde proton is not abstracted as a hydride. This mechanistic limitation can be overcome by providing a nucleophilic cofactor that transiently attacks the carbonyl group, turning it into an alcohol containing a proton that can be abstracted hydrodically. Aldehyde dehydrogenases (AlDHs) exploit this approach through a cysteine ​​moiety in the enzyme active site.

[0113] Based on the above considerations, it will be customary to have at least one mediator compound present in the reaction mixture that enhances the oxidative activity of the enzyme, which should preferably be present in the reaction mixture in an amount of 0.001-10 mg / kg of 4-acetoxy-2-methylene-butan-1-ol.

[0114] In general, such mediator compounds selectively bind to the compound to be oxidized. The extent of binding of the mediator compound (A) to the compound to be oxidized (B) is determined by the chemical equilibrium constant (K d ) can be quantified by:

number

[0115] Chemical equilibrium constant (K d ) is given by:

number

[0116] As used herein, the intermediate compound has a chemical equilibrium constant (K d ) is 1x10 -4 Less than 1x10 -6 It is preferable that it is less than 1000 .mu.m.

[0117] Without intending to limit the present invention, suitable classes of mediator compounds include oxidized nicotinamide cofactors; antibodies and fragments thereof that retain their binding properties; peptides; and peptidomimetics, in which peptides are modified by the incorporation of non-natural amino acids and / or non-natural chemical bonds between amino acids. Furthermore, systems containing laccase can include known compounds of the laccase mediator system (LMS), of which HBT (1-hydroxybenzotriazole); ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)]; NHA (N-hydroxyacetanilide); NEIAA (N-acetyl-N-phenylhydroxylamine); HBTO (3-hydroxyl,2,3-benzotriazin-4(3H)-one); syringaldazine; and VIO (violuric acid). Also useful disclosures regarding mediator compounds include: WO 94 / 04678 A (Casterman et al.); WO-94 / 25591 A (Unilever PLC et al.); and WO 94 / 29457 A (Unilever PLC).

[0118] As described above, 4-acetoxy-2-methylene-butan-1-ol (III) is contacted with the enzyme and mediator under aerobic conditions. The most economical source of oxygen is air, which has the advantages of being readily available from the atmosphere at no cost, being non-toxic, and not needing to be removed after the reaction. Alternatively, molecular oxygen itself may be used, or a molecular oxygen liberating system such as a mixture of catalase enzyme and hydrogen peroxide may be used.

[0119] In an exemplary implementation, introduced air or oxygen, or in situ generated oxygen, is mixed with the reaction mixture with stirring for a time sufficient to convert substantially all of the 4-acetoxy-2-methylene-butan-1-ol (III) to 4-acetoxy-2-methylenebutyric acid (V). A typical reaction period is less than 24 hours, for example 1 to 5 hours.

[0120] The reaction mixture may further include a buffer solution to ensure that its pH remains within an appropriate range for the enzyme to be active. For example, most laccases exhibit a pH optimum in the acidic pH range, but show low activity or inactivity at pH 7 or above. That being said, the pH of the reaction mixture is generally kept in the range of 5 to 9, for example, 5.5 to 8.5. While there is no intention to limit the buffers used in such ranges, mention may be made of imidazole; 1,4-piperazinediethanesulfonic acid (PIPES); 4-morpholinepropanesulfonic acid (MOPS); 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES); triethanolamine; tris(hydroxymethyl)aminomethane (TRIS); alkali metal phosphates; citric acid; and trisodium citrate. The reaction mixture is maintained at a temperature that allows the reaction to proceed substantially to completion. The enzyme may be active at temperatures ranging from 0°C to 80°C, although it will be more common to use temperatures ranging from 10°C to 50°C. Use of a temperature in the range of 15° C. to 35° C. is advantageous as it eliminates the need to substantially heat or cool the reaction mixture from room temperature.

[0121] The progress of the reaction in step c) is as follows: 1 H NMR, Fourier transform infrared spectroscopy, ultra-performance liquid chromatography (UPLC), gas chromatography or thin layer chromatography (TLC) The activity can be monitored by known techniques such as:

[0122] The 4-acetoxy-2-methylenebutyric acid (V) product of step c) can be isolated from the reactor. The isolated compound (V) may be purified, if necessary, using methods known in the art, including, but not limited to, solvent extraction, filtration, evaporation, distillation, crystallization, and chromatography.

[0123] Step c) Whole cell biotransformation For the implementation of this step by whole cell biotransformation, two options are considered. In a first embodiment for producing 4-acetoxy-2-methylene-butyric acid, the following substeps are carried out: c) i) contacting the cells (CC) with either a medium containing 4-acetoxy-2-methylene-butan-1-ol (III) or a medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-ol under conditions that allow the cells to form 4-acetoxy-2-methylene-butyric acid (V) from 4-acetoxy-2-methylene-butan-1-ol (III); optionally c) ii) isolating the resulting 4-acetoxy-2-methylene-butyric acid (V); Here, said cells (CC) exhibit the activity of at least one enzyme exhibiting oxidative activity.

[0124] As a preliminary point regarding this embodiment, it is not excluded that the cell (CB) of step b) and the cell (CC) of step c) are identical, but a single cell may have the relevant enzymatic activities of both steps. Apart from that, the cell (CC) used in step c) i) may be a wild-type cell or a non-recombinant laboratory cell carrying a gene encoding at least one enzyme exhibiting oxidative activity, in this respect the cell may optionally carry an Alk operon, in particular an Alk operon containing the gene products AlkB, AlkH and AlkJ. In this context, reference may again be made to Pseudomonas putida. Alternatively, the cell (CC) used in this embodiment may be genetically modified with respect to its wild-type so that it is able to produce more 4-acetoxy-2-methylenebutyric acid starting from 4-acetoxy-2-methylene-butan-1-ol compared to the wild-type. This comparison is intended to encompass both: α) when wild-type genetically modified cells produce detectable amounts of 4-acetoxy-2-methylenebutyric acid; and β) when wild-type genetically modified cells are unable to produce detectable amounts of 4-acetoxy-2-methylenebutyric acid. Thus, for the second category of cells β), detectable amounts of 4-acetoxy-2-methylenebutyric acid are only formed when genetically modified relative to the wild-type to create the cells used in step c).

[0125] In a second embodiment of step c) for producing 4-acetoxy-2-methylene-butyric acid, the following substeps are carried out: c) i) contacting cells (CC1) with a culture medium containing 4-acetoxy-2-methylene-butan-1-ol (III) or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-ol under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-al (IV) from 4-acetoxy-2-methylene-butan-1-ol; c) ii) optionally isolating the obtained 4-acetoxy-2-methylene-butan-1-al (IV); c) iii) contacting the cells (CC2) with a culture medium containing 4-acetoxy-2-methylene-butan-1-al (IV) or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-al (IV) under conditions that allow the cells to form 4-acetoxy-2-methylenebutyric acid (V) from 4-acetoxy-2-methylene-butan-1-al; and c) iv) isolating the resulting 4-acetoxy-2-methylenebutyric acid (V); Here, said cells (CC1, CC2) each exhibit an increased activity of at least one enzyme exhibiting oxidative activity.

[0126] As mentioned above, it is not necessary to carry out step c) ii). If the cells (CC1) secrete 4-acetoxy-2-methylene-butan-1-al (IV) into the culture medium, the cells (CC2) may be placed in the same bioreactor and there is no need to carry out intermediate separation of 4-acetoxy-2-methylene-butan-1-al. If either cell (CC2) is placed in a separate downstream bioreactor or if the host cell (CC1) that produces 4-acetoxy-2-methylene-butan-1-al does not secrete it, step c) ii) may be carried out to isolate the aldehyde.

[0127] As a preliminary point regarding this embodiment, it is not excluded that the cell (CB) of step b) and the cell (CC1) of step c) i) are identical, but rather a single cell may have the relevant enzymatic activities of both steps. Apart from that, the cell used (step c) i) may be a wild-type cell or a non-recombinant laboratory cell carrying a gene encoding at least one enzyme exhibiting oxidative activity, in this regard the cell (CC1) may optionally carry an Alk operon, in particular an Alk operon containing the gene products AlkH and AlkJ. In this context, reference may again be made to Pseudomonas putida. Alternatively, the cell used (CC1) may be genetically modified with respect to its wild-type so that it is able to produce more 4-acetoxy-2-methylene-butan-1-al starting from 4-acetoxy-2-methylene-butan-1-ol compared to the wild-type.

[0128] The cell (CC2) used may be a wild-type cell or a non-recombinant laboratory cell carrying a gene encoding at least one enzyme exhibiting oxidative activity, and in this regard, the cell (CC2) may optionally carry an Alk operon, in particular an Alk operon containing the gene products AlkH and AlkJ. Alternatively, the cell (CC2) used may be genetically modified relative to its wild-type so that it is able to produce more 4-acetoxy-2-methylenebutyric acid starting from 4-acetoxy-2-methylene-butan-1-al compared to the wild-type.

[0129] As mentioned above, any comparison between recombinant and wild-type cells is intended to encompass both: α) cases where the wild-type genetically modified cells produce detectable amounts of target product; and β) cases where the wild-type genetically modified cells are unable to form detectable amounts of target product. Thus, for said second category of cells β), detectable amounts of target product are only formed upon genetic modification to the wild-type to create the cells used in steps c) i) and c) iii).

[0130] Each genetically modified cell (CC, CC1, CC2) is preferably genetically modified such that it forms at least 10 times, for example at least 100 times or at least 1000 times more target product than wild-type cells in a defined time interval of 24 hours. The increase in product formation can be measured by culturing the cells used according to step c) of the present disclosure and the wild-type cells separately at the same initial cell density, nutrient medium and culture conditions for a specific time interval, and then measuring the amount of target product in each nutrient medium.

[0131] The cells (CC, CC1, CC2) used in step c) of the present disclosure can be prokaryotic or eukaryotic, independently. They can be mammalian cells (including human cells), plant cells or microorganisms, such as fungi, molds or bacteria, with preferred microorganisms being those deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Yeasts can be important microorganisms (fungi) in this context, but it is preferred to use bacteria, and therefore reference can be made to http: / / www.dsmz.de / species / bacteria.htm.

[0132] Without intending to limit the present invention, the cells (CC, CC1, CC2) that may be genetically modified and used in step c) of the present disclosure are preferably independently selected from the following genes: Corynebacterium; Brevibacterium; Bacillus; Lactobacillus; Lactococcus; Candida; Pichia; Kluveromyces; Saccharomyces; Escherichia; Zymomonas; Yarrowia; Methylobacterium; Ralstonia; Pseudomonas; Burkholderia; and Clostridium. A particular preference may be mentioned for the use of cells independently selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida (CC, CC1, CC2). Good results have been obtained with the E. coli BL21 strain.

[0133] Compared to its wild type, the genetically modified cell (CC) according to step c) should show an increase in at least one activity of the following enzymes: alcohol dehydrogenase (ADH); alcohol oxidase (AlcOx); aldehyde dehydrogenase (AlDH); and laccase. Compared to the wild type, the genetically modified cell (CC1) should show an increase in at least one activity of the following enzymes: alcohol dehydrogenase (ADH); alcohol oxidase (AlcOx); and laccase. Also, compared to the wild type, the genetically modified cell (CC2) should show an increase in at least one activity of the following enzymes: aldehyde dehydrogenase (AlDH) and laccase.

[0134] Without intending to limit the invention, the enzyme alcohol dehydrogenase can be encoded by a homologue of the AlkJ gene, and the aldehyde dehydrogenase can be encoded by the AlkH gene from Pseudomonas putida GP01. DNA sequence information for AlkJ alcohol dehydrogenase and AlkH dehydrogenase can be obtained from the Pseudomonas putida OCT plasmid alk gene cluster identified as GenBank AJ 245436.1. And, enzymes encoded by nucleic acids having at least 40%, such as at least 75%, preferably at least 90% identity to the listed sequences are suitable for use in the methods of the invention.

[0135] To achieve increased intracellular activity of the aforementioned enzymes in genetically modified cells (CC, CC1, CC2), one or more of the following measures can be used: increasing the copy number of the gene sequence encoding the enzyme; using a stronger promoter of the gene; using a stronger ribosome binding site; using codon optimization; and utilizing genes or alleles encoding the corresponding enzymes with increased activity.

[0136] Where applicable, genetically modified cells (CC, CC1, CC2) are created by transformation, transduction and / or conjugation with a vector containing the desired gene, an allele of this gene or a portion thereof, and a vector allowing expression of the gene. Heterologous expression is achieved by integration of the gene or its allele in the chromosome of the cell or an extrachromosomally replicating vector. While not intending to limit this disclosure, WO2009 / 077461 (Evonik Degussa GmbH) provides a useful reference for cell genetic transformation.

[0137] The cells utilized (CC, CC1, CC2) can be contacted with said medium in one or more bioreactors: as described above, cells CC1 and CC2 can be placed in the same or separate bioreactors depending on the secretion of the target product by cells CC1, the process suitability of the independent strains CC1 and CC2, and / or the isolation of the intermediate aldehyde before the second oxidation step c) iii). In the one or more bioreactors, cells (CC, CC1, CC2) are cultivated continuously or discontinuously in a batch process, a fed-batch process, or a repeated fed-batch process, as described above.

[0138] In the or each bioreactor defined in these embodiments of step c), the bioproduction system is maintained within an appropriate temperature range and within a controlled dissolved oxygen concentration range for a time sufficient to obtain the desired conversion of the starting alcohol or aldehyde to the target product. A temperature range of 20°C to 50°C, for example 20°C to 40°C, is cited as being suitable. Anaerobic conditions may be maintained in the bioreactor used in this step, but it is more typical to maintain aerobic conditions therein via the addition of oxygen or an oxygen-containing gas such as air. The desired aerobic, microaerobic or anaerobic conditions can be maintained or confirmed by monitoring the dissolved oxygen level of the liquid culture containing the nutrient medium.

[0139] It will again be clear to those skilled in the art that the culture medium used in this step must be suitable for the requirements of the microorganism used to produce the desired end product. To form the culture medium, at least one growth factor or precursor thereof; at least one nitrogen source; at least one phosphorus source; and at least one trace metal source selected from the group consisting of manganese, boron, cobalt, copper, molybdenum, zinc, calcium, magnesium, iron, nickel and combinations thereof may be appropriately combined. Suitable growth factors include, but are not limited to, amino acids and vitamins, such as biotin, vitamin B12, derivatives of vitamin B12, thiamine and pantothenic acid. Nitrogen sources include, but are not limited to, peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Phosphorus sources include, but are not limited to, phosphoric acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate. It will further be appreciated that the medium may contain adjuvants such as inorganic acids and bases, e.g., pH adjusters including sodium hydroxide, potassium hydroxide, ammonia, aqueous ammonia; antifoaming agents; antibiotics to maintain plasmid stability; and the like.

[0140] The medium may be a defined medium. Also, step b) may be carried out using a minimal medium or a supplemented minimal medium. Furthermore, the use of commercially prepared media is not excluded, and may in particular include: Luria-Bertani (LB) broth; M9 minimal medium; Sabouraud dextrose (SD) broth; Yeast medium (YM) broth, and yeast synthetic minimal medium (Ymin).

[0141] In the aforementioned final sub-step c) ii) or c) iv), respectively, of each of the above embodiments, the obtained 4-acetoxy-2-methylenebutyric acid may be isolated from the bioreactor. Depending on the cells used, the isolation of 4-acetoxy-2-methylenebutyric acid is either from the culture medium if the host cell secretes it into the medium, or directly from the host cell producing it if it is not secreted. The isolated 4-acetoxy-2-methylenebutyric acid may be optionally purified using methods known in the art, including but not limited to solvent extraction, filtration, evaporation, distillation, crystallization and chromatography.

[0142] Process D) This process provides for the conversion of 4-acetoxy-2-methylenebutyric acid to α-methylene-γ-butyrolactone. Although there is no intention to limit the manner in which this process is carried out, two preferred embodiments are worth mentioning.

[0143] In a first embodiment, this step d) can be carried out in a one-pot synthesis, in which 4-acetoxy-2-methylenebutyric acid is subjected to acidic conditions at temperatures between 30° C. and 300° C., for example between 30° C. and 150° C., under which hydrolysis occurs to give γ-hydroxy-α-methylenebutyric acid as intermediate product, which is cyclized under such acidic conditions to give α-methylene-γ-butyrolactone. [ka]

[0144] According to this embodiment of step d), either a Lewis acid or a Bronsted acid can be used, and typically the molar ratio of said Lewis acid or said Bronsted acid to 4-acetoxy-2-methylenebutyric acid should be in the range of 0.15 to 1:1. Suitable Lewis acids include BF 3 ;AlCl 3 ;t-BuCl / Et 2 AlCl;Cl 2 / BCl 3 ;AlBr 3 ;AlBr 3.TiCl 4 ;ZrCl 4 ;I 2 ;SbCl 5 ;WCl 6 ;AlEt 2 Cl;PF 5 ;VCl 4 ;AlEtCl 2 ;B.F. 3 Et 2 O;PCl 5 ;PCl 3 ;POCl 3 ;TiCl 6 ;FeCl 3 ;NiCl 2 ; and SnCl 4 Examples of suitable Bronsted acids include, but are not limited to, those mentioned above. Suitable Bronsted acids may be organic or inorganic acids, but it is preferable to use Bronsted acids having a pKa value of at most 2.5, and in particular Bronsted acids having a pKa value of 2.5 to -10. Examples of suitable Bronsted acids include sulfuric acid; phosphoric acid; hydrochloric acid, hydrobromic acid; hydroiodic acid; methanesulfonic acid; p-toluenesulfonic acid; perfluoroalkanesulfonic acids, such as trifluoromethanesulfonic acid (or triflic acid, CF 3 SO 3 H), C 2 F 5 SO 3 H, C 4 F 9 SO 3 H, C 5 F 11 SO 3 H, C 6 F 13 SO 3 H and C 8 F 17 SO 3 Mention may be made of Bronsted acids selected from the group consisting of H; tetrafluoroboric acid; trifluoroacetic acid; trichloroacetic acid; and oxalic acid.

[0145] The progress of the above reaction is as follows: 1The conversion can be monitored by known techniques, which can include H NMR, Fourier transform infrared spectroscopy, ultra-performance liquid chromatography (UPLC), gas chromatography or thin layer chromatography (TLC). At an appropriate level of conversion, the reaction mixture can first be cooled to room temperature. The α-methylene-γ-butyrolactone is then isolated and may be used in crude form in any subsequent synthetic steps or may be further purified. Effective isolation and purification methods include, but are not limited to, solvent extraction, filtration, evaporation, distillation, crystallization and chromatography.

[0146] In a second embodiment, α-methylene-γ-butyrolactone is synthesized in a two-step process via α-hydroxy-γ-methylenebutyric acid (VI), which can be summarized in the following reaction scheme: [ka]

[0147] The first step conversion of the acetoxy (OAc) group to a hydroxyl group can be carried out reductively, by basic hydrolysis, or enzymatically using a suitable lipase or carboxylesterase enzyme.

[0148] In the reductive transformation, 4-acetoxy-2-methylenebutyric acid (V) is contacted with at least one hydride donor selected from borohydride and aluminum hydride compounds. The hydride donor is, for example, potassium borohydride (KBH 4 ); Sodium borohydride (NaBH 4 ); Sodium triacetoxyborohydride (NaBH(OAc) 3 ); Diborane (B 2 H 6 ); Sodium cyanoborohydride (NaBH 3 CN); Zinc borohydride (ZnBH 4 ); Aluminum hydride (AlH 3 ); and lithium aluminum hydride (LiAlH 4) may be selected from the group consisting of

[0149] Preferably, the hydride donor(s) is added to the entire reaction mixture in an amount of at least 0.35 molar equivalents relative to the amount of 4-acetoxy-2-methylenebutyric acid, for example, the hydride donor may be added in an amount of 0.5 to 2 molar equivalents relative to the amount of 4-acetoxy-2-methylenebutyric acid. As will be appreciated by those skilled in the art, the latter may be added in an amount of 0.5 to 2 molar equivalents relative to the amount of 4-acetoxy-2-methylenebutyric acid. 4 The equivalent range of 2 to 8 fold excess of hydride (H) helps ensure complete reaction.

[0150] The choice of hydride donor and / or the desired selectivity of the reaction will determine the solvent in which this reduction step is carried out. Exemplary solvents for this stage of step d) are water; 1 -C 8 Alkanols; Acetonitrile; N,N-Di(C 1 -C 4 ) alkyl acylamides, such as N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMAc); hexamethylphosphoramide; N-methylpyrrolidone; pyridine; esters, such as (C 1 -C 8 ) alkyl acetates, ethoxydiglycol acetate, dimethyl glutarate, dimethyl maleate, dipropyl oxalate, ethyl lactate, benzyl benzoate, butyl octyl benzoate and ethylhexyl benzoate; ketones, such as acetone, ethyl ketone, methyl ethyl ketone (2-butanone) and methyl isobutyl ketone; ethers, such as tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF) and 1,2-dimethoxyethane; 1,3-dioxolane; dimethyl sulfoxide (DMSO); and dichloromethane (DCM).

[0151] The reductive conversion of 4-acetoxy-2-methylenebutyric acid (V) to γ-hydroxy-α-methylenebutyric acid (VI) does not require special equipment to exclude water or air, and the reduction can be carried out at temperatures between 0 and 40°C, with room temperature being the preferred temperature condition.

[0152] In the conversion of 4-acetoxy-2-methylenebutyric acid (V) to α-hydroxy-γ-methylenebutyric acid (VI) under basic conditions, a reaction mixture is provided that includes water, 4-acetoxy-2-methylenebutyric acid, and a base. The base should be present in the reaction mixture in an amount of 1 to 5 molar equivalents relative to the amount of 4-acetoxy-2-methylenebutyric acid. Preferably, the base is present in an amount of 1.5 to 3 molar equivalents or 2 to 3 molar equivalents relative to the amount of 4-acetoxy-2-methylenebutyric acid.

[0153] Although not limiting the present invention, alkali metals (C 1 -C 4 It is preferable that the catalyst is composed of at least one compound selected from the group consisting of alkoxides, alkali metal carbonates, alkaline earth metal carbonates and alkali metal hydroxides. It is preferable to use at least one of potassium carbonate, sodium carbonate and calcium carbonate.

[0154] The basic hydrolysis may be carried out under reflux conditions. As will be appreciated by those skilled in the art, the reflux temperature will depend on the reactants and solvents present. It is recognized that the reflux temperature (at atmospheric pressure) for this step is typically between 100°C and 2250°C, for example between 125°C and 2100°C. In another methodology, the basic hydrolysis may be carried out in a closed autoclave where autogenous pressure is generated depending on the temperature at which the reaction is carried out. Temperatures between 100°C and 2500°C, for example between 100°C and 2250°C, may be established, such reaction temperatures generating autogenous pressures in the range of 0.5 to 40 bar.

[0155] The progress of the above reaction is as follows: 1The conversion can be monitored by known techniques, which can include H NMR, Fourier transform infrared spectroscopy, ultra-performance liquid chromatography (UPLC) or thin layer chromatography (TLC). At the appropriate level of conversion, the reaction mixture can be first cooled to room temperature. The α-hydroxy-γ-methylenebutyric acid (VI) can then be isolated and used in the subsequent synthetic steps in crude form or can be further purified. Effective isolation and purification methods include, but are not limited to, solvent extraction, filtration, evaporation, distillation, crystallization and chromatography.

[0156] The second step of the aforementioned scheme can be carried out enzymatically: for example, WO 2002101013 A2 (EI DuPont de Nemours and Company) discloses the formation of α-methylene-γ-butyrolactone from α-methylene-γ-hydroxybutyrate via a glucosylated intermediate under the action of the UDP-glucose glucosyltransferase enzyme.

[0157] In carrying out the second step of the scheme chemically, α-hydroxy-γ-methylenebutyric acid (VI) is subjected to strong acidic conditions at temperatures between 30° C. and 300° C., for example between 30° C. and 150° C., whereby the hydroxy acid cyclizes to give α-methylene-γ-butyrolactone (VII). Either Lewis or Bronsted acids may be used in this step, and typically the molar ratio of said Lewis or Bronsted acid to 4-acetoxy-2-methylenebutyric acid (V) should be in the range of 0.15 to 1:1. Suitable Lewis acids include, but are not limited to, aluminum trichloride; tin tetrachloride; titanium tetrachloride; zirconium tetrachloride; iron trichloride; and nickel dichloride. Suitable Bronsted acids may be organic or inorganic, but it is preferred to use Bronsted acids with a pKa value of at most 2.5, particularly Bronsted acids with a pKa value of 2.5 to -10. Mention may be made of Bronsted acids selected from the group consisting of sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, methanesulfonic acid, p-toluenesulfonic acid, tetrafluoroboric acid, trifluoroacetic acid, trichloroacetic acid, and oxalic acid.

[0158] The progress of the second reaction can be monitored by the techniques referenced above. At the appropriate level of conversion, the reaction mixture can first be cooled to room temperature. The α-methylene-γ-butyrolactone can then be isolated and used in crude form in any subsequent synthetic steps or can be further purified. Effective isolation and purification methods include, but are not limited to, solvent extraction, filtration, evaporation, distillation, crystallization and chromatography.

[0159] Application of α-methylene-γ-butyrolactone Formation of polymers with pendant lactone rings The present disclosure provides for the polymerization of α-methylene-γ-butyrolactone (hereinafter MBL) as defined above to form a polymer having a pendant lactone ring. Generally, the polymerization is carried out under anionic conditions, and one skilled in the art will select appropriate conditions such that the vinyl addition route of polymerization dominates over the competing ring-opening polymerization route. The polymer or copolymer thus obtained (p-MBL) retains the lactone structure in its repeating unit.

[0160] The monomer (MBL) may be incorporated into a copolymer (p-MBL) with at least one comonomer. Most broadly, a viable comonomer is one that provides a reasonable polymerization reaction rate under suitable practical anionic polymerization conditions. Preferably, the at least one comonomer is a non-carbonyl-donating olefinically unsaturated monomer selected from the group consisting of (meth)acrylonitrile; alkyl (meth)acrylate esters; (meth)acrylic acid; vinyl esters; and vinyl monomers.

[0161] Suitable vinyl monomers include ethylene; 1,3-butadiene; isoprene; styrene; divinylbenzene; heterocyclic vinyl compounds; and vinyl halides, such as chloroprene. Suitable vinyl esters include vinyl acetate, vinyl propionate, vinyl versatate, and vinyl laurate. Suitable alkyl esters of acrylic and methacrylic acid include C 1 -C 14 and di(meth)acrylate esters of alkanediols, such as 1,6-hexanediol diacrylate, and non-limiting examples thereof include methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, n-butyl acrylate, n-butyl methacrylate, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, isopropyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, isopropyl methacrylate, n-propyl acrylate, n-propyl methacrylate, and di(meth)acrylate esters of alkanediols, such as 1,6-hexanediol diacrylate.

[0162] The anionic polymerization of the α-methylene-γ-butyrolactone (MBL) and any comonomers present is carried out in the presence of an initiator selected from the group consisting of alkali metal organyls; alkali metal alkoxides; alkali metal thiolates; alkali metal amides; and compounds of elements of Group 3a of the Periodic Table of the Elements, preferably aluminum or boron organyls and aluminum alkoxides, such as aluminum trimethoxide, aluminum triethoxide, aluminum tripropoxide and aluminum tributoxide.

[0163] The amount of initiator used is not particularly limited, but is usually 0.0001 to 5 parts by weight, preferably 0.05 to 1 part by weight, based on 100 parts by weight of monomer. Furthermore, anionic polymerization can be carried out in solution or in the melt without the use of a solvent. If used, a suitable solvent for polymerization should be a non-reactive organic liquid capable of dissolving at least 1 wt.%, preferably more than 10 wt.%, of the polymer at 25°C. Exemplary solvents include dichloromethane and tetrahydrofuran (THF).

[0164] The anionic polymerization process is desirably carried out in the presence of a Lewis acid, particularly an "aprotic" Lewis acid that cannot function as a source of protons (H+). Furthermore, the anionic homopolymerization or copolymerization of α-methylene-γ-butyrolactone (MBL) should be carried out under anhydrous conditions and in the absence of compounds with active hydrogen atoms, except when the initiator compound is intentionally included.

[0165] The process pressure is not critical in that the reaction can be carried out at subatmospheric, atmospheric or superatmospheric pressure, but anionic polymerizations should typically be carried out at a temperature of at least 25° C. The reaction temperature may be 200° C. or higher, but it is preferred that the temperature not exceed 200° C., 175° C., or 150° C., particularly to maintain workable reactor pressures, to minimize the rate of polymer decomposition and the associated production of volatile impurities or other by-products, and, in some cases, to maintain adequate catalyst activity without deactivating or decomposing the catalyst.

[0166] The reaction product may be isolated and purified using methods known in the art. In this context, extraction, evaporation, distillation and chromatography may be mentioned as suitable techniques, but the reaction product is most conveniently isolated by distilling the solvent and unreacted starting materials under reduced pressure. If the (optionally purified) reaction product is intended to be stored at the time of production, the polymer should be placed in a container with an airtight and moisture-resistant seal.

[0167] Exemplary homopolymers or copolymers derived by anionic polymerization of α-methylene-γ-butyrolactone (p-MBL) obtained according to the present invention have i) a number average molecular weight (Mn) measured by gel permeation chromatography (GPC) in tetrahydrofuran using polystyrene standards of at least 2500 g / mol, e.g., 10000-150000 g / mol, preferably 10000-100000 g / mol; ii) a glass transition temperature (Tg) of 50-200° C., e.g., 100-200° C.; iii) a polydispersity index (PDI) of 1.1-2.0, e.g., 1.10-1.90, preferably 1.10-1.80.

[0168] The polymers of the present invention (p-MBL) are versatile and therefore are believed to have many applications. For example, polymers bearing lactones can be used to prepare ionic complexes with drugs bearing cationic moieties (including therapeutic agents such as peptides). The lactone ring(s) present in these polymers can also be opened by alkali hydroxides to form the alkali metal salts of the corresponding hydroxycarboxylic acids. Furthermore, polymers containing lactone groups (p-MBL) can be crosslinked by functional compounds capable of reacting with lactones. Unsubstituted primary or secondary amines and polyfunctional amines, such as hydroxyl-substituted mono-, di- or tri-(C 1 -C 12 ) alkylamines are particularly preferred in this regard. It is therefore expected that the functional polymers (p-MBL) may find utility as curable, crosslinkable or other reactive components in coating, sealant or adhesive compositions.

[0169] Polyester derivatives of α-methylene-γ-butyrolactone (MBL) or its homopolymers and copolymers (p-MBL) The present disclosure also provides for the formation of polyesters by ring-opening polymerization of either the pendant lactone groups of the above-mentioned homo- and copolymers of α-methylene-γ-butyrolactone (MBL) obtained according to the defined methods, or of methylene-γ-butyrolactone (p-MBL) obtained by anionic polymerization. In this respect, said (co)polymer p-MBL can be considered as a reactive macromonomer.

[0170] The present disclosure further provides for the formation of block or random copolyesters, whereby the lactone functionality of said methylene-γ-butyrolactone (MBL) or its (co-)polymer (p-MBL) is used to regulate the ring-opening polymerization of at least one further monomer (M2) selected from the group consisting of cyclic carbonates; cyclic anhydrides; oxalates; and cyclic esters having 5-, 6- and / or 7-membered rings. In particular, the at least one further monomer (M2) may be selected from the group consisting of lactide; glycolide; ε-caprolactone; para-dioxanone; trimethylene carbonate; 1,4-dioxepan-2-one; 1,5-dioxepan-2-one; γ-butyrolactone; γ-methyl-α-methylene-γ-butyrolactone; α-bromo-γ-butyrolactone; α-hydroxy-γ-butyrolactone; α-acetyl-γ-butyrolactone; spirocyclic-γ-butyrolactone; γ-valerolactone; α-angelica lactone; and β-angelica lactone.

[0171] There is no particular intent to limit the mechanism of ring-opening polymerization utilized in the present invention, and thus ring-opening polymerization of cyclic monomers by an anionic route via base catalyst is not strictly excluded, however, it is preferred herein that the polymerization proceeds by a cationic route via an acid catalyst. Generally, any suitable acidic ring-opening polymerization catalyst may be utilized herein, as well as mixtures of catalysts may be used.

[0172] Both Lewis and Bronsted acids are suitable in this situation, although the latter are preferred as they tend to be effective at temperatures below 150°C, and are usually effective at temperatures between 50°C and 100°C.

[0173] An example of a suitable Lewis acid is BF 3 ;AlCl 3 ;t-BuCl / Et 2 AlCl;Cl 2 / BCl 3 ;AlBr 3 ;AlBr 3 .TiCl 4 ;I 2 ;SbCl 5 ;WCl 6 ;AlEt 2 Cl;PF 5 ;VCl 4 ;AlEtCl 2 ;B.F. 3 Et 2 O;PCl 5 ;PCl 3 ;POCl 3 ;TiCl 6 ; and SnCl 4 Examples of Bronsted acid or protonic acid type catalysts (optionally disposed on a solid inorganic support) include, but are not limited to, HCl; HBr; HI; H 2 SO 4 ;HClO 4 para-toluenesulfonic acid; trifluoroacetic acid; and perfluoroalkanesulfonic acids, such as trifluoromethanesulfonic acid (or triflic acid, CF 3 SO 3 H), C 2 F 5 SO 3 H, C 4 F 9 SO 3 H, C 5 F 11 SO 3 H, C 6 F 13 SO 3 H and C 8 F 17 SO 3Among these strong acids, the most preferred is trifluoromethanesulfonic acid (triflic acid, CF 3 SO 3 H).

[0174] The catalyst for the ring-opening polymerization may be used at a concentration of 1 to 1000 ppm by weight based on the total weight of the monomers to be polymerized. Preferably, 5 to 150 ppm by weight, and most preferably, 5 to 50 ppm, is used. Increasing the contact temperature between the monomer and the catalyst may reduce the amount of catalyst.

[0175] The ring-opening polymerization may be conveniently carried out at a temperature ranging from 10° C. to 150° C. However, the temperature range is preferably 20° C. or 50° C. to 100° C., since avoiding high temperatures limits the loss of volatile monomers from the reaction mixture due to their low boiling points.

[0176] The process pressure is not critical, and thus the polymerization reaction can be carried out at subatmospheric, atmospheric or superatmospheric pressures, although atmospheric and superatmospheric pressures are preferred. It is important, however, that the reaction be carried out under anhydrous conditions and in the absence of compounds having active hydrogen atoms.

[0177] The duration of the reaction depends on the time it takes for the system to reach equilibrium. However, it is equally understood that the desired product can be obtained by stopping the equilibrium at exactly the desired time. For example, the reaction can be monitored by analyzing the viscosity over time or by analyzing the monomer conversion using gas chromatography, and the reaction can be stopped when the desired viscosity or monomer conversion is achieved. Apart from these considerations, the polymerization reaction is generally carried out for 0.5 hours to 72 hours, more commonly 1 hour to 30 hours or 1 hour to 20 hours. The acid catalyst present in the reaction mixture at the end of the polymerization reaction can be easily neutralized to stabilize the reaction product.

[0178] Once the polymerization is complete, the solid suspended compounds can be removed, for example by filtration, cross-flow filtration or centrifugation.Furthermore, the output of the polymerization can be processed using methods known in the art to isolate and purify the hydroxyl-functional polyester.In this regard, extraction, evaporation, distillation and chromatography can be mentioned as suitable techniques.Upon isolation, the typical yield of the hydroxyl-functionalized polyester is found to be at least 40%, and often at least 60%.

[0179] Exemplary polyesters that may be derived by this ring-opening polymerization method have molecular weights (Mn) of at least 5000, preferably 10,000 to 200,000 g / mol, as measured by gel permeation chromatography (GPC) in tetrahydrofuran using polystyrene standards. Additionally, the polymers may be characterized by a polydispersity index in the range of 1.0 to 2.5, preferably 1.0 to 2.0.

[0180] Further polyester formation Methylene-γ-butyrolactone (MBL) or its (co-)polymer (p-MBL) may further be useful as a monomer in esterification, where the resulting copolymer comprises non-lactoyl units derived from at least two comonomers capable of forming ester bonds. More specifically, these comonomers comprise: i) at least one diol; and (ii) at least one dicarboxylic acid or its ester-forming derivative.

[0181] Diols (i) suitable for use in this context include saturated and unsaturated aliphatic and cycloaliphatic dihydroxy compounds, as well as aromatic dihydroxy compounds. These diols preferably have a molecular weight of 250 Daltons or less. As used herein, the term "diol" should be interpreted as including its equivalent ester-forming derivatives, with the exception that the molecular weight requirement relates only to the diol, not to its derivatives. Exemplary ester-forming derivatives include the acetate of the diol, as well as, for example, the ethylene oxide or ethylene carbonate of ethylene glycol.

[0182] Preferred diols are those having 2 to 10 carbon atoms. Examples of these diols include ethylene glycol, propylene glycol, 1,3-propanediol, 1,2-butanediol, 2-methylpropanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, neopentyl glycol, hexanediol, decanediol, hexamethylene glycol, cyclohexanedimethanol, resorcinol, and hydroquinone. Mixtures of such diols can be used, but in this regard, generally, at least about 60 mol %, preferably at least 80 mol %, based on the total diol content, are the same diol.

[0183] Dicarboxylic acids (ii) suitable for use in the above context include aliphatic, cycloaliphatic, and / or aromatic dicarboxylic acids. These acids should preferably have a molecular weight of less than 300 Daltons. The term "dicarboxylic acid" as used herein includes equivalents of dicarboxylic acids having two functional carboxyl groups that react with glycols and diols in forming polyesters to function substantially similarly to dicarboxylic acids. These equivalents include esters and ester-forming reactive derivatives such as acid halides and acid anhydrides, although the above molecular weight preference is with respect to the acid and not its equivalent ester or ester-forming derivatives. Thus, esters of dicarboxylic acids having a molecular weight greater than 300 Daltons, or acid equivalents of dicarboxylic acids having a molecular weight greater than 300 Daltons, are included when the acid has a molecular weight less than 300 Daltons. Additionally, the dicarboxylic acids may contain any substituents or combinations that do not substantially interfere with the formation and use of the polymers of the present invention.

[0184] Preferred dicarboxylic acids are those selected from the group consisting of alkyl dicarboxylic acids having a total of 2 to 16 carbon atoms and aryl dicarboxylic acids having a total of 8 to 16 carbon atoms. Representative alkyl dicarboxylic acids include glutaric acid, adipic acid, pimelic acid, succinic acid, sebacic acid, azelaic acid, and malonic acid. Representative aryl dicarboxylic acids include terephthalic acid, phthalic acid, isophthalic acid, dimethyl derivatives of the foregoing acids, and mixtures thereof.

[0185] Further embodiments of the present disclosure The present invention also relates to a method for preparing a compound of formula (BIII) from a compound of formula (BII) by whole cell biotransformation: [ka] [In the formula, n is an integer from 0 to 8; R 1 is C 1 -C 4is alkyl; R 2 is H or C 1 -C 4 is alkyl; and R 3 is H or C 1 -C 4 is alkyl] And, iii) contacting cells (CB) with a culture medium containing the compound of formula (BII), or a culture medium in contact with an organic phase containing the compound of formula (BII), under conditions that allow the cells to form the compound of formula (BIII) from the compound of formula (BII); and, optionally iv) isolating the resulting compound of formula (BIII); Including, wherein the cell (CB) is a compound of formula (BII) n+3 -hydroxylation of at least one alkane monooxygenase enzyme.

[0186] The cell (CB) used in this process has a gene for alkane monooxygenase, optionally as part of an Alk operon. In one embodiment, the cell (CB) used can be a wild-type cell or a non-recombinant laboratory cell that has a gene for alkane monooxygenase, optionally as part of an Alk operon, in particular an Alk operon containing gene products AlkB, AlkH and AlkJ. The wild-type genotype of Pseudomonas putida has two Alk operons, the first operon encoding the gene products AlkB, AlkF, AlkG, AlkH, AlkJ, AlkK and AlkL, and the second operon encoding AlkS and AlkT, where AlkS functions to regulate the expression of the first alk operon.

[0187] Alternatively, the cells (CB) used may be genetically modified relative to their wild type such that they are able to produce more of the compound of formula (BIII) starting from the compound of formula (BII) compared to the wild type. This comparison is intended to encompass both: i) when the wild type genetically modified cells produce detectable amounts of the compound of formula (BIII); ii) when the wild type genetically modified cells are unable to form detectable amounts of the compound of formula (BIII). Thus, with regard to said second category of cells ii), detectable amounts of the compound of formula (BIII) are only formed when the wild type genetically modified cells are used in the steps of the method.

[0188] The cell (CB) utilized is preferably genetically modified such that it forms at least 10 times, for example at least 100 times or at least 1000 times more of the compound of formula (BIII) than the wild-type cell in a defined time interval of 24 hours. The increase in product formation can be measured by culturing the cells used according to step b) of the present disclosure and the wild-type cell separately at the same initial cell density, nutrient medium and culture conditions for a specific time interval, and then measuring the amount of the target product in each nutrient medium.

[0189] The cells (CB) used in this step of the present disclosure can be prokaryotic or eukaryotic. They can be mammalian cells (including human cells), plant cells or microorganisms, such as fungi, molds or bacteria, with preferred microorganisms being those deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Yeasts can be important microorganisms (fungi) in this context, but it is preferred to use bacteria, and therefore, reference can be made to http: / / www.dsmz.de / species / bacteria.htm.

[0190] The potential use of wild-type Pseudomonas putida in the present invention has been described above. Without intending to limit the present invention, preferred cells (CB) that may be genetically modified and used in this embodiment of the present disclosure may be selected from the following genes: Corynebacterium; Brevibacterium; Bacillus; Lactobacillus; Lactococcus; Candida; Pichia; Kluveromyces; Saccharomyces; Escherichia; Zymomonas; Yarrowia; Methylobacterium; Ralstonia; Pseudomonas; Burkholderia; and Clostridium. It may be mentioned that the use of cells (CB) selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida is particularly preferred. Good results have been obtained with the E. coli BL21 strain.

[0191] Compared to its wild type, the genetically modified cell (CB) according to this embodiment has a C n+3 -hydroxylation of the compound of formula (BII) n+3 -hydroxylation leads to compounds of formula (BIII).

[0192] The enzyme alkane monooxygenase may be encoded by the AlkB gene from Pseudomonas putida GPO1, its variants and homologues. For example, the enzyme alkane monooxygenase may be encoded by the AlkB gene from Pseudomonas putida GPO1, selected from the group consisting of the AIkBGT gene cluster; the AlkBGTJH gene cluster; and the AlkBGTJHL gene cluster. The isolation of the respective gene sequences is described, for example, in van Beilen et al., "Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria", Journal of Bacteriology, Vol. 184 (6), pages 1733-1742 (2002). Additionally, DNA sequence information encoding AlkB, AlkF, AlkG and AlkT was obtained from the Pseudomonas putida OCT plasmid alk gene cluster identified as GenBank AJ-245436.1; and the plasmid sequence of the pCom10 vector into which AlkBGT was cloned can be found in GenBank 302087.1.

[0193] Enzymes encoded by nucleic acid sequences having at least 40%, preferably at least 50%, more preferably at least 75% identity to the identified sequences are suitable for use in the methods of the invention. For completeness, further exemplary homologs of the AlkB gene include, but are not limited to, AlkB-P1 obtained from Pseudomonas putida P1; Alk1-MO obtained from Marinobacter hydrocarbonoclasticus; and AlkB1 obtained from Alcanivorax borkumensis.

[0194] To achieve increased intracellular activity of the aforementioned enzymes in genetically modified cells (CB), one or more of the following measures can be adopted: increasing the copy number of the gene sequence encoding the enzyme; using a strong promoter for the gene; using a stronger ribosome binding site; using codon optimization; using genes or alleles encoding the corresponding enzymes with increased activity; and using modified amino acid sequences of the above enzymes that show increased activity, as described, for example, in Koch et al., “In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6”, Applied and Environmental Microbiology, Vol 75 (2), pages 337-344 (2009).

[0195] The genetically modified cells used in the methods of the present invention are produced by transformation, transduction and / or conjugation with an expression vector containing the desired gene, an allele of this gene or a part thereof, and a vector allowing the expression of this gene. Heterologous expression is achieved by integration of the gene or its allele in the chromosome of the cell or in an extrachromosomally replicating vector. Without intending to limit the disclosure, WO2009 / 077461 (Evonik Degussa GmbH) provides a useful reference for cell genetic transformation.

[0196] The cells (CB) can be contacted with said culture medium in a bioreactor and can thus be cultured continuously or discontinuously in a batch process, a fed-batch process or a repeated fed-batch process, as described above in relation to step b).

[0197] In the bioreactor, the bioproduction system performs the desired conversion (C n+3-hydroxylation) to obtain a chemical product (BIII). Suitable temperature ranges for this step include 20°C to 50°C, for example 20°C to 40°C. Anaerobic conditions may be maintained in the bioreactor used in this step, but aerobic conditions are more typically maintained therein via the addition of oxygen or an oxygen-containing gas such as air. The dissolved oxygen level of the liquid culture containing the nutrient medium can be monitored to maintain or confirm the desired aerobic, microaerobic or anaerobic conditions.

[0198] It will be clear to those skilled in the art that the culture medium used in this step must be suitable for the requirements of the microorganism used to produce the desired end product (BIII). To form the culture medium, at least one growth factor or its precursor; at least one nitrogen source; at least one phosphorus source; at least one trace metal source selected from the group consisting of manganese, boron, cobalt, copper, molybdenum, zinc, calcium, magnesium, iron, nickel and combinations thereof may be appropriately combined. Suitable growth factors include, but are not limited to, amino acids and vitamins, such as biotin, vitamin B12, derivatives of vitamin B12, thiamine and pantothenic acid. Nitrogen sources include, but are not limited to, peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Phosphorus sources include, but are not limited to, phosphoric acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate. It will be further appreciated that the medium may contain adjuvants such as inorganic acids and bases, e.g., pH adjusters including sodium hydroxide, potassium hydroxide, ammonia, aqueous ammonia; antifoaming agents; and antibiotics to maintain plasmid stability.

[0199] The medium may be a defined medium. The present embodiment may also be practiced utilizing minimal or supplemental minimal medium. Furthermore, the use of commercially prepared media is not precluded, and may include, among others: Luria-Bertani (LB) broth; M9 minimal medium; Sabouraud dextrose (SD) broth; Yeast medium (YM) broth, and yeast synthetic minimal medium (Ymin).

[0200] Compound (BIII) can optionally be isolated from the bioreactor, either from the culture medium if the host cells secrete it into the medium, or directly from the host cells that produce it if they do not. The isolated compound (BIII) may then be purified using methods known in the art, including, but not limited to, solvent extraction, filtration, evaporation, distillation, crystallization, and chromatography.

[0201] The following examples are illustrative of the present invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0202] Unless otherwise noted, all chemicals and compounds described below were purchased of the highest purity commercially available from Merck / Sigma-Aldrich, TCI chemicals or Carl Roth.

[0203] NMR spectroscopy : 1 H- and 13 C-Spectrum is Avance TM The data were recorded using a III 300 MHz FT NMR spectrometer, and 5–10 mg of the analyte was dissolved in CDCl 3 and the spectral data obtained were compared with the literature.

[0204] Gas Chromatography-Mass Spectrometry (GC-MS)This analysis was used to qualitatively confirm the formation of the desired product. The analytical conditions were set using pure compounds. All measurements were performed using a Shimadzu GCMS-QP2010 SE instrument equipped with an AOC-20i / s autosampler and injector unit and a Zebron ZB-5MSi capillary column (30 m x 0.25 mm x 0.25 μm, Phenomenex). The details of the test parameters are shown in Table 1 below: [Table 1]

[0205] Gas Chromatography-Flame Ionization Detector (GC-FID): This analysis was used to quantitatively confirm the formation of the desired product. The analytical conditions were established using pure compounds. All measurements were performed on a Shimadzu Nexis GC-2030 equipped with an AOC-20i Plus autosampler and injector unit and a Zebron ZB-5MSi capillary column (30 m x 0.25 mm x 25 μm, Phenomenex). The concentrations of the analytes were calculated from a calibration curve made by measuring the samples with known concentrations of pure compounds (0-6 mM, standardized with the internal standard ISTD) extracted from resting cell buffer. In particular, the calibration curve for isoprenyl acetate was applied to 4-acetoxy-2-methylene-butan-1-ol. Detailed test parameters are shown in Table 2 below: [Table 2]

[0206] Example 1: Synthesis of isoprenyl acetate(II) In a 50 mL round bottom flask equipped with a magnetic stirrer, add 60.9 mmol of acetic anhydride (Ac 2 O) and 0.58 mmol Mg(ClO) 4 4 was added. The reaction was started by dropwise addition of isoprenol (I, 58 mmol). The reaction was monitored by thin layer chromatography (cyclohexane:ethyl acetate (EtOAc) 3:1).

[0207] After 30 min, the reaction was complete and aqueous NaHCO 3 The product was dissolved in diethyl ether (Et 2 The organic layer was extracted with MgSO 4 The mixture was dried at 40° C. and the solvent was evaporated to give the pure ester (yield: 88%). The formation of isoprenyl acetate (II) was confirmed by GC-MS and NMR-spectroscopy in accordance with the published literature [5].

[0208] Example 2 2a) Cloning and expression of AlkBFGT AlkBFGT was cloned into the broad-host vector pCom10_AlkL [3] by excising AlkL in a manner similar to that described by Schrewe et al. [1] and van Nuland et al. [2], resulting in the vector pBT10. Synthetic DNA fragments encoding alkBFG and alkT were obtained from Integrated DNA Technologies. The fragments were subcloned into the pJET1.2 vector using the Thermo Scientific CloneJET PCR Cloning Kit. The alkBFG and alkT fragments were inserted into the pCom10 vector backbone by the FastCloning method [4]. Correct assembly of pBT10 was confirmed by colony PCR and Sanger sequencing.

[0209] E. coli BL21(DE3) carrying the pBT10 plasmid was used to express AlkBFGT under conditions similar to those described in the literature ([1], [2]). E. coli BL21(DE3) pCom10_AlkL was used for the empty vector control experiment. The strains were cultured at 50 μg mL -1 Luna-Bertani (LB) medium supplemented with 100 mg of kaneimicin or M9 minimal medium (1x M9 salts, 0.5% glucose, 2 mM MgSO 4 , 1ml L -1 of the US FeA single colony was inoculated into 5 mL of LB medium and incubated overnight at 30°C under constant agitation (120 rpm). 500 μL of the overnight culture (ONC) was transferred to 50 mL of M9 minimal medium in a 100 mL shake flask and the culture was again incubated overnight at 30°C.

[0210] For expression, 100–400 mL of M9 minimal medium was incubated at 37°C for 1 h at an optical density of 600 nm (OD 600 Inoculate M9-preculture until the OD was 0.15. 600 The cells were cultured at 30°C until the β-terminal tail reached 0.4-0.5. Expression of the recombinant gene was induced by adding 0.05% (v / v) dicyclopropyl ketone (DCPK). After 4 hours of expression at 30°C, the cells were harvested by centrifugation (4400xg, 4°C, 15 min).

[0211] 2b) Whole-cell biotransformation and isolation of 4-acetoxy-2-methylene-butan-1-ol (III) After expression and harvesting, cells were cultured at OD 600 Add 10 mL of resting cell buffer (50 mM KPi, pH 7.4, 1% glucose, 2 mM MgSO) 4 ) and resuspended in 10 mL of 10% ethanol. The cells were allowed to adapt to the reaction conditions for 5–10 min. The following reactions were then performed in biological triplicates:

[0212] The reaction was carried out in a total volume of 300 μL in 1.5 mL glass vials. The reaction was started by adding 5 mM substrate isoprenyl acetate (II), stock 200 mM in EtOH, 2.5% (v / v) EtOH to the reaction. The cells were incubated at 25° C. under constant agitation (180 rpm) for 24 h. To stop the reaction, the mixture was stored at −20° C. until further use. Substrate conversion was analyzed by GC-MS and GC-FID.

[0213] As a negative control, E. coli pCom10_AlkL was used instead of E. coli pBT10. To confirm the general activity of the AlkBFGT system, the conversion of n-octane to 1-octanol and the further peroxidation to octanal and octanoic acid by AlkBFGT were performed as model reactions.

[0214] To confirm the formation of 4-acetoxy-2-methylene-butan-1-ol (III) by NMR spectroscopy, the reaction was scaled up to a total volume of 20 mL and carried out in a 100 mL shake flask for a reaction time of 24 h. The reaction mixture was stored at -20 °C until further use.

[0215] The product was extracted from the cell suspension with ethyl acetate (EtOAc). The organic layer was washed with MgSO 4 The extract was dried at 37° C. and the solvent was evaporated. The crude extract was subjected to column chromatography (stationary phase: silica 60; solvent: cyclohexane / EtOAc 4:1) to obtain purified 4-acetoxy-2-methylene-butan-1-ol (III). The solvent of the fractions containing compound III was evaporated to obtain pure III, the yield of which is reported in Table 3 below. The fractions were analyzed by TLC (cyclohexane:EtOAc, 2:1; detection by KMnO4 staining). The spectral data were compared with those in the literature ([6], [7]).

[0216] For analysis of whole-cell biotransformation, 250 μL of the reaction mixture was acidified with 25 μL of 2 M HCl and extracted with 250 μL of EtOAc containing 1 mM methyl benzoate as an internal standard by vigorous shaking. Phase separation was achieved by centrifugation (16,000 g, 4 °C, 7 min). The organic phase was removed with MgSO 4 The extract was dried at 4°C for 10 min at 4°C for 20 min at 4°C for 1 h. 200 μL of the extract was directly subjected to GC-MS or GC-FID analysis for qualitative or quantitative analysis, respectively.

[0217] Yield data reported in Table 3 below are presented as arithmetic means and standard deviations (SD) of triplicate reactions. [Table 3]

[0218] Example 3 3a) Cloning and expression of AlkBFGT with co-expression of AlkL AlkBFGTL was similarly cloned into the broad-host vector pCom10_AlkL [3] in a manner similar to that described by Schrewe et al. [1] and van Nuland et al. [2] to obtain the vector pBTL10. Synthetic DNA fragments encoding alkBFG and alkT were obtained from Integrated DNA Technologies. The gene encoding AlkL was amplified from pCom10_AlkL. The fragments were subcloned into the pJET1.2 vector using the Thermo Scientific CloneJET PCR Cloning Kit. The alkBFG, alkT and alkL fragments were inserted into the pCom10 vector backbone by the FastCloning method [4]. Correct assembly of pBTL10 was confirmed by colony PCR and Sanger sequencing.

[0219] E. coli BL21(DE3) carrying the pBTL10 plasmid was used to express AlkBFGTL under conditions similar to those described in the literature ([1], [2]). E. coli BL21(DE3) pCom10_AlkL was used as an empty vector control. Strains were cultured at 50 μg mL -1 Luna-Bertani (LB) or M9 minimal medium (1x M9 salts, 0.5% glucose, 2 mM MgSO) supplemented with 100 µL of kanamycin 4 , 1ml / L US Fe A single colony was inoculated into 5 mL of LB medium and incubated overnight at 30°C with constant agitation (120 rpm). 500 μL of the overnight culture (ONC) was transferred to 50 mL of M9 minimal medium in a 100 mL shake flask and the culture was again incubated overnight at 30°C.

[0220] For expression, 100–400 mL of M9 minimal medium was incubated at 37°C for 1 h at an optical density of 600 nm (OD 600 Inoculate M9-preculture until the OD is 0.15. 600 The cells were cultured at 30°C until the β-terminal tail reached 0.4-0.5. Expression of the recombinant gene was induced by adding 0.05% (v / v) dicyclopropyl ketone (DCPK). After 4 hours of expression at 30°C, the cells were harvested by centrifugation (4400xg, 4°C, 15 min).

[0221] 3b) Whole-cell biotransformation and isolation of 4-acetoxy-2-methylene-butan-1-ol (III) After expression and harvesting, cells were cultured at OD 600 Add 10 mL of resting cell buffer (50 mM KPi, pH 7.4, 1% glucose, 2 mM MgSO) 4 ) and resuspended in 10 mL of 10% ethanol. The cells were allowed to adapt to the reaction conditions for 5–10 min. The following reactions were then performed in biological triplicates:

[0222] The reactions were carried out in 1.5 mL glass vials in a total volume of 300 μL. The reactions were started by the addition of 5 mM substrate (isoprenyl acetate (II), stock 200 mM in EtOH, 2.5% (v / v) EtOH). The cells were incubated at 25° C. under constant agitation (180 rpm) for 24 h. To stop the reaction, the mixture was stored at −20° C. until further use. Substrate conversion was analyzed by GC-MS and GC-FID.

[0223] As a negative control, E. coli pCom10_AlkL was used instead of E. coli pBTL10. To confirm the general activity of the AlkBFGTL system, the conversion of n-octane to 1-octanol and further peroxidation to octanal and octanoic acid by AlkBFGTL was carried out as a model reaction.

[0224] To confirm the formation of 4-acetoxy-2-methylene-butan-1-ol (III) by NMR spectroscopy, the reaction was scaled up to a total volume of 20 mL and carried out in a 100 mL shake flask for a reaction time of 24 h. The reaction mixture was stored at -20 °C until further use.

[0225] The product was extracted from the cell suspension with ethyl acetate (EtOAc). The organic layer was washed with MgSO 4 The crude extract was dried at 37° C. and the solvent was evaporated. The crude extract was subjected to column chromatography (stationary phase: silica 60; solvent: cyclohexane / EtOAc 4:1) to obtain purified 4-acetoxy-2-methylene-butan-1-ol (III). The solvent of the fractions containing compound III was evaporated to obtain pure III, the yield of which is reported in Table 4 below. The fractions were analyzed by TLC (cyclohexane:EtOAc, 2:1; KMnO 4 The spectral data were compared with those in the literature ([6], [7]).

[0226] For analysis of whole-cell biotransformation, 250 μL of the reaction mixture was acidified with 25 μL of 2 M HCl and extracted with 250 μL of EtOAc containing 1 mM methyl benzoate as an internal standard by vigorous shaking. Phase separation was performed by centrifugation (16,000 g, 4 °C, 7 min). The organic phase was removed with MgSO 4 The extract was dried at 4°C for 10 min at 4°C for 20 min at 4°C for 1 h. 200 μL of the extract was directly subjected to GC-MS or GC-FID analysis for qualitative or quantitative analysis, respectively.

[0227] Yield data reported in Table 4 below are presented as arithmetic means and standard deviations (SD) of triplicate reactions. [Table 4]

[0228] Example 4 4a) Cloning and expression of AlkB mutants Two AlkBFGT DNA fragments, each carrying an AlkB mutant, were cloned into the broad-host vector pCom10_AlkL[3] by excising AlkL as described by Schrewe et al.[1] and van Nuland et al.[2] to obtain the vector pBT10_AlkB mutant. The following AlkB mutants are identified as follows: AlkB(I233V) (mutant disclosed in Koch et al.[8]); AlkB(F164L) (single mutant identified from docking studies using isoprenyl acetate as a ligand, in which the amino acid phenylalanine (Phe) of wild-type AlkB is exchanged for leucine (Leu)).

[0229] Synthetic DNA fragments encoding alkBFG and alkT were obtained from Integrated DNA Technologies. Where appropriate, the gene encoding AlkL was amplified from pCom10_AlkL. The fragments were subcloned into the pJET1.2 vector using the Thermo Scientific CloneJET PCR Cloning Kit. The alkBFG, alkT, and, where applicable, AlkL fragments were inserted into the pCom10 vector backbone via the FastCloning method [4], resulting in the pBT10 vector. Mutations were introduced by site-directed mutagenesis. The correct assembly of pBT10_AlkB mutants and successful mutagenesis were verified by colony PCR and Sanger sequencing.

[0230] E. coli BL21(DE3) carrying the pBT10_AlkB mutant plasmid was used to express AlkB(mut)FGT or, where applicable, AlkB(mut)FGTL under conditions similar to those described in the literature ([1], [2]). E. coli BL21(DE3) pCom10_AlkL was used as an empty vector control. Strains were cultured at 50 μg mL -1 Luna-Bertani (LB) or M9 minimal medium (1x M9 salts, 0.5% glucose, 2 mM MgSO) supplemented with 100 µL of kanamycin 4 , 1ml / L US FeThe strains were grown and selected in either 0.1% or 1.0% M9 medium (min. 0.01% ethanol) or 0.1% M9 medium (min. 0.01% ethanol). A single colony was inoculated into 5 mL of LB medium and incubated overnight at 30°C under constant agitation (120 rpm). 500 μL of the overnight culture (ONC) was transferred to 50 mL of M9 minimal medium in a 100 mL shake flask and the culture was again incubated overnight at 30°C.

[0231] For expression, 100–400 mL of M9 minimal medium was incubated at 37°C for 1 h at an optical density of 600 nm (OD 600 Inoculate M9-preculture until the OD is 0.15. 600 The cells were cultured at 30°C until the β-terminal affinity reached 0.4 to 0.5. Expression of the recombinant gene was induced by adding 0.05% (v / v) dicyclopropyl ketone (DCPK). After 4 hours of expression at 30°C, the cells were harvested by centrifugation (4400xg, 4°C, 15 minutes).

[0232] 4b) Whole-cell biotransformation and isolation of 4-acetoxy-2-methylene-butan-1-ol (III) After expression and harvesting, cells were cultured at OD 600 Add 10 mL of resting cell buffer (50 mM KPi, pH 7.4, 1% glucose, 2 mM MgSO) 4 ) and resuspended in 10 mL of 10% PBS. The cells were allowed to adapt to the reaction conditions for 5-10 minutes. The following reactions were then performed in biological triplicate:

[0233] The reaction was carried out in a total volume of 300 μL in 1.5 mL glass vials. The reaction was started by adding 5 mM substrate isoprenyl acetate (II), stock 200 mM in EtOH, 2.5% (v / v) EtOH into the reaction. The cells were incubated at 25° C. under constant agitation (180 rpm) for 24 h. To stop the reaction, the mixture was stored at −20° C. until further use. Substrate conversion was analyzed by GC-MS and GC-FID.

[0234] As a negative control, E. coli pCom10_AlkL was used instead of the E. coli pBT10_AlkB mutant.To confirm the general activity of the AlkBFGT or, where applicable, the AlkBFGTL system, the conversion of n-octane to 1-octanol and further overoxidation to octanal and octanoic acid by AlkBFGT(L) was carried out as a model reaction.

[0235] To confirm the formation of 4-acetoxy-2-methylene-butan-1-ol (III) by NMR spectroscopy, the reaction was scaled up to a total volume of 20 mL and carried out with 100 mL shaking for 24 h reaction time. The reaction mixture was stored at -20 °C until further use.

[0236] The product was extracted from the cell suspension with ethyl acetate (EtOAc). The organic layer was washed with MgSO 4 The crude extract was dried at 37° C. and the solvent was evaporated. The crude extract was subjected to column chromatography (stationary phase: silica 60; solvent: cyclohexane / EtOAc 4:1) to obtain purified 4-acetoxy-2-methylene-butan-1-ol (III). The solvent of the fractions containing compound III was evaporated to obtain pure III, the yield of which is reported in Table 5 below. The fractions were analyzed by TLC (cyclohexane:EtOAc, 2:1; KMnO 4 The spectral data were compared with those in the literature ([6], [7]).

[0237] For analysis of whole-cell biotransformation, 250 μL of the reaction mixture was acidified with 25 μL of 2 M HCl and extracted with 250 μL of EtOAc containing 1 mM methyl benzoate as an internal standard by vigorous shaking. Phase separation was performed by centrifugation (16,000 g, 4 °C, 7 min). The organic phase was removed with MgSO 4 The extract was dried at 4°C for 10 min at 4°C for 20 min at 4°C for 1 h. 200 μL of the extract was directly subjected to GC-MS or GC-FID analysis for qualitative or quantitative analysis, respectively.

[0238] Yields of reactions reported in Table 5 are presented as the mean and standard deviation (SD) of triplicate reactions. [Table 5]

[0239] Example 5 5a) Cloning and expression of AlkB homologues The following homologs (a), (b), and (c)) were cloned into the broad-host vector pCom10_AlkL[3] by excising AlkL independently, in a manner similar to that described by Schrewe et al.[1] and van Nuland et al.[2], to obtain the vector pBT10: a) AlkB from Pseudomonas putida P1 (PpP1_AlkB; accession number: P12691.1), which has 90% protein sequence identity with AlkB from P. putida GPo1 (PpGPo1); b) AlkMO from Marinobacter hydrocarbonclasticus (Mh_AlkMO; RCW72391.1), which has 78% protein sequence identity with AlkB from P. putida GPO1; and c) AlkMO from P. putida AlkB1 from Alcanivorax borkumensis (Ab_AlkB1; AB110225.1), which shares 77% protein sequence identity with AlkB from GPo1.

[0240] Synthetic DNA fragments encoding alkBFG, alkT, and independently the aforementioned homologs were obtained from Integrated DNA Technologies. The alkBFGT and alkT fragments were subcloned into the pJET1.2 vector using the Thermo Scientific CloneJET PCR Cloning Kit. The alkBFG and alkT DNA fragments were inserted into the pCom10 vector backbone by the FastCloning method [4] to obtain the pBT10 plasmid. Homologs a)-c) were inserted into the pBT10 backbone by replacing the alkB gene from P. putida GPO1. The correct assembly of pBT10_AlkBhomolog was confirmed by colony PCR and Sanger sequencing.

[0241] E. coli BL21(DE3) carrying the pBT10_AlkBhomolog plasmid was used independently to express AlkFGT and its homologs under conditions similar to those described in the literature ([1], [2]). E. coli BL21(DE3)pCom10_AlkL was used as an empty vector control. Strains were grown in Luna-Bertani (LB) or M9 minimal medium (1x M9 salts, 0.5% glucose, 2 mM MgSO) supplemented with 50 μg / mL kanamycin. 4 , 1ml / L US Fe The strains were grown and selected in either 0.1% M9 or 1.0% M9 medium (min. 0.01 mg / mL, 100% ethanol) or 0.1% M9 medium (min. 0.01 mg / mL, 100% ethanol). A single colony was inoculated into 5 mL of LB medium and incubated overnight at 30°C with constant agitation (120 rpm). 500 μL of the overnight culture (ONC) was transferred to 50 mL of M9 minimal medium in a 100 mL shake flask and the culture was again incubated overnight at 30°C.

[0242] For expression, 100–400 mL of M9 minimal medium was incubated at 37°C for 1 h at an optical density (OD) of 600 nm. 600 Inoculate M9-preculture to an OD of 0.15. 600The cells were grown at 30°C until the ΔP reached 0.4 to 0.5. Recombinant gene expression was induced by adding 0.05% (v / v) dicyclopropyl ketone (DCPK). After 4 h of expression at 30°C, the cells were harvested by centrifugation (4400xg, 4°C, 15 min).

[0243] 5b) Whole-cell biotransformation and isolation of 4-acetoxy-2-methylene-butan-1-ol (III) After expression and harvesting, the three engineered cells were cultured at OD 600 Add 10 mL of resting cell buffer (50 mM KPi, pH 7.4, 1% glucose, 2 mM MgSO) 4 ) and resuspended in 10 mL of 10% CO. Cells were allowed to adapt to reaction conditions for 5-10 min. The following reactions were then performed in biological triplicates:

[0244] Reactions were carried out in 1.5 mL glass vials with a total volume of 300 μL. The reactions were started by the addition of 5 mM substrate (isoprenyl acetate (II), stock 200 mM in EtOH, 2.5% (v / v) EtOH). The cells were incubated independently at 25 °C for 24 h under constant agitation (180 rpm). To stop the reaction, the mixture was stored at -20 °C until further use. Substrate conversion was analyzed by GC-MS and GC-FID.

[0245] As a negative control, E. coli pCom10_AlkL was used.To confirm the general activity of the AlkFGT and homologue ((a), (b) or c)) systems, the conversion of n-octane to 1-octanol and its further peroxidation to octanal and octanoic acid were performed as model reactions.

[0246] To confirm the formation of 4-acetoxy-2-methylene-butan-1-ol (III) by NMR spectroscopy, the reaction was scaled up to a total volume of 20 mL and carried out with 100 mL shaking for 24 h reaction time. The reaction mixture was stored at -20 °C until further use.

[0247] The product was extracted from the cell suspension with ethyl acetate (EtOAc). The organic layer was washed with MgSO 4The extract was dried at 37° C. and the solvent was evaporated. The crude extract was subjected to column chromatography (stationary phase: silica 60; solvent: cyclohexane / EtOAc 4:1) to obtain purified 4-acetoxy-2-methylene-butan-1-ol (III). The solvent of the fractions containing compound III was evaporated to obtain pure III, the yield of which is reported in Table 6 below. The fractions were analyzed by TLC (cyclohexane:EtOAc, 2:1; detection by KMnO4 staining). The spectral data were compared with those in the literature ([6], [7]).

[0248] For analysis of whole-cell biotransformation, 250 μL of the reaction mixture was acidified with 25 μL of 2 M HCl and extracted with 250 μL of EtOAc containing 1 mM methyl benzoate as an internal standard by vigorous shaking. Phase separation was performed by centrifugation (16,000 g, 4 °C, 7 min). The organic phase was removed with MgSO 4 The extract was dried at 4°C for 10 min and 200 μL of the extract was directly subjected to GC-MS or GC-FID analysis for qualitative or quantitative analysis, respectively.

[0249] The yields of the reactions are reported herein below in Table 6. Data are presented as the mean and standard deviation of triplicate reactions. [Table 6]

[0250] Example 6 6a) Cloning and expression of an AlkB homologue by co-expression of the transporter protein AlkL. The following homologs (a), (b), and (c)) were cloned independently into the broad-host vector pCom10_AlkL[3] in a manner similar to that described by Schrewe et al. [1] and van Nuland et al. [2], resulting in vector pBTL10: a) AlkB from Pseudomonas putida P1 (PpP1_AlkB; accession number: P12691.1), which shares 90% protein sequence identity with AlkB from P. putida GPo1; b) AlkMO from Marinobacter hydrocarbonclasticus (Mh_AlkMO; RCW72391.1), which shares 78% protein sequence identity with AlkB from P. putida GPo1; and c) AlkMO from P. putida AlkB1 from Alcanivorax borkumensis (PpGPo1_AlkB), which shares 77% protein sequence identity with AlkB from GPo1 (Ab_AlkB1; AB110225.1).

[0251] Synthetic DNA fragments encoding alkBFG, alkT, and independently the aforementioned homologs were obtained from Integrated DNA Technologies. The gene encoding the transporter AlkL was amplified from the pCom10_AlkL plasmid. The alkBFGT and alkT fragments were subcloned into the pJET1.2 vector using the Thermo Scientific CloneJET PCR Cloning Kit. The DNA fragments alkBFG, alkL, and alkT were inserted into the pCom10 vector backbone via the FastCloning method [4], resulting in the pBTL10 plasmid. Homologs a)-c) were inserted into the pBTL10 backbone by replacing the alkB gene from P. putida GPO1. The correct assembly of pBTL10_AlkBhomolog was confirmed by colony PCR and Sanger sequencing.

[0252] E. coli BL21(DE3) carrying the pBTL10 plasmid was used independently to express AlkFGTL and its homologs under conditions similar to those described in the literature ([1], [2]). E. coli BL21(DE3)pCom10_AlkL was used as an empty vector control. Strains were grown in Luna-Bertani (LB) or M9 minimal medium (1x M9 salts, 0.5% glucose, 2 mM MgSO) supplemented with 50 μg / mL kanamycin. 4 , 1ml / L US Fe The strains were grown and selected in either 0.1% M9 or 1.0% M9 medium (min. 0.01 mg / mL, 100% Tween 100 and 1.0 mg / mL, respectively). A single colony was inoculated into 5 mL of LB medium and incubated overnight at 30°C with constant agitation (120 rpm). 500 μL of the overnight culture (ONC) was transferred to 50 mL of M9 minimal medium in a 100 mL shake flask and the culture was again incubated overnight at 30°C.

[0253] For expression, 100–400 mL of M9 minimal medium was incubated at 37°C for 1 h at an optical density (OD) of 600 nm. 600 Inoculate M9-preculture to an OD of 0.15. 600 The cells were grown at 30°C until the ΔP reached 0.4 to 0.5. Recombinant gene expression was induced by adding 0.05% (v / v) dicyclopropyl ketone (DCPK). After 4 h of expression at 30°C, the cells were harvested by centrifugation (4400xg, 4°C, 15 min).

[0254] 6b) Whole-cell biotransformation and isolation of 4-acetoxy-2-methylene-butan-1-ol (III) Following expression and harvest, each of the three engineered cell types was independently cultured at OD 600 Add 10 mL of resting cell buffer (50 mM KPi, pH 7.4, 1% glucose, 2 mM MgSO) 4 The cells were then resuspended in 10 mL of PBS. The cells were allowed to adapt to the reaction conditions for 5-10 minutes. The following reactions were then performed in biological triplicate:

[0255] Reactions were carried out in 1.5 mL glass vials in a total volume of 300 μL. The reactions were started by the addition of 5 mM substrate (isoprenyl acetate (II), stock 200 mM in EtOH, 2.5% (v / v) EtOH). The cells were incubated independently at 25 °C for 24 h under constant agitation (180 rpm). To stop the reaction, the mixture was stored at -20 °C until further use. Substrate conversion was analyzed by GC-MS and GC-FID.

[0256] As a negative control, E. coli pCom10_AlkL was used.To confirm the general activity of the AlkFGTL and homologue ((a), (b) or c)) systems, the conversion of n-octane to 1-octanol and further peroxidation to octanal and octanoic acid were carried out as model reactions.

[0257] To confirm the formation of 4-acetoxy-2-methylene-butan-1-ol (III) by NMR spectroscopy, the reaction was scaled up to a total volume of 20 mL and carried out with 100 mL shaking for 24 h reaction time. The reaction mixture was stored at -20 °C until further use.

[0258] The product was extracted from the cell suspension with ethyl acetate (EtOAc). The organic layer was washed with MgSO 4 The extract was dried at 37° C. and the solvent was evaporated. The crude extract was subjected to column chromatography (stationary phase: silica 60; solvent: cyclohexane / EtOAc 4:1) to obtain purified 4-acetoxy-2-methylene-butan-1-ol (III). The solvent of the fractions containing compound III was evaporated to obtain pure III, the yield of which is reported in Table 7 below. The fractions were analyzed by TLC (cyclohexane:EtOAc, 2:1; detection by KMnO4 staining). The spectral data were compared with those in the literature ([6], [7]).

[0259] For analysis of whole-cell biotransformation, 250 μL of the reaction mixture was acidified with 25 μL of 2 M HCl and extracted with 250 μL of EtOAc containing 1 mM methyl benzoate as an internal standard by vigorous shaking. Phase separation was performed by centrifugation (16,000 g, 4 °C, 7 min). The organic phase was removed with MgSO 4 The extract was dried at 4°C for 10 min and 200 μL of the extract was directly subjected to GC-MS or GC-FID analysis for qualitative or quantitative analysis, respectively.

[0260] The yields of the reactions are shown below in Table 7. Data are presented as the mean and standard deviation (SD) of triplicate reactions. [Table 7]

[0261] Example 7: Synthesis of 4-acetoxy-2-methylene-butan-1-al The conversion of the precursor 4-acetoxy-2-methylene-butan-1-ol (III) to 4-acetoxy-2-methylene-butanal (IV) was achieved by chemical oxidation with Jones reagent. CrO 3 and concentrated H 2 SO 4 The Jones reagent was synthesized from. The crude extract of 4-acetoxy-2-methylene-butan-1-ol (III) (615 mg containing approximately 0.7 mmol of (III)) was dissolved in 6 mL of acetone in a round-bottom flask equipped with a magnetic stirrer, placed on ice, and Jones reagent (0.345 mmol, 0.5% eq.) was added dropwise. The reaction was followed by TLC. After 45 min, the reaction was quenched by the addition of 2-propanol, and the reaction mixture was first diluted with double distilled water (ddH2O) and then diluted with 100% ethyl acetate (100% ethyl acetate). 2 O), then extracted with EtOAc. The organic layer was washed with brine and saturated NaHCO 3 (1:2) and then washed twice with NaSO 4 The solvent was evaporated and the resulting residue was subjected to NMR spectroscopy, which confirmed the formation of 4-acetoxy-2-methylene-butanal (IV). 1 H NMR (500 MHz, CDCl 3): δ 9.51 (s, 1H), 6.31 (s, 1H), 6.07 (s, 1H), 4.15 (t, J= 6.6 Hz, 2H), 2.56 (t, J= 6.5 Hz, 2H), 1.99 (s, 3H) ppm.

[0262] Example 8: Synthesis of 4-acetoxy-2-methylenebutyric acid(V) 4-Hydroxy-2-methylenebutyric acid (200 mg, 1.7 mmol) was acetylated with acetic anhydride (5.0 eq.) in pyridine (5.7 eq.) in a 10 mL round flask equipped with magnetic stirring at 0° C. overnight. The reaction was monitored by thin layer chromatography (TLC): once the reaction was complete, the reagents were first removed by evaporation under reduced pressure. The resulting residue was washed with 1 M HCl (5 mL) and extracted with EtOAc (3 times, 5 mL). The organic layers were combined and concentrated under reduced pressure in a water bath at 50° C. The remaining traces of reagent were removed by evaporation under inert gas to give the title compound (V) in pure form as a yellow viscous oil (167 mg, 62% yield). The title compound was subjected to NMR spectroscopy, the results of which were in full agreement with the data reported in the literature. 1 H NMR (300 MHz, CDCl 3 ): δ 6.37 (s, 1H), 5.73 (s, 1H), 4.23 (t, J= 6.6 Hz, 2H), 2.65 (t, J= 6.6 Hz, 2H), 2.03 (s, 3H) ppm.

[0263] Example 9: Lactonization of 4-acetoxy-2-methylenebutyric acid to α-methylene-γ-butyrolactone (Tulipalin A) 10 mg of 4-acetoxy-2-methylenebutyric acid was dissolved in HCl (10.0 eq.) and double distilled water was added to a total volume of 1 mL. The reaction mixture was incubated at 80° C. and 800 rpm. 20 μL samples were taken at the beginning of the incubation, after 3 h and after 20 h and analyzed by thin layer chromatography (TLC) (EtOAc:DCM, 3:1) and GC-FID, respectively. Thus, samples were extracted with 100 μL of EtOAc containing 1 mM methylbenzoate as an internal standard (ISTD) and further diluted to 200 μL with solvent. Prior to GC-FID analysis, samples were derivatized by silylation with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, 2 μL) and incubation at 60° C. for 1 h.

[0264] Reference sample [1] YM van Nuland et al., Appl. Environ. Microbiol. 2016, 82, 3801-3807. [2] M. Schrewe et al., Advanced Synthesis & Catalysis 2011, 353, 3485-3495. [3] MK Julsing et al., Appl. Environ. Microbiol. 2012, 78, 5724-5733. [4] C. Li et al. BMC Biotechnol. 2011, 11, 92. [5] N. Iranpoor et al., Journal of Sulfur Chemistry 2007, 28, 581-587. [6] P. Ferraboschi et al. Tetrahedron: Asymmetry 1994, 5, 691-69 [7] Y. Kim et al., Tetrahedron: Asymmetry 2011, 22, 1658-1661. [8] Koch et al. Applied Environmental Microbiology 2009, 75, 337-344.

[0265] In view of the above description and examples, it will be apparent to one skilled in the art that equivalent modifications or combinations thereof can be made without departing from the scope of the claims.

Claims

1. 1. A process for preparing α-methylene-γ-butyrolactone, comprising the steps of: a) acetylating the C1-hydroxyl group of isoprenol to obtain isoprenyl acetate; b) forming 4-acetoxy-2-methylene-butan-1-ol from said isoprenyl acetate by whole cell biotransformation, i) contacting cells (CB) with a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; ii) optionally isolating the resulting 4-acetoxy-2-methylene-butan-1-ol; wherein said cells (CB) exhibit at least one alkane monooxygenase enzyme activity that catalyzes the formation of 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; c) oxidizing the 4-acetoxy-2-methylene-butan-1-ol to produce 4-acetoxy-2-methylenebutyric acid; and d) converting the 4-acetoxy-2-methylenebutyric acid to α-methylene-γ-butyrolactone by hydroxylysis to γ-hydroxy-α-methylenebutyric acid and subsequent cyclization of the γ-hydroxy-α-methylenebutyric acid; The method includes:

2. The isoprenol of step a) is obtained by a fermentation step, said step comprising: providing a fermentation medium comprising a fermentable carbohydrate; and introducing into said medium an inoculum comprising a culture of one or more microorganisms selected from the group consisting of bacteria, molds and yeasts; Including, 2. The method of claim 1, wherein the one or more microorganisms ferment the carbohydrate to form isoprenol.

3. 3. The method of claim 1 or 2, wherein step a) comprises treating isoprenol with an acetylating agent selected from the group consisting of acetic acid, acetic anhydride and acetyl chloride at a temperature between 20 and 150° C.

4. 3. The method of claim 1 or 2, wherein step a) comprises treating isoprenol with an acetylation reagent in the presence of an enzyme capable of catalyzing an acetylation reaction.

5. The method according to any one of claims 1 to 4, wherein the cell (CB) comprises a gene for an alkane monooxygenase, optionally as part of the Alk operon.

6. The cell (CB) has been genetically modified to exhibit increased activity of an alkane monooxygenase enzyme encoded by a homologue of the AlkB gene from Pseudomonas putida GPO1, or The cell (CB) has been genetically modified to exhibit increased activity of the alkane monooxygenase enzyme encoded by the AIkBGT gene cluster from Pseudomonas putida GPO1, or The cell (CB) has been genetically modified to exhibit increased activity of the alkane monooxygenase enzyme encoded by the AIkBGTJH gene cluster from Pseudomonas putida GPO1, or The cell (CB) has been genetically modified to exhibit increased activity of the alkane monooxygenase enzyme encoded by the AIkBGTJHL gene cluster from Pseudomonas putida GPO1. The method according to any one of claims 1 to 5.

7. Step c) comprises a two-step oxidation, wherein: In the first step, 4-acetoxy-2-methylene-butan-1-ol is oxidized to 4-acetoxy-2-methylene-butan-1-al; 7. The process according to any one of claims 1 to 6, wherein in a second step 4-acetoxy-2-methylene-butan-1-al is oxidized to 4-acetoxy-2-methylene-butyric acid.

8. Step c) is a step of reacting the 4-acetoxy-2-methylene-butan-1-ol with chromic acid (H 2 CrO 4 );Na 2 CrO 4 ;K 2 CrO 4 ;K 2 Cr 2 O 7 ;K 2 Cr 2 O 7 8. The process of claim 7, wherein the process is carried out without intermediate isolation of the 4-acetoxy-2-methylene-butan-1-al by reacting it with a stoichiometric excess of an oxidizing agent selected from: a) a 4-acetoxy-2-methylene-butan-1-al; b) a permanganate salt; and c) a Jones reagent.

9. Step c) comprises reacting 4-acetoxy-2-methylene-butan-1-ol under aerobic conditions with at least one enzyme exhibiting oxidative activity; Optionally, at least one mediator compound that enhances the oxidative activity of the enzyme; The method according to any one of claims 1 to 6, characterized in that it is an enzymatic oxidation process comprising contacting the

10. 10. The method according to claim 9, wherein the reaction mixture of step c) comprises 0.001 to 10 mg of the at least one enzyme per kg of 4-acetoxy-2-methylene-butan-1-ol.

11. 11. The method according to claim 9 or 10, wherein the reaction mixture of step c) comprises 0.001 to 10 mg of said at least one mediator compound per kg of 4-acetoxy-2-methylene-butan-1-ol.

12. step c) is carried out by whole cell biotransformation; i) contacting cells (CC) with either a culture medium containing 4-acetoxy-2-methylene-butan-1-ol or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-ol under conditions that allow the cells to form 4-acetoxy-2-methylene-butyric acid from said 4-acetoxy-2-methylene-butan-1-ol; and ii) isolating the resulting 4-acetoxy-2-methylene-butyric acid; Including, The method according to any one of claims 1 to 6, wherein said cells (CC) exhibit the activity of at least one enzyme exhibiting oxidative activity.

13. 13. The method according to any one of claims 9 to 12, wherein the at least one enzyme exhibiting oxidative activity is selected from the group consisting of alcohol dehydrogenase (ADH); alcohol oxidase (AlcOx); aldehyde dehydrogenase (AlDH); and laccase.

14. step c) is carried out by whole cell biotransformation; i) contacting cells (CC1) with a culture medium containing 4-acetoxy-2-methylene-butan-1-ol or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-ol under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-al from 4-acetoxy-2-methylene-butan-1-ol; ii) optionally isolating the resulting 4-acetoxy-2-methylene-butan-1-al; iii) contacting cells (CC2) with a culture medium containing 4-acetoxy-2-methylene-butan-1-al or a culture medium in contact with an organic phase containing 4-acetoxy-2-methylene-butan-1-al under conditions that allow the cells to form 4-acetoxy-2-methylenebutyric acid from said 4-acetoxy-2-methylene-butan-1-al; and iv) isolating the resulting 4-acetoxy-2-methylenebutyric acid; Including, The method according to any one of claims 1 to 6, wherein said cells (CC1, CC2) each exhibit an increased activity of at least one enzyme exhibiting oxidative activity.

15. the cell (CC1) is genetically modified to exhibit increased activity of at least one enzyme exhibiting oxidative activity selected from the group consisting of alcohol dehydrogenase (ADH), alcohol oxidase (AlcOx) and laccase; 15. The method of claim 14, wherein the cells (CC2) are genetically engineered to exhibit increased activity of at least one enzyme exhibiting oxidative activity selected from the group consisting of aldehyde dehydrogenase (AlDH) and laccase.

16. the alcohol dehydrogenase (ADH) enzyme is encoded by a homologue of the AlkJ gene from Pseudomonas putida GPO1; and / or 16. The method of claim 13 or 15, wherein the aldehyde dehydrogenase (AlDH) enzyme is encoded by a homolog of the AlkH gene from Pseudomonas putida GP01.

17. 17. The process according to any one of claims 1 to 16, wherein step d) comprises subjecting the 4-acetoxy-2-methylenebutyric acid to acidic conditions at a temperature between 30°C and 300°C, preferably between 30°C and 150°C.

18. 1. A process for preparing 4-acetoxy-2-methylene-butan-1-ol, comprising the steps of: a) acetylating the C1-hydroxyl group of isoprenol to obtain isoprenyl acetate; b) forming 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate by whole cell biotransformation, i) contacting cells (CB) with a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; ii) isolating the resulting 4-acetoxy-2-methylene-butan-1-ol; Including, wherein the cells (CB) exhibit at least one alkane monooxygenase enzyme activity that catalyzes the formation of 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate, The method includes:

19. 1. A process for preparing 2-methylenebutane-1,2-diol, comprising the steps of: a) acetylating the C1-hydroxyl group of isoprenol to obtain isoprenyl acetate; b) forming 4-acetoxy-2-methylene-butan-1-ol from said isoprenyl acetate by whole cell biotransformation, i) contacting cells (CB) with a culture medium containing isoprenyl acetate or a culture medium in contact with an organic phase containing isoprenyl acetate under conditions that allow the cells to form 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; ii) isolating the resulting 4-acetoxy-2-methylene-butan-1-ol; Including, wherein the cells (CB) exhibit at least one alkane monooxygenase enzyme activity that catalyzes the formation of 4-acetoxy-2-methylene-butan-1-ol from isoprenyl acetate; and c) hydrolyzing the 4-acetoxy-2-methylene-butan-1-ol to form 2-methylenebutane-1,2-diol; The method includes:

20. Method for preparing a compound of formula (BIII) from a compound of formula (BII) by whole cell biotransformation: 【Chemistry 1】 [In the formula, n is an integer from 0 to 8; R 1 is C 1 -C 4 is alkyl; R 2 is H or C 1 -C 4 is alkyl; R 3 is H or C 1 -C 4 is alkyl] And, v) contacting cells (CB) with a culture medium containing the compound of formula (BII), or a culture medium in contact with an organic phase containing the compound of formula (BII), under conditions that allow the cells to form the compound of formula (BIII) from the compound of formula (BII); and vi) isolating the resulting compound of formula (BIII), Including, wherein the cell (CB) is a compound of formula (BII) n+3 - The method of claim 1, wherein the method further comprises:

21. The method according to any one of claims 18 to 20, wherein the cell (CB) comprises a gene for an alkane monooxygenase, optionally as part of the Alk operon.

22. The cell (CB) has been genetically modified to exhibit increased activity of an alkane monooxygenase enzyme encoded by a homologue of the AlkB gene from Pseudomonas putida GPO1, or The cell (CB) has been genetically modified to exhibit increased activity of the alkane monooxygenase enzyme encoded by the AIkBGT gene cluster from Pseudomonas putida GPO1, or The cell (CB) has been genetically modified to exhibit increased activity of the alkane monooxygenase enzyme encoded by the AIkBGTJH gene cluster from Pseudomonas putida GPO1, or The method according to any one of claims 18 to 21, wherein the cell (CB) has been genetically modified to exhibit increased activity of the alkane monooxygenase enzyme encoded by the AIkBGTJHL gene cluster from Pseudomonas putida GP01.

23. Use of 4-acetoxy-2-methylene-butan-1-ol obtained according to the process defined in any one of claims 18 and 21 to 22 for the synthesis of 2-methylenebutane-1,2-diol.

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