RU2784878C2 - Method for production of 2-oxo-4-valerolactone - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention provides processes of conversion of pyruvate obtained from sugars or other carbon sources into valuable C5 materials, such as levulinic acid, levulinate composite esters, valerolactone, and derivatives thereof. In particular, the invention relates to a method for the production of 2-oxo-4-valerolactone, while the method includes conversion of pyruvate into 4-hydroxy-2-oxopentanic acid by aldol attachment, and conversion of 4-hydroxy-2-oxopentanic acid into 2-oxo-4-valerolactone by lactonization, where lactonization is carried out at pH from low to neutral. The invention also relates to a method for the production of methylenemethylbutyrolactone.

EFFECT: obtaining valuable C5 materials.

14 cl, 13 tbl, 9 dwg

Description

A PRIORITY

[0001] This application claims priority over US Provisional Application No. 61/378199, filed August 30, 2010, which is incorporated herein by reference.

BACKGROUND

[0002] Levulinic acid or 4-oxopentanoic acid is an organic compound with the formula CH ₃ C(O)CH ₂ CH ₂ CO ₂ H. It is a keto acid. Levulinic acid is usually obtained chemically, for example by heating sucrose with concentrated hydrochloric acid. The process proceeds through the intermediate formation of glucose, which isomerizes into fructose and then hydroxymethylfurfural.

Levulinic acid is a potential precursor for nylon-like polymers, synthetic rubbers and plastics. Levulinic acid is a versatile synthetic intermediate, for example in the synthesis of pharmaceuticals, and is a precursor in the industrial production of other chemicals such as methyltetrahydrofuran, valerolactone, and ethyllevulinate.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

[0003] In certain aspects and embodiments, the present invention provides a chemical route for converting pyruvate derived from sugars or other carbon sources into valuable C5 materials such as levulinic acid. Exemplary C5 compounds are shown in Figures 1 and 2. When used with sugars as the carbon source, the key to this pathway is the conversion of C6 sugars (such as, but not limited to, glucose, fructose, galactose) and/or C5 sugars (such as, but not limited to , xylose, arabinose) to pyruvate, and then the conversion of pyruvate to one or more valuable C5 compounds through chemical or biochemical reactions of aldol addition, oxidation, reduction, dehydration and cyclization. When used with another carbon source, such as, but not limited to, fatty acids and glycerol, the carbon source is first converted to pyruvate and then converted to one or more valuable C5 compounds that include linear C5 keto acids or esters, or their cyclized derivatives of the following of the general formula: C ₅ C ₄ (X)C ₃ C ₂ (Y)C ₁ (=O)(Z), where X is either hydroxyl or ketone oxygen, Y is either hydrogen, or hydroxyl or ketone oxygen, the bond between the C3 and C2 carbons is either single or double (eg saturated or unsaturated) and Z is an alkoxy, sulfide or phenoxy group so as to give either an ester or thioester or carboxylic acid functional group. In some embodiments, the C5 compound is C ₅ C ₄ (O ₁ )C ₃ C ₂ (Y)C ₁ (=O)(O ₁ ), where the index “O ₁ ” is the same oxygen atom, such that a cyclic ester or lactone is formed and Y is either hydrogen or hydroxyl or ketone oxygen. All other atomic valences or bonds are assumed to be occupied by hydrogen atoms unless otherwise noted.

[0004] In one aspect, the present invention provides a process for making a compound that is a C5 keto acid or ester, or a C5 hydroxy acid or ester, or a cyclic derivative thereof. The method includes converting pyruvate to a C5 intermediate by aldol addition and converting the C5 intermediate to said compound by chemical or enzymatic steps, or a combination thereof. In certain embodiments, the C5 compound has the general formula C ₅ C ₄ (X)C ₃ C ₂ (Y)C ₁ (=O)(Z) or C ₅ C ₄ (O ₁ )C ₃ C ₂ (Y)C ₁ (=O)(O ₁ ), which are described above. In certain embodiments, the compound is obtained from 5-carbon and/or 6-carbon sugars or a feedstock suitable as a carbon source for the microbial host. In these embodiments, the method includes generating pyruvate from a sugar or feedstock (e.g., via a microbial host) and aldol addition of acetylaldehyde to pyruvate (e.g., in a microbial host or in a cell-free system), thereby producing a 5-carbon keto acid as an intermediate. product to obtain the desired C5 compound. Acetylaldehyde for aldol addition can be obtained by decarboxylation of pyruvate in a microbial host. The aldol addition product may further undergo one or more reduction, oxidation, dehydration, group transfer, hydrolysis, and/or lactonization reactions (eg, each independently in a microbial host or cell-free system) to produce the desired C5 product.

[0005] Such products can be used as building blocks to produce commercially valuable chemicals and fuels. For example, lactones such as 2-oxovalerolactone (compound L7 in Fig. 2), 2-hydroxyvalerolactone (compound L6 in Fig. 2), angelicalactones (compound L2, L3 and L10 in Fig. 2) and 4-valerolactone (γ- valerolactone, compound L1 in figure 2) can be used as a solvent. Angelicalactone and 4-valerolactone can also be converted chemically to methylenemethylbutyrolactone (MeMBL) (see eg WO/2006/015023, WO/2006/015024 for methods to catalyze this conversion). Methylenemethylbutyrolactone can be used as a monomer or copolymer to increase the thermal stability of polymethyl acrylate (PMMA) polymers commonly used in electronics and internal combustion engine applications, or to produce polymers in general (such as Poly(MeMBL), see e.g. WO/2005/028529). In addition, 4-valerolactone can be converted by chemical catalysis to valeric acid and, additionally, to valerate esters, as well as isomeric butenes, butadiene and other alkenes, including alkenes with eight carbons or more, as discussed in Bozell J., Connecting Biomass petroleum Processing with a chemical bridge, Science 329:522-523 (2010). Levulinic acid (compound P1 in Fig. 1) can be converted to 1,4-pentanediol and diphenolic acid, both of which can be used to make polymers. δ-amminolevulic acid (a derivative of levulinic acid) is a herbicide with an estimated demand of over 300 pounds per year. Moreover, levulinic acid can be converted into pyrrolidones (WO/2004/085048), pyrrolidinone (WO/2010/065833, WO/2004/085390, WO/2004/085349, WO/2004/084633), angelicalactone (WO/2005/ 097723), 4-valerolactone and 2-methyl-THF, which are end products or can be further converted into other compounds with various useful properties such as anionic liquids (WO/2010/065833), biofuels and fuel additives. Levulinic acid can be further used as a material for batteries (for example, patent document JP09190820), paints (patent document US5769929), coatings (patent document JP06280041), anti-corrosion coatings (patent document EP496555). Levulinate esters (or levulinate esters, compounds P9 in Figure 1) are polymeric building blocks proper, and after conversion to ketals (patent document US 2008/0242721) and can also be used as fuel additives (as described in patent US 7,153,996, which is incorporated herein by reference in its entirety). In addition, levulinic esters can be used in personal care products (eg Japanese patent JP 05320023), surfactants and lubricants (patent document EP 882745), absorbents (see WO/1998/9843684). All reference materials mentioned in this paragraph are incorporated into this document by reference.

[0006] Both levulinic acid and levulinic esters and some of the lactones listed in FIG. 2 can also be used in the manufacture of pharmaceutically active ingredients and pharmaceutical applications, some of which are listed in Bozell J., Production of levulinic acid and use as a platform chemical for derived products, Resources, Conservation and Recycling 28:227-239 (2000 ). For example, WO/1995/022524 reports the use of the levulinate methyl ester for the synthesis of novel indole derivatives useful as anticancer agents. Levulinic acid and 4-hydroxypentanoic acid can also be used as a chiral reagent with a wide range of potential applications (see e.g. Meyers et al., Stereoselective alkylations in rigid systems. Effect of remote substituents on p-facial additions to lactam enolates. Stereoelectronic and steric effects, J. Am. Chem. Soc. 120:7429-7438 (1998) Pharmaceutical applications of the C5 produced by the present invention may include the use of butyro- and valero-lactone derivatives as antibiotics and anti-formation agents. biofilms, by interfering with the molecular mechanism of "quorum sensing" in bacteria (see, for example, patent documents EP 1716131 and WO/2006/117113) Additional applications may stem from the biologically active protoanemone (compound L4 in Fig. 2). To top it all off, levulinic acid and esters have been applied to food, flavor and aroma. congestion (patent document EP1533364), as well as additives in a variety of consumer products. For example, levulinic acid is used as an additive in cigarettes (patent document WO/2010/051076). All references in this paragraph are incorporated herein by reference.

[0007] In certain embodiments, the implementation of the present invention, the method includes the conversion of pyruvate in 4-valerolactone. In another embodiment, the method includes converting pyruvate to levulinic acid. In another embodiment, the method includes converting pyruvate to levulinic esters (levulinates), such as, without limitation, ethyl levulinate and propyl levulinate. In another alternative embodiment of the present invention, the method comprises converting pyruvate to angelicalactone, alpha and alpha'-angelicalactones. In still other embodiments, the method includes converting pyruvate to 2,4-dihydroxypentanoic acid or its cyclized form, 2-hydroxy-4-valerolactone. In yet another embodiment, the method includes converting pyruvate to 2-oxo-4-hydroxypentanoic acid or its cyclized form, 2-oxo-4-valerolactone.

In some embodiments of the present invention, the method includes multiple enzymatic steps combined into a single metabolic pathway in a eukaryotic, prokaryotic, or archaea fermenting host, including, without limitation, Saccharamyces sp., Pichia sp., Pseudomonas sp., Bacillus sp., Chrysosporium sp. and Escherichia coli. In these and other embodiments, the method includes one or more enzymatic steps carried out in a cell-free system, or chemical catalysis steps, or a combination thereof, wherein various pathway intermediates are optionally isolated and/or purified from the fermentation broth as necessary to complete the process.

[0008] The advantage of certain embodiments of the present invention is that they are built on top of the main metabolism. For example, both C5 and C6 metabolism in eukaryotes, prokaryotes, and archea may involve glycolysis to produce pyruvate. Pyruvate is one of the most important basal metabolic intermediates and, in addition to glycolysis, can be obtained from lipid metabolism as well as amino acid metabolism. According to the method of the present invention, pyruvate is taken and two pyruvate molecules are converted into one C5 molecule such as levulinic acid and 4-valerolactone. In the case of C6 sugars, the carbon yield can be up to 80%. In the case of C5 sugars, the carbon yield could theoretically be up to 100%. If the method uses a microbial strain capable of simultaneously fermenting C5 and C6, such as, but not limited to, engineered by Saccharomyces cerevisae and Pichia stipitis, then this allows the direct fermentation of sugars to levulinic acid, 4-valerolactone, or any of the C5 compounds depicted in Fig. 1 and FIG. 2. This high achievable yield represents a distinct industrial advantage over alternative thermochemical processes for the production of levulinic acid or gamma-valerolactone, which typically yield molar yields of 40% or less.

[0009] In one embodiment of the present invention, the process converts a stream of sugars into one or more of the C5 compounds listed in Figures 1 and 2. In another embodiment, starch is used as a feedstock to the process. In another embodiment, the method converts lignocellulosic feedstock (including, but not limited to, corn stover, wood chips, household waste, pulp and paper mill waste) into at least one of the C5 compounds listed in Figures 1 and 2.

[0010] In one embodiment of the present invention, the method converts C6 sugars to one or more of the C5 compounds listed in Figures 1 and 2, preferably in fermentation strains highly efficient at consuming and fermenting C6 sugars, such as, without limitation, Saccharomyces cerevisiae, strain Cargill CB1 (as described in WO/2007/106524), Pseudomonas, Chrysosporium and Escherichia coli (E. coli). In another embodiment of the present invention, the method converts C5 sugars to one or more of the C5 compounds listed in Figures 1 and 2, preferably in fermentation strains highly efficient at consuming and fermenting C5 sugars, such as, but not limited to, those engineered by Saccharomyces cerevisiae and Pichia stipitis. In an embodiment of the present invention, the method simultaneously converts C5 and C6 sugars to a C5 compound, preferably in fermentation strains highly efficient at consuming and fermenting both C5 and C6 sugars (eg Saccharomyces cerevisiae). In another embodiment of the present invention, the fermentation strains exhibit a high level of resistance to biomass hydrolysate inhibitors, such as, but not limited to, furans, and to low pH or high organic acid titer environments.

[0011] In certain embodiments, the feed contains one or more C6 sugars selected from allose, altrose, glucose, mannose, gulose, idose, talose, galactose, fructose, psicose, sorbose, and tagatose. In these or other embodiments, the feed contains one or more C5 sugars selected from xylose, arabinose, ribose, lyxose, xylulose, and ribulose.

[0012] If the method of the present invention is used to convert C5 and C6 sugars to 4-hydroxypentanoic acid or 4-valerolactone, the pathway is designed to be redox (reduction-oxidation) balanced: two reducing equivalents (formation of NAD(P)H) are formed during glycolysis with the release of pyruvate (one molecule of glucose to two molecules of pyruvate) and two reducing equivalents are consumed (formation of NAD(P)) in the further process from pyruvate to 4-hydroxypentanoic acid or 4-valerolactone (γ-valerolactone). The fact that this pathway is redox balanced with respect to the formation of these two molecules will result in an optimized conversion in the event of a fermentation process and will reduce or eliminate the need for additional fermentation host design to counter the imbalance. With respect to the fermentation of sugars directly into all other compounds or building blocks (FIG. 1), the fermenting host will depend on individual side reactions to balance the pathway or an external source of redox equivalent suitable to balance the pathway.

BRIEF DESCRIPTION OF GRAPHICS

[0013] Figure 1 shows the molecular formulas for levulinic acid (compound P1 in Figure 1), as well as valuable derivatives that can be obtained at various stages along the way and in various embodiments of the processes described herein (compounds P1 - P16 in Fig. 1).

[0014] Figure 2 shows the molecular formulas for various C5 lactones that can be made at various stages along the route according to various process embodiments (compounds L1 - L10).

[0015] Figure 3 provides an overview of the biochemical processes for converting pyruvate to any of the C5 compounds of Figure 1 or Figure. 2 highlighting certain stages. Some possible chemical intermediates and sub-routes are not shown here. See Figure 5 for a more comprehensive depiction of the possibilities of the various paths.

[0016] Figure 4 provides an overview of the biochemical pathway and processes in accordance with certain embodiments of the present invention, where the order of the oxidation/reduction steps (corresponding to steps 3 and 4 in Figure 3) is inverted. As in Fig. 3, some possible chemical intermediates and sub-routes are not shown here. See Fig. 6 for a more comprehensive depiction of the possibilities of the various paths.

[0017] Figure 5 provides an overview of the biochemical pathway/processes of FIG. 3 where step 4 and step 5 are reduced to one step using oxidative dehydratase.

[0018] Figure 6 provides a detailed overview of the biochemical pathway/processes for converting pyruvate to any of the C5 compounds or building blocks of FIG. 1 or 2. In addition to the chemical steps depicted in FIG. 3, 4 and 5 depict the various cyclic intermediates that can result from the cyclization reaction from intermediates from the main route, as well as the chemical transformation that leads from cyclic lactone intermediates to various C5 compounds or building blocks. Additionally, various CoA intermediates are also provided that can be obtained from intermediates in the main pathway. The route can be used to produce levulinyl-CoA, from which either levulinic acid and 4-valerolactone, or levulinate and/or other pentane esters, such as 4-oxo-pentane ester (compound P9) in FIG. one.

[0019] Figure 7 shows the principle of obtaining levulinic esters (levulinates) and levulinic acid from the levulinic-CoA intermediate by the action of either a thioesterase or a transferase. The side chain R can be any functional group such as, without limitation, methyl, ethyl, propyl, aryl, phenyl, naphthyl and other aromatic groups, as well as an alkyl group with oxygen and nitrogen substituents, such as ketones, primary, secondary and tertiary alcohols, primary, secondary and tertiary amines, etc.

[0020] Figure 8 shows the kinetic curves obtained by reacting two enoate reductase enzymes (Genbank accession numbers AAA64522 and AAD16106 designated 6001 and 6002 in Figure 8) with the substrate 4-oxo-2-pentenoic acid (also known as acetylacrylic acid). acid, see compound P2 in Figure 1) and cyclohexenone substrate as control. The curve labeled “6001 aceto” and “6002 aceto” demonstrates the activity of proteins in the presence of 100 μM NADPH and the substrate 4-oxo-2-pentenoic acid. The curve labeled “6001 cycles” and “6002 cycles” demonstrates the activity of the proteins in the presence of 100 μM NADPH and the substrate cyclohexenone. A decrease in absorbance at 340 nm is observed, reflecting the conversion of NADPH to the oxidized form of NADP+. This curve shows the conversion of the substrate 4-oxo-2-pentenoic acid to levulinic acid (compound P1 in Figure 1) by both proteins. The control curves (labeled "6001 buffer" and "6002 buffer" and "cyclo buffer" and "aceto buffer") show a decrease in absorbance with substrates alone in buffer or proteins alone in buffer. No significant activity was found under these conditions. All curves were obtained in 100 mM potassium phosphate buffer, pH 7.0 and at room temperature (25ºC). Initial concentration of NADPH 100 μM, initial concentration of 4-oxo-2-pentenoic acid 100 mM, initial concentration of cyclohexenone 50 mM. The protein concentration varied.

[0021] Figure 9 shows the activity of the class II aldolase enzyme from Pseudomonas putida, HpaI aldolase (Genbank accession number ADA63518) on the substrates acetaldehyde and pyruvate. Assay conditions were as follows: protein was expressed and purified for Ni from E. coli and reacted with a mixture of acetaldehyde and pyruvate at an initial concentration of 100 mg/ml, in Tris buffer, pH 8.0, supplemented with 100 mM MnCl ₂ . Protein and substrates were incubated at room temperature for 30 min. before quenching with HCl and running on HPLC with a polar EPIC column. Figure 9 shows the resulting HPLC curves, with peaks corresponding to substrates and products indicated in the figure by a black arrow. The chemical identity of the product, 4-hydroxy-2-oxopentanoic acid, was confirmed by LC/MS (data not shown).

DETAILED DESCRIPTION OF THE INVENTION

[0022] In certain aspects and embodiments, the present invention provides a chemical route for converting pyruvate derived from sugars or other carbon sources into valuable C5 materials such as levulinic acid. In essence, the method of the present invention provides a path that is organized into at least two stages, and in some embodiments 4-8 stages, such as 7-8 stages (see 8 main stages depicted in Fig. 3), with up to 4 additional cyclization steps of intermediates obtained along the way. Attaching the intermediate in multiple steps to the coenzyme A (CoA) moiety allows the pathway to lead to the formation of CoA intermediates such as levulinyl-CoA (see Fig. 6). In addition, four optional steps can lead to the formation of cyclized variants of key intermediates in this pathway (see again Figure 6).

[0023] According to various embodiments, the first step is glycolysis, in which sugars (eg, from biomass) are converted to pyruvate, or alternatively, any chemical conversion from sugars to pyruvate. In the second step, two pyruvate molecules are converted into one molecule of 4-hydroxy-2-oxopentanoic acid and CO ₂ . An optional cyclization step gives the corresponding lactone, 2-oxo-4-valerolactone. An optional CoA addition step may result in the formation of 4-hydroxy-2-oxo-pentanoyl-CoA. In the third step, 4-hydroxy-2-oxopentanoic acid is reduced to 2,4-dihydroxypentanoic acid, or 4-hydroxy-2-oxopentanoyl-CoA to 2,4-dihydroxy-pentanoyl-CoA, or 2-oxo-4-valerolactone – to 2-hydroxy-4-valerolactone. An optional cyclization step yields the corresponding lactone, 2-hydroxy-4-valerolactone, either from 2,4-dihydroxy-pentanoic acid or 2,4-dihydroxy-pentanoyl-CoA. An optional CoA addition step results in the formation of 2,4-dihydroxy-pentanoyl-CoA from 2,4-dihydroxy-pentanoic acid. At the fourth stage, 2,4-dihydroxypentanoic acid is oxidized to 2-hydroxy-4-oxopentanoic acid, or 2,4-dihydroxy-pentanoyl-CoA to 2-hydroxy-4-oxo-pentanoyl-CoA. In an optional CoA addition step, 2-hydroxy-4-oxopentanoic acid is converted to 2-hydroxy-4-oxo-pentanoyl-CoA. At the fifth stage, 2-hydroxy-4-oxopentanoic acid is dehydrated to 4-oxo-2-pentenoic acid, or 2-hydroxy-4-oxopentanoyl-CoA to 4-oxo-2-pentenoyl-CoA. In an optional CoA addition step, 4-oxo-2-pentenoic acid is converted to 4-oxo-2-pentenoyl-CoA. In an optional step, 4-oxo-2-pentenoic acid is further reduced to 4-hydroxy-2-pentenoic acid, or 4-oxo-2-pentenoic acid to 4-hydroxy-2-pentenoyl-CoA, both of which may optionally be cyclized with the formation of angelicalactone. Another optional CoA addition step results in the formation of 4-hydroxy-2-pentenoyl-CoA from 4-hydroxy-2-pentenoic acid, which can again optionally be cyclized to form angelicalactone. An alternative embodiment of the present invention "reduces" the fourth and fifth steps into one single step. In the sixth step, levulinic acid (4-hydroxypentanoic acid) is formed as a result by reduction of 4-oxo-2-pentenoic acid in a similar manner as described above. In an optional step, coenzyme A (CoA) is attached to levulinic acid, resulting in the formation of levulinyl-CoA. Levulinyl-CoA can then be converted to a variety of levulinic esters by transferase by reacting with the appropriate alcohol. In some embodiments, in the seventh step, levulinic acid is further reduced to form 4-hydroxypentanoic acid. In the eighth step, 4-hydroxypentanoic acid is cyclized to yield 4-valerolactone.

[0024] In certain embodiments, steps 4 and 5 can be performed in a single transformation, oxidative dehydration. In another embodiment of the present invention, steps 3 and 4 are reversed so that 2-hydroxy-4-oxo-pentanoic acid is initially oxidized to 2,4-dioxo-pentanoic acid and then reduced to 2-oxo-4-hydroxy-pentanoic acid, so that the path of FIG. 3 becomes as shown in FIG. four.

[0025] In another embodiment of the present invention, steps 3, 5 and 6 (Fig. 3) are carried out directly on the respective lactones L1, L2, L6, L7, L8, L9 and L10, where the offshoot from linear intermediates derived originally from pyruvate and acetaldehyde occurs in any of the cyclization steps described in Figure 6. This embodiment of the present invention can be used either to produce lactones directly or after hydrolysis to produce back any of the compounds P1 - P16 (including levulinic acid).

[0026] In yet another aspect of the present invention, steps 2, 3, 4, 5, and 6 (FIG. 3) are performed on CoA intermediates where branching from linear intermediates derived originally from pyruvate and acetaldehyde occurs in any of the addition steps. CoA described in the paragraph of Figure 6. This embodiment of the present invention can be used to obtain any of the compounds P1 - P16 (including levulinic acid) using thioesterases. Levulin esters (P9) can be prepared from levulinyl-CoA and the corresponding alcohol using a transferase.

Step 1: Conversion of Sugars to Pyruvate

[0027] The conversion of sugars to pyruvate is part of a well-studied metabolic pathway, glycolysis. In glycolysis, the action of many enzymes results in the conversion of each C6 sugar molecule, such as glucose, into two pyruvate molecules, two ATP molecules, and two reducing equivalents in the form of two NAD(P)H molecules.

[0028] In one embodiment of the present invention, pyruvate is produced from glycolysis in the fermentation organism and subsequently used in a further pathway in the fermentation host. In an alternative embodiment, the pyruvate is isolated from the fermentation broth and then processed according to the further path.

Step 2: Conversion of pyruvate to 4-hydroxy-2-oxopentanoic acid

[0029] 4-Hydroxy-2-oxopentanoic acid can be obtained by aldol addition of acetaldehyde (aldehyde) to pyruvate (α keto acid). In addition, one equivalent of acetaldehyde reacts with one equivalent of pyruvate. Acetylaldehyde can be obtained in various ways. For example, pyruvate decarboxylase catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde. Therefore, pyruvate decarboxylase from a variety of eukaryotic or prokaryotic sources (eg Saccharomcyes cerevisiae) can be used. In a preferred embodiment of the present invention, acetylaldehyde is produced from pyruvate with the enzyme pyruvate decarboxylase.

[0030] Many aldolases have been isolated and shown to catalyze the aldol addition of pyruvate with acetaldehyde. Class I aldolase, 4-hydroxy-2-keto-pentanoic acid aldolase (HKP-aldolase), is an aldolase that uses a Schiff base with a lysine residue and catalyzes the forward and reverse reactions. In one embodiment of the present invention, the aldol addition of pyruvate with acetaldehyde is catalyzed by an E. coli HKP aldolase described in Pollard, JR et al., Substrate selectivity and biochemical properties of 4-hydroxy-2-keto-pentanoic acid aldolase from E. coli , Appl. and Environ. Microbiology, 64(10):4093-4094 (1998), or its homologue, or its mutants (these mutants are optionally obtained by protein engineering methods using computer calculation, directed development techniques or rational mutagenesis, or a combination thereof). Computer calculation techniques are disclosed in US 2009-0191607 and WO 2010/077470, which are incorporated herein by reference in their entirety.

[0031] At least two class II aldolases are known to catalyze the addition of pyruvate to acetaldehyde, and two (BphI and HpaI) have been characterized in some detail in Wang W et al., Comparison of two metal-dependent pyruvate aldolases related by convergent evolution: substrate specificity, kinetic mechanism and substrate channeling, Biochemistry, 49:3774-3782 (2010). These enzymes use a metal co-factor (mainly either Zn or Mn). BphI and HpaI have no detectable overall sequence similarity. Since BphI is stereoselective and results in the formation of a 4S adduct, HpaI, due to its very open active site, produces a racemic mixture (4R and 4S adducts). BphI is allosterically linked to BphJ, an acetaldehyde dehydrogenase, and is inactive and stable when expressed in isolation. HpaI, however, can be expressed by itself in E. coli and is active. In an alternative embodiment of the present invention, the aldol addition of pyruvate to acetaldehyde is catalyzed by HpaI, or BphI, or mutants thereof (whereby these mutants are optionally obtained by protein engineering methods using computer calculation, directed development techniques or rational mutagenesis, or a combination of the three).

[0032] Continuing, any suitable pyruvate aldolase and other similar aldolases (eg, KDPG-aldolase) that catalyze the aldol addition of an aldehyde to a ketone can presumably be redesigned to catalyze the aldol addition of acetaldehyde to pyruvate. Reconstruction may include, without limitation, achieving the desired substrate specificity for both pyruvate and acetaldehyde, controlling the desired stereoselectivity to form either racemic or enantiopure adducts ((R)4-hydroxy-2-oxopentanoic acid and (S)4-hydroxy -3-oxopentanoic acid), stabilizing the enzyme to obtain the desired catalytic activity under the industrial conditions in which the present invention is practiced (e.g., thermal stabilization or high titer organic stabilization), and/or increasing the expressibility and solubility of the enzyme in the context of industrial conditions. in which the present invention is practiced (eg, in the metabolic pathway in Saccharomyces cerevisiae). In another embodiment of the present invention, the aldol addition of pyruvate to acetaldehyde is catalyzed by pyruvate aldolase or any of its homologues and mutants (these mutants are optionally obtained using protein engineering methods using computer calculation, directed development techniques or rational mutagenesis, or a combination of the three).

[0033] Finally, using a de novo enzyme design technique such as that described by Zanghellini, A et al, New Algorithms and an in silico Benchmark for Computational Enzyme Design, Protein Science 15:2785-2794 (2006), one can design new aldolase enzymes for substrates that may or may not exist in nature. Up to 70 of these aldolases have been constructed de novo as described in US Patent Application 2009-0191607, which is incorporated herein by reference in its entirety. Application of this methodology to the substrates pyruvate and acetaldehyde can lead to the production of aldolases with the desired activity. In another embodiment of the present invention, the aldol addition of pyruvate to acetaldehyde is catalyzed by a de novo engineered aldolase.

Step 2': cyclization of 4-hydroxy-2-oxo-pentanoic acid to 2-oxo-4-valerolactone

[0034] 4-Hydroxy-2-oxo-pentanoic acid is cyclized to 2-hydroxy-4-valerolactone (compound L7 in Figure 1). In acid-neutral solutions, thermodynamic equilibrium favors cyclization to lactone. Cyclization to lactone can be kinetically increased by either chemical or biochemical catalysis. Homogeneous and heterogeneous catalysts for lactonization include strong acidic conditions (eg sulfuric acid), metal catalysts (eg palladium, rubidium). Biochemical catalysis can be generated by the action of lipases, esterases, proteases, and lactonases under conditions that favor the direct lactonation reaction (low-neutral pH/high titer organic solvent) as demonstrated, for example, in Martin CH, et al, Integrated bioprocessing for pH- dependent of 4-valerolactone from levulinate in Pseudomonas Putida KT2440, Appl. and Environ, Microbiology 76(2):417-424.

[0035] In one embodiment of the present invention, 2-oxo-4-valerolactone is prepared from 4-hydroxy-2-oxo-pentanoic acid in the presence of a catalyst after isolation of 4-hydroxy-2-oxo-pentanoic acid from the fermentation broth or cell-free solution. In another embodiment of the present invention, the lactonization of 4-hydroxy-2-oxo-pentanoic acid to 2-oxo-4-valerolactone is catalyzed directly by a lipase, or an esterase, or a protease, or a lactonase, or their mutants (these mutants are optionally produced using protein-coil methods). engineering using computer calculation, directed development techniques, rational mutagenesis, or a combination of the three).

Step 3: reduction of 4-hydroxy-2-oxo-pentanoic acid to 2,4-dihydroxy-pentanoic acid

[0036] Among the wide variety of naturally occurring dehydrogenases, dehydrogenases with substrate specificity that allows for 4-hydroxy-2-oxopentanoic acid and 2,4-dihydroxypentanoic acid can be selected by in silico and/or experimental screening. In addition, computer calculation, directed development techniques, or rational mutagenesis, or a combination of the three can be used to alter or increase the substrate specificity of an existing dehydrogenase for 4-hydroxy-2-oxo-pentanoic acid and 2,4-dihydroxy-pentanoic acid. Examples of suitable starting points for dehydrogenases include L- and D-lactate dehydrogenases (NAD(P)H- or heme-dependent, eukaryotic or bacterial origin), malate, aspartate and glutamate dehydrogenases (NAD(P)H-dependent eukaryotic or bacterial origin), as well as alcohol dehydrogenases (such as NAD(P)H-dependent dehydrogenases of alkyl and phenyl alcohols). Examples of such dehydrogenases are listed in the examples section.

[0037] In one embodiment of the present invention, 4-hydroxy-2-oxo-pentanoic acid is selectively reduced to 2,4-dihydroxy-pentanoic acid using homogeneous or heterogeneous chemical catalysis. To complete this step, 2,4-dihydroxypentanoic acid may or may not be isolated/purified from the enzymatic or cell-free solution. Preferably, 2,4-dihydroxypentanoic acid is isolated from the solution or fermentation broth prior to being subjected to said reduction.

[0038] In one embodiment of the present invention, NAD(P)H-dependent dehydrogenase is used to catalyze the reduction of the ketone at the 2-position of 4-hydroxy-2-oxo-pentanoic acid. In another embodiment, said dehydrogenase reduces a ketone with a high degree of substrate specificity for 4-hydroxy-2-oxo-pentanoic acid and high regioselectivity for the 2-position ketone. In one embodiment of the present invention, said dehydrogenase is not stereoselective and can accept both the 4R and 4S enantiomers. In another embodiment of the present invention, said dehydrogenase selectively reduces either the 4R or 4S enantiomeric form of 4-hydroxy-2-oxopentanoic acid.

[0039] In another embodiment of the present invention, instead of NAD(P)H-dependent dehydrogenase, a FAD-dependent dehydrogenase is used, preferably with a high degree of substrate and regioselectivity. In one embodiment of the present invention, said dehydrogenase is not stereoselective and can accept both the 4R and 4S enantiomers. In another embodiment of the present invention, said dehydrogenase selectively reduces either the 4R or 4S enantiomeric form of 4-hydroxy-2-oxopentanoic acid.

[0040] In another embodiment of the present invention, instead of NAD(P)H-dependent dehydrogenase, an FMN-dependent dehydrogenase is used, preferably with a high degree of substrate and regioselectivity. In one embodiment of the present invention, said dehydrogenase is not stereoselective and can accept both the 4R and 4S enantiomers. In another embodiment of the present invention, said dehydrogenase selectively reduces either the 4R or 4S enantiomeric form of 4-hydroxy-2-oxopentanoic acid.

[0041] In yet another embodiment of the present invention, instead of NAD(P)H-dependent dehydrogenase, a ferricytochrome-dependent dehydrogenase is used, preferably with a high degree of substrate and regioselectivity. In one embodiment of the present invention, said dehydrogenase is not stereoselective and can accept both the 4R and 4S enantiomers. In another embodiment of the present invention, said dehydrogenase selectively reduces either the 4R or 4S enantiomeric form of 4-hydroxy-2-oxopentanoic acid.

[0042] In another embodiment of the present invention, instead of NAD(P)H-dependent dehydrogenase, a quinone-dependent dehydrogenase is used, preferably with a high degree of substrate and regioselectivity. In one embodiment of the present invention, said dehydrogenase is not stereoselective and can accept both the 4R and 4S enantiomers. In another embodiment of the present invention, said dehydrogenase selectively reduces either the 4R or 4S enantiomeric form of 4-hydroxy-2-oxopentanoic acid.

Step 3': cyclization of 2,4-dihydroxypentanoic acid to 2-hydroxy 4-valerolactone

[0043] 2,4-Dihydroxypentanoic acid is cyclized to 2-hydroxy-4-valerolactone (compound L6 in Figure 1). In acid-neutral solutions, thermodynamic equilibrium favors cyclization to 4-valerolactone. Similar notes about thermodynamic equilibrium and chemical and biochemical catalysis remain valid as described above.

[0044] In one embodiment of the present invention, 2-hydroxy-4-valerolactone is obtained from 2,4-dihydroxypentanoic acid in the presence of a catalyst after isolation of 2,4-dihydroxypentanoic acid from a fermentation broth or cell-free solution. In another embodiment of the present invention, the lactonization of 2,4-dihydroxypentanoic acid to 2-hydroxy-4-valerolactone is catalyzed directly by a lipase, or an esterase, or a protease, or a lactonase, or their mutants (these mutants are obtained using protein engineering methods using computer calculation, directed development techniques, or rational mutagenesis, or a combination of the three).

Step 4: Oxidation of 2,4-dihydroxypentanoic acid to 4-oxo-2-hydroxypentanoic acid

[0045] In one embodiment of the present invention, 2,4-dihydroxypentanoic acid is selectively oxidized to 4-oxo-2-hydroxypentanoic acid using homogeneous or heterogeneous chemical catalysis. To complete this step, 2,4-dihydroxypentanoic acid may or may not be isolated/purified from the enzymatic or cell-free solution. Preferably, the 4-oxo-2-hydroxypentanoic acid is isolated from the solution or fermentation broth before being subjected to said oxidation.

[0046] In a preferred embodiment of the present invention, NAD(P)H-dependent dehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4-position of 2,4-dihydroxypentanoic acid. In a preferred embodiment, said dehydrogenase oxidizes a hydroxyl with a high degree of substrate specificity for 2,4-dihydroxypentanoic acid and a high regioselectivity for the 4-hydroxyl. Preferably, said dehydrogenase accepts four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of 2,4-dihydroxypentanoic acid. In an alternative embodiment, said dehydrogenase selectively oxidizes either the 2R (2R4R, 2R4S) or 2S (2S4R, 2S4S) enantiomers of 2,4-dihydroxypentanoic acid, depending on which is the most numerous enantiomer resulting from the previous reduction of 4-hydroxy-2- oxopentanoic acid. Examples of such dehydrogenases are listed in the examples section.

[0047] In another embodiment of the present invention, a FAD-dependent dehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4-position of 2,4-dihydroxypentanoic acid. In a preferred embodiment, said dehydrogenase oxidizes a hydroxyl with a high degree of substrate specificity for 2,4-dihydroxypentanoic acid and a high regioselectivity for the 4-hydroxyl. Preferably, said dehydrogenase accepts four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of 2,4-dihydroxypentanoic acid. In an alternative embodiment, said dehydrogenase selectively oxidizes either the 2R (2R4R, 2R4S) or 2S (2S4R, 2S4S) enantiomers of 2,4-dihydroxypentanoic acid, whichever is the most numerous enantiomer resulting from the reduction of 4-hydroxy-2-oxopentanoic acid. acids.

[0048] In another embodiment of the present invention, an FMN-dependent dehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4-position of 2,4-dihydroxypentanoic acid. In a preferred embodiment, said dehydrogenase oxidizes a hydroxyl with a high degree of substrate specificity for 2,4-dihydroxypentanoic acid and a high regioselectivity for the 4-hydroxyl. Preferably, said dehydrogenase accepts four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of 2,4-dihydroxypentanoic acid. In an alternative embodiment, said dehydrogenase selectively oxidizes either the 2R (2R4R, 2R4S) or 2S (2S4R, 2S4S) enantiomers of 2,4-dihydroxypentanoic acid, whichever is the most numerous enantiomer resulting from the reduction of 4-hydroxy-2-oxopentanoic acid. acids.

[0049] In yet another embodiment of the present invention, a ferricytochrome-dependent dehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4-position of 2,4-dihydroxypentanoic acid. In a preferred embodiment, said dehydrogenase oxidizes a hydroxyl with a high degree of substrate specificity for 2,4-dihydroxypentanoic acid and a high regioselectivity for the 4-hydroxyl. Preferably, said dehydrogenase accepts four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of 2,4-dihydroxypentanoic acid. In an alternative embodiment, said dehydrogenase selectively oxidizes either the 2R (2R4R, 2R4S) or 2S (2S4R, 2S4S) enantiomers of 2,4-dihydroxypentanoic acid, whichever is the most numerous enantiomer resulting from the reduction of 4-hydroxy-2-oxopentanoic acid. acids.

[0050] In yet another embodiment of the present invention, a quinone-dependent dehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4-position of 2,4-dihydroxypentanoic acid. In a preferred embodiment, said dehydrogenase oxidizes a hydroxyl with a high degree of substrate specificity for 2,4-dihydroxypentanoic acid and a high regioselectivity for the 4-hydroxyl. Preferably, said dehydrogenase accepts four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of 2,4-dihydroxypentanoic acid. In an alternative embodiment, said dehydrogenase selectively oxidizes either the 2R (2R4R, 2R4S) or 2S (2S4R,2S4S) enantiomers of 2,4-dihydroxypentanoic acid, whichever is the most numerous enantiomer resulting from the reduction of 4-hydroxy-2-oxopentanoic acid. acids.

Step 5: Dehydration of 4-oxo-2-hydroxypentanoic acid to 4-oxo-2-pentenoic acid

[0051] Typically, chemical dehydration is achieved by either homogeneous or heterogeneous catalysis, such as temperature > 100°C, concentrated acid (4.0 M sulfuric acid), and/or a metal oxide catalyst (zinc or aluminum oxides). In one embodiment of the present invention, the 4-oxo-2-hydroxy-pentanoic acid obtained from the reduction and oxidation steps is chemically dehydrated to 4-oxo-2-pentenoic acid using homogeneous or heterogeneous catalysis. To complete this step, 4-oxo-2-hydroxypentanoic acid may or may not be isolated/purified from the enzymatic or cell-free solution. Preferably, the 4-oxo-2-hydroxypentanoic acid is isolated from the solution or fermentation broth prior to being subjected to said dehydration.

[0052] The dehydration of organic compounds may alternatively be catalyzed by the enzyme dehydratase. Several classes of dehydratase have been characterized and depend on different mechanisms: a radical mechanism, such as in vitamin B12-dependent or SAM-dependent dehydratases (eg, diol dehydratase, glycerol dehydratase), a Lewis acid-based mechanism, such as in iron-sulfur-containing dehydratases (eg dihydroxy acid dehydratase, aconitase), and an enolate ion intermediate mechanism such as diacid dehydratase (eg tartrate dehydratase). Given that all mechanisms are applicable for the dehydration of 4-oxo-2-hydroxypentanoic acid, mechanisms based on the enolate intermediate are preferred, since the pKa of the α-proton decreases when the enolate anion is formed on the carbonyl β to form the hydroxyl to be cleaved off, thus allowing it is easy for him to go to the main acid/base group. The additional main acid/base group protonates the leaving water molecule. This mechanism is used by a wide range of natural dehydratases: magnesium-dependent dehydratases from the enolase superfamily, such as tartrate dehydratase, gluconate dehydratase, use this mechanism to dehydrate structurally different diacids with high substrate specificity, as described, for example, in Gerlt et al., Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity, Biochemistry, 433:59-70 (2005). Fumarase (also known as fumarate hydratase) catalyzes the enolate-based reversible hydration of malate to fumarate. Enoil dehydratase (also known as crotonase) employs the thioester-CoA enolate anion to catalyze the reversible hydration of various CoA substrates (see e.g. Holden et al., The Crotonase Superfamily: divergently related enzymes that catalyze different reactions involving acyl Coenzyme A thioesters, Acc Chem Res 34:145-157 (2001)).

[0053] In one embodiment of the present invention, the dehydration of 4-oxo-2-hydroxypentanoic acid to 4-oxo-2-pentenoic acid is catalyzed by dehydratase. In a preferred embodiment of the present invention, said dehydratase employs an enolate intermediate to catalyze the dehydration. Preferably, said dehydratase is a member of the enolase superfamily, the fumarase or enoyl-coA dehydratase superfamily, or protein engineered mutants thereof. In a preferred embodiment of the present invention, said dehydratase exhibits a high level of substrate specificity for 4-oxo-2-hydroxypentanoic acid. In another preferred embodiment of the present invention, said dehydratase dehydrates equally the 2R and 2S enantiomers of 4-oxo-2-hydroxypentanoic acid. In an alternative embodiment of the present invention, said dehydratase selectively dehydrates either the 2R or 2S enantiomer of 4-oxo-2-hydroxypentanoic acid.

Alternative to steps 4 and 5:

[0054] As an alternative to the 2-step conversion of 2,4-dihydroxypentanoic acid to 4-oxo-2-pentenoic acid, the 1-step conversion can be accomplished by oxidative dehydration. Oxidative dehydration is typical in sugar metabolism. So-called 4,6-dehydratase enzymes, such as UDP-GlcNAc-inverting 4,6-dehydratase, whose structural details are described in Ishiyama et al., Structural studies of FlaA1 from helicobacter pylori reveal the mechanism for inverting 4,6-dehydratase activity , J. Bio. Chem. 281(34):24489-24495 (2006). In one embodiment of the present invention, such a 4,6-dehydratase is used to catalyze the oxidative dehydration of 2,4-dihydroxypentanoic acid to 4-oxo-2-pentanoic acid. In one aspect of the present invention, said 4,6-dehydratase is enantioselective and preferentially dehydrates one of the enantiomers of 2,4-dihydroxypentanoic acid (any of 2R4R, 2R4S, 2S4R, or 2S4S). In other aspects of the present invention, said 4,6-dehydratase is not enantioselective and dehydrates two or more enantiomers of 2,4-dihydroxypentanoic acid with similar catalytic efficiency. In a preferred embodiment of the present invention, 4,6-dehydratase is highly active on 2,4-dihydroxypentanoic acid, derived from natural 4,6-dehydratase by protein engineering methods using computer calculation, directed development techniques or rational mutagenesis, or a combination thereof.

Step 6: reduction of 4-oxo-2-pentenoic acid to 4-oxo-pentanoic acid (levulinic acid)

[0055] Double bonds on substituted alkenes can be reduced (hydrogenated) to give the corresponding saturated alkanes. Substituted alkenes can be reduced using chemical catalysis or, in general, asymmetrically, using biocatalysts such as enoate reductases, as discussed in Stuermer et al., Asymmetric bioreduction of activated C=C bonds using enoat reductases from the old yellow enzyme family, Curr. Opin. In Chem. Bio. 11:203-213 (2007). Enoate reductases have been characterized both from eukaryotic organisms such as Sacharomyces cerevisiae and marchantia and from prokaryotic organisms such as Clostridium. The enoate reductase enzyme family depends on the flavin cofactor (FMN), which is oxidized in each enzyme cycle. With the exception of one known case, which is nicotinamide-independent, the flavin cofactor is in turn reduced by the nicotinamide cofactor, either NADH or NADPH, which also binds at the active site. At the end of one cycle, the substrate was reduced due to the fact that the NAD(P)H cofactor was oxidized to NAD(P)+. Enoate reductases differ in their substrate specificity. However, several enoate reductases, such as yeast and Clostridium enoate reductases, have broad substrate specificities and can give rise to linear substituted alkenes (with acid or ketone functionality) as well as substituted lactones such as 4-valerolactone.

[0056] In one embodiment of the present invention, 4-hydroxy-2-oxopentanoic acid is isolated from the separation broth or cell-free solution and the double bond is selectively reduced using homogeneous or heterogeneous catalysis.

[0057] In another embodiment of the present invention, the enzyme enoate reductase is used to reduce 4-hydroxy-2-oxo-pentanoic acid to levulinic acid. In a preferred embodiment, said enoate reductase is dependent on both FMNH2 and NAD(P)H cofactors, with said NAD(P)H cofactor being used at the active site to regenerate FMNH2 to its redox state prior to catalysis. In a preferred embodiment of the present invention, said enoate reductase is cloned and expressed in a fermenting host. In an alternative embodiment, said enoate reductase is administered extracellularly or in a cell-free system with an appropriate cofactor regeneration system. In another alternative embodiment, said reduction is catalyzed by a whole cell catalyst expressing one or more enoate reductases such that said cell is distinct from the fermenting host cell(s) in which part of the pathway and the entire pathway are involved. .

Step 7: Reduction of 4-oxo-pentanoic acid (levulinic acid) to 4-hydroxy-pentanoic acid

[0058] Similar to step 3 (paragraph [0037] - [0043]), the reduction of the 4-position ketone on levulinic acid can be accomplished either by chemical catalysis or by a dehydrogenase biocatalyst. In the context of the metabolic pathway, this last reduction (and the corresponding oxidation of one reduction equivalent) ensures the redox balance of the entire pathway from C5 and/or C6 sugars.

[0059] In one embodiment of the present invention, levulinic acid is isolated from the broth or cell-free solution and the 4-position ketone is selectively reduced by homogeneous or heterogeneous catalysis to give 4-hydroxypentanoic acid.

[0060] In an alternative embodiment of the present invention, NAD(P)-dependent dehydrogenase is used to catalyze the reduction of a 4-position ketone on levulinic acid to the corresponding hydroxyl to give 4-hydroxypentanoic acid. In a preferred embodiment, said dehydrogenase reduces a ketone with a high degree of substrate specificity for levulinic acid and high regioselectivity for the ketone at the 4-position. Preferably, said dehydrogenase is the same enzyme as for the oxidation of hydroxyl at the 4-position of 4-oxo-2-hydroxypentanoic acid, or a mutant thereof (the mutant being obtained by computer calculation, or experimental mutagenesis, or a combination of the two). In a preferred embodiment of the present invention, said dehydrogenase selectively produces one of the (4R or 4S) enantiomers of 4-hydroxypentanoic acid. In an alternative embodiment, said dehydrogenase produces a racemic mixture of the 4R and 4S enantiomers of 4-hydroxypentanoic acid.

Step 8: Cyclization of 4-hydroxypentanoic acid to 4-valerolactone

[0061] 4-Hydroxypentanoic acid is cyclized to 4-valerolactone (also known as γ-valerolactone, compound L1 in Fig. 1). In acidic solutions, thermodynamic equilibrium favors cyclization to 4-valerolactone. The same notes about thermodynamic equilibrium and chemical and biochemical catalysis remain valid as in paragraph [0035].

[0062] In one embodiment of the present invention, 4-valerolactone is obtained from 4-hydroxypentanoic acid in the presence of a catalyst after isolation of 4-hydroxypentanoic acid from the fermentation broth or cell-free solution. In a preferred embodiment of the present invention, enantio-pure 4-hydroxypentanoic acid (either the 4R or 4S enantiomer) is converted with said catalyst to enantio-pure 4-valerolactone. In an alternative embodiment, a racemic mixture of the two enantiomers of 4-hydroxypentanoic acid (4R and 4S) is converted with said catalyst to a racemic mixture of 4-valerolactone.

[0063] In another embodiment of the present invention, the lactonization of 4-hydroxypentanoic acid to 4-valerolactone is catalyzed directly by a lipase, or an esterase, or a protease, or a lactonase, or mutants thereof (whereby these mutants are obtained using protein engineering methods using computer calculation, directed development techniques or rational mutagenesis or a combination of the three) inside or outside the cell. In a preferred embodiment of the present invention, said lipase or esterase or protease or lactonase acts on an enantiopure 4-hydroxypentanoic acid substrate to produce enantiopure 4-valerolactone. In an alternative embodiment, said lipase or esterase or protease or lactonase acts on a racemic mixture of the 4R and 4S enantiomers of 4-hydroxypentanoic acid to produce a racemic mixture of the 4R and 4S enantiomers of 4-valerolactone.

EXAMPLES

[0064] Examples of pyruvate decarboxylase enzymes: An enzyme of the pyruvate decarboxylase family (EC number EC 4.1.1.1), such as the enzyme pyruvate decarboxylase, can be used to catalyze the first step of the pathway, the conversion of pyruvate to acetaldehyde. Table 1 below lists examples of such enzymes (along with their source organisms) that have been studied and characterized in the literature, listing their GenBank Access Number (NCBI). Homologous enzymes may also be used, e.g., protein and DNA sequences derived from the sequences in Table 1 (or reverse translation thereof) using alignment software such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with a value of e for alignment <0.1.

TABLE 1

[0065] Examples of aldolase enzymes that catalyze the production of 4-hydroxy-2-keto-pentanoic acid: Homologous enzymes can also be used, for example, protein and DNA sequences obtained from the sequences in the tables below (or their reverse translation) using software for an alignment such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an e value for alignment <0.1.

TABLE 2: Class I aldolase: EC 4.1.3.39, official name: 4-hydroxy-2-oxovalerate aldolase

TABLE 3: class II aldolases: EC 4.1.3.39, official name: 4-hydroxy-2-oxovalerate aldolase

TABLE 4: examples of additional pyruvate aldolases suitable to catalyze the reaction, either as WT or after protein engineering

[0066] Examples of dehydrogenase enzymes capable of reducing the ketone at position 4 of pentanoic acid derivatives to secondary alcohol (hydroxyl) / oxidizing the secondary alcohol (hydroxyl) at position 4 of pentanoic acid derivatives to ketone: homologous enzymes can also be used, for example, protein and DNA sequences obtained from the sequences in the tables below (or reverse translation thereof) using alignment software such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an alignment e value of <0.1.

[0067] A wide variety of dehydrogenases are capable of oxidizing/reducing secondary alcohols/ketones with varying degrees of substrate specificity. The dehydrogenase sequences listed below are some examples of dehydrogenases described in the literature as being active on secondary alcohol/ketone substituents on alkyl chains of three carbons or more.

TABLE 5

[0068] Examples of dehydrogenase enzymes to reduce 2,4-dioxo-pentanoic acid to 4-oxo-2-hydroxy-pentanoic acid: Homologous enzymes can also be used, for example, protein and DNA sequences derived from the sequences in the tables below (or their reverse translation) with using alignment software such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an alignment e value of <0.1.

[0069] Lactate dehydrogenase enzymes with broad substrate specificity have been demonstrated in the literature to accept a 2,4-dioxopentanoic acid substrate. The two sequences below have different stereoselectivities.

TABLE 6

[0070] An example of dehydratase enzymes catalyzing the conversion of 4-oxo-2-hydroxypentanoic acid to 4-oxo-2-pentenoic acid: homologous enzymes can also be used, for example, protein and DNA sequences derived from the sequences in the tables below (or their inverse translation) using alignment software such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an alignment e value of <0.1.

[0071] Enolate superfamily dehydratases: These dehydratase enzymes, which are structurally related to the "enolase" family of enzymes, stabilize the enolate ion formed after elimination of one of the α hydrogens in the acid functional group. Because these enzymes depend on the stabilization of the enolate anion to reduce the activation energy for the dehydration reaction, they can be active on a hydroxyl leaving β substrate with any of the carboxylic acid, ketone, or ester functionalities. A few examples of this class of dehydratases are shown in the table below:

TABLE 7

[0072] Dehydratases of the enoyl-coA-hydratase or “crotonase” family: these enzymes can catalyze the reversible addition/removal of water molecules to/from α,β-unsaturated thioesters (coenzyme A derivatives). Because they depend on the stabilization of the enolate anion formed after proton elimination, enzymes are also able to catalyze the hydration (and reversible dehydration) of α,β-unsaturated carboxylic acids and ketones. In contrast to dehydratase from the enolase superfamily, these enzymes do not require any cofactor.

TABLE 8

[0073] Fumarase C family dehydratases (fumarase A and B family enzymes use an iron-sulfur cassette): Regarding the enoyl-coA hydratase family, these enzymes stabilize enolate without the need for any cofactor. Substrate binding and transition state stabilization is achieved by amino acids from the active site.

TABLE 9

[0074] Other dehydratases: All other known dehydratases (EC number 4.2.1.*) can also be used to catalyze the dehydration of 4-oxo-2-hydroxypentanoic acid to 4-oxo-2-pentenoic acid, such as the dehydratase enzymes on based on an iron-sulfur cluster (eg, dihydroxydiol dehydratase, fumarase A and C) or vitamin B12-dependent and SAM-dependent dehydratases such as glycerol and propanediol dehydratase.

[0075] Examples of oxidase/epimerase enzymes capable of catalyzing the oxidative dehydration/conversion of 2,4-dihydroxypentanoic acid to 4-oxo-2-pentenoic acid: Homologous enzymes may also be used, e.g. protein and DNA sequences derived from the sequences in the tables below (or reverse translation thereof) using alignment software such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an alignment e value of <0.1.

TABLE 10

[0076] Examples of enzymes that catalyze the reduction of 4-oxo,2-hydroxopentanoic acid to levulinic acid: Homologous enzymes can also be used, for example, protein and DNA sequences obtained from the sequences in the tables below (or their reverse translation) using software for an alignment such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an e value for alignment <0.1.

[0077] A family of enzymes called enoate reductases or, more informally, the old yellow enzymes, are NAD(P)H- and FMN-dependent enzymes that catalyze the reversible reduction of α,β-unsaturated thioesters, carboxylic acids, and ketones. They exhibit broad substrate specificities, and the following sequences have been successfully shown experimentally (see data) to catalyze the reduction of 4-oxo, 3-hydroxypentanoic acid to levulinic acid.

TABLE 11

It has also been experimentally shown that multiple site mutants of the NCR enzyme from Pseudomonas syringae exhibit different catalytic activities towards 4-oxo,2-hydroxopentanoic acid as a substrate. These mutants correspond to Y178A, P242Q, D338Y and F315Y in the amino acid sequence numbering of AAD16106.

[0078] Examples of enzymes capable of catalyzing the lactonization of 4-hydroxy acids to their respective cyclic esters (lactones): Homologous enzymes can also be used, for example, protein and DNA sequences derived from the sequences in the tables below (or their reverse translation) using software an alignment tool such as, but not limited to, Blast, PSI-Blast, or HMMER3, and with an alignment e value of <0.1.

[0079] Many types of lactonases (eg, lactonohydrolases) are known that can be used to catalyze the reversible formation of 1,4-cyclic esters from 4-hydroxy acids. In particular, 1,4-lactonases (EC 3.1.1.25) show some specificity for 4-hydroxy acids and are therefore the preferred sequences for catalyzing the step 8 reactions in Figures 3, 4 and 5 and the multiple lactonization reactions in Figure 6. In particular, some 1,4-lactonases have been analyzed with 4-hydroxypentanoic acid and confirmed to catalyze their reversible cyclization to gamma-valerolactone. The table below lists some of the lactonase enzymes that have been reported in the literature to catalyze this reaction.

TABLE 12

[0080] A wide range of other characterized lactonases are suitable for catalysis of 4-hydroxy acid cyclization. Below is a table listing the EC numbers corresponding to existing lactonases (a subclass of carboxyesterases).

TABLE 13

[0081] Ultimately, esterases, lipases and peptidases/amidases were observed to catalyze lactonization reactions under suitable experimental conditions (non-alkaline pH and typically room temperature). For example, lipases are mentioned in PCT/US2010/055524 for lactonization, and amidase/peptidase has been used successfully for the synthesis of lactones in WO/2009/142489, both of which are incorporated herein by reference.

[0082] Examples of non-biocatalytic methods for catalyzing the lactonization of 4-hydroxy acids to their corresponding cyclic esters (lactones): There are many non-biocatalytic methods for catalyzing 1,4-lactonization of hydroxy acids. For example, it is well known that such lactonization is acid-catalyzed and therefore lowering the pH of the medium (either inside or outside living cells) increases the rate of the lactonization reaction. Additionally, it has been reported in PCT/US2010/055524 (which is incorporated herein by reference) that activation via group transfer to a 4-hydroxy acid functional group is sufficient under acceptable conditions, such as pH 2.5-7.0 and room temperature, to quantitative yield of the lactone form. For example, PCT/US2010/055524 provides (1) activation with a phosphate group (to form, in this case, 4-hydroxylbutyryl phosphate) and (2) activation with coenzyme A (to form 4-hydroxylbutyryl-CoA). Synthesis of 4-hydroxylpentanoyl phosphate or 4-hydroxylpentanoyl-CoA intermediates using a natural or engineered kinase or CoA synthetase enzyme, respectively, or chemical synthesis, is expected to result in similar activation and random lactonization under appropriate conditions.

[0083] All reference materials mentioned in this document are incorporated herein by reference for all purposes.

Claims (14)

1. A method for preparing 2-oxo-4-valerolactone, the method comprising: converting pyruvate to 4-hydroxy-2-oxo-pentanoic acid by aldol addition and converting 4-hydroxy-2-oxo-pentanoic acid to 2-oxo-4-valerolactone by lactonization, where the lactonization is carried out at a low to neutral pH. 2. The method of claim 1 wherein the pyruvate is obtained from a carbon source containing one or more C6 sugars and/or one or more C5 sugars. 3. The method of claim 2, wherein the carbon source contains one or more of glycerol, a fatty acid, or an amino acid. 4. The method according to any one of paragraphs. 1-3, where pyruvate is produced at least in part by glycolysis in a microbial system. 5. The method of claim 2 wherein the one or more C6 sugars are selected from the group consisting of: allose, altrose, glucose, mannose, gulose, idose, talose, galactose, fructose, psicose, sorbose, and tagatose. 6. The method of claim 2, wherein the one or more C5 sugars are one or more of xylose, arabinose, ribose, lyxose, xylulose, and ribulose. 7. The method according to any one of paragraphs. 1-6, further comprising aldol addition catalysis with engineered pyruvate aldolase. 8. The method according to any one of paragraphs. 1-6, where the lactonization is catalyzed by the engineered biochemical catalyst. 9. The method of claim 8, wherein the aldol addition is carried out in the fermentation broth or cell-free solution, and wherein lactonization catalysis is carried out after isolation of 4-hydroxy-2-oxopentanoic acid from the fermentation broth or cell-free solution. 10. The method of claim 8, wherein the aldol addition is carried out in a fermentation broth or cell-free solution, and wherein the lactonization catalysis is carried out directly in the fermentation broth or cell-free solution using an engineered biochemical catalyst. 11. The method of claim 9 or 10, wherein the engineered biochemical catalyst is a lipase, esterase, protease, or lactonase mutant. 12. The method according to any one of paragraphs. 7-11, wherein the engineered pyruvate aldolase and/or the engineered biochemical catalyst is produced by protein engineering techniques using computer calculation, directed development techniques, rational mutagenesis, or a combination thereof. 13. The method according to any one of paragraphs. 8-12, where the engineered biochemical catalyst is 1,4-lactonase. 14. A method for producing methylenemethylbutyrolactone, the method comprising: converting pyruvate to 4-hydroxy-2-oxopentanoic acid by aldol addition; conversion of 4-hydroxy-2-oxopentanoic acid to 2-oxo-4-valerolactone via lactonization; conversion of 2-oxo-4-valerolactone to 2-hydroxy-4-valerolactone; converting 2-hydroxy-4-valerolactone to angelicalactone via reduction; and converting angelicalactone to methylenemethylbutyrolactone, thereby obtaining methylenemethylbutyrolactone.

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