# Patent

## Title
Methods for producing dicarboxylic acids

## Abstract

The present invention provides methods of producing dicarboxylic acids. The methods involve incubating a fatty acid or hydrocarbon substrate with an enzyme to produce a dicarboxylic acid product. The enzyme acts on the substrate to produce a product that has been both over-oxidized and has undergone cleavage of a C—C bond. In some embodiments the enzymes having these useful characteristics are mutants of a cytochrome P450 enzyme, for example an enzyme of the class CYP102 or a mutant thereof. The invention provides enzymes where these desirable characteristics can be found in a single enzyme, and thus in some embodiments the methods can be performed through the action of a single enzyme.

## Background
This application claims the benefit of U.S. provisional application Ser. No. 61/906,819, filed Nov. 20, 2013, which is hereby incorporated by reference in its entirety, including all Tables, Figures, and claims.


FIELD OF THE INVENTION
The invention relates to the enzymatic production of dicarboxylic acids from hydrocarbons or fatty acids.
INCORPORATION OF SEQUENCE LISTING
The materials in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name SGI1760_1_Sequence Listing_ST25, was created on Nov. 18, 2014 and is 53 KB. The file can be assessed using Microsoft Word on a computer that uses Windows OS.
BACKGROUND
Long chain di-carboxylic acids are versatile chemical intermediates that are used in the synthesis of perfumes, polymers, adhesives and lubricants, as well as by the pharmaceutical industry for the synthesis of antibiotics. Diacids with carbon chains longer than C10-C12 carbons, offer potential advantages over shorter diacids but current methods of synthesis of these compounds is commercially inadequate. One of the most important uses of such compounds is in the synthesis of polymers. In these uses long-chain di-carboxylic acids provide greater flexibility and strength than is available with short-chain di-carboxylic acids due to their ability to bend, which minimizes breakage and reduces the number of links in a polymers.
Current methods for the synthesis of long chain dicarboxylic acids involve either the chemical oxidation of unsaturated fatty acids, or the enzymatic oxidation of alkanes or fatty acids using whole cell fermentations (Huf, S. et al. Eur. J Lipid Sci. Technol. (2011), 113: 548-561). Chemical methods for the synthesis of dicarboxylic acids require derivatized fatty acids, such as unsaturated fatty acids (e.g., oleic, palmitoleic), hydroxylated fatty acids (e.g., ricinoleic acid), and the like. Typical chemistries that are used for these transformations include ozonolysis, which oxidizes an alkene to two carboxylic acids. Because ozonolysis is expensive and difficult to practice at the industrial scale, other chemical methods for the oxidation of unsaturated fatty acid have been developed. But these methods often suffer from lower yields (Warwel S, et al Lipid Technol 1997, 9:10-14). Various methods developed for the synthesis of dicarboxylic acids from fatty acids are described in Metzger, J. O., Eur. J. Lipid Sci. Technol. 2009, 111, 865-876. Other chemical methods such as cross metathesis technologies have been reported, but are not currently used in the commercial manufacturing of long fatty acids.
Some biocatalytic approaches for the synthesis of large (>C12) di-carboxylic acids utilize omega hydroxylase in the P450 family. These approaches utilize yeasts such as Candida, which oxidize the alpha and omega positions saturated wax hydrocarbons such as tridecane to tridecanedioic acid (Shuchen, L; et al Enz. Microb. Technol. 2004, 34, 73; Liu, S C; et al Pertochem Technol. 2002, 31, 558). But these methods rely on the use of non-renewable petrochemical waxes and require complicated fermentations of engineered bacteria with limited titers yields and productivities (Huf, S. et al Eur. J Lipid Sci. Technol. 2011, 113, 548). These methods also are limited to di-carboxylic acids having the same number of carbons as the starting wax or fatty acid. (Huf, S et al Eur. J Lipid Sci. Technol. (2011), 113, 548-561).
The P450 enzyme CYP107H1 (Biol) oxidizes fatty acids with C12 to C16 carbons to produce a single product, pimelic acid (heptanedioic acid C7). Besides being very selective for its products, CYP107H1 requires acyl-carrier-protein (ACP)-bound fatty acid as substrate. When free fatty acids are used as substrates, CYP107H1 gives a number of mono-hydroxylation products. Another disadvantage of this enzyme is the requirement for two additional enzymes, a ferredoxin and a feredoxin reductase, that decrease the activity and catalytic turnover of the enzyme (Lawson, R. J et al Biochemistry 2004, 43, 12390; Cryle, M J, Schlichting I. PNAS, 2008, 105, 15696).
It would be useful to have enzymes that could produce dicarboxylic acid products by over-oxidation of the substrates and also perform cleavage of the C—C bonds. This would be particularly useful if it could be done with a single enzyme, and from renewable resources such as fatty acids.
SUMMARY
The present invention provides methods and compositions for producing dicarboxylic acids. The methods involve contacting a fatty acid or hydrocarbon substrate with one or more enzymes to produce a dicarboxylic acid product. The one or more enzymes act on the substrate to produce a product that has been both over-oxidized and has undergone cleavage of a C—C bond. In some embodiments the one or more enzymes having these useful properties are mutants of a cytochrome P450 enzyme, for example a mutant an enzyme of the class CYP102 (cytochrome P450BM-3). The invention provides enzymes where these desirable characteristics can be found in a single enzyme, and thus in some embodiments the methods can be performed through the action of a single enzyme.
In a first aspect the invention provides a method of producing a dicarboxylic acid. The method involves contacting a hydrocarbon or fatty acid substrate with an enzyme that oxidizes the hydrocarbon or fatty acid substrate and breaks a C—C bond of the hydrocarbon or fatty acid substrate to produce a dicarboxylic acid product. In one embodiment the substrate is a fatty acid, which can be a saturated or unsaturated fatty acid. In various embodiments the saturated fatty acid can have an aliphatic chain of 4-28 carbon atoms, or an aliphatic chain of 15-20 carbon atoms.
In other embodiments the substrate is a hydrocarbon, for example a cyclic hydrocarbon. In some particular embodiments the cyclic hydrocarbon can be cyclooctane or cyclodecane, or can be selected from the group consisting of cyclohexane, cyclohexanol, cyclooctane, and cyclodecane.
In one embodiment of the invention the dicarboxylic acid product has a smaller number of carbons than the fatty acid substrate, for example the dicarboxylic acid product can have 3 carbons fewer than the fatty acid substrate. Hydroxylated fatty acid products can be produced in addition to the dicarboxylic acid product when the enzyme is incubated with the fatty acid. In some embodiments the enzyme is a CYP102 family enzyme. In some of these embodiments the enzyme can have between 55% and 99% amino acid sequence identity with BM3 (CYP102A1). The enzyme can be BM3-F87A.
In some embodiments the substrate is a fatty acid and the enzyme over-oxidizes the fatty acid. The reaction can involve the over-oxidation of adjacent diols. Oxidizing the fatty acid and breaking the C—C bond can comprise the reaction mechanism:




wherein m+n=14-20. In some embodiments m and n are positive integers between 4 and 7 and m+n=9-14, and in some specific embodiments the substrate is tridecanedioic acid (C19) and the dicarboxylic acid is palmitic acid (C16); or the substrate is cyclodecane and the dicarboxylic acid is sebacic acid (C7).
In some embodiments of the invention the substrate is a hydrocarbon and is incubated with the enzyme in a cell free reaction. The enzyme can be immobilized on a solid support. The enzyme can have at least 50% sequence identity to CYP102A1 wt (BM3) or CYP102A2 wt (YetO) or CYP102A3 wt (yrhJ) enzymes, and/or the enzyme can have at least one amino acid residue different from CYP102A1 wt (BM3) and/or CYP102A2 wt (YetO) and/or CYP102A3 wt (yrhJ) enzymes. In other embodiments the enzyme has at least 50% sequence identity to CYP102A1 wt (BM3) or CYP102A2 wt (YetO) and has at least one amino acid residue different from CYP102A1 wt (BM3) and/or CYP102A2 wt (YetO). And in more embodiments the enzyme is selected from the group consisting of: BM3 (F87A), YetO (F89A), YetO (F89I), YetO (F89S), YetO (F89V).
These and other objects, aspects, and features of the invention will become more fully apparent to those of ordinary skill in the art upon review of the following detailed description of the invention and the claims in conjunction with the accompanying figures.



DESCRIPTION OF THE DRAWINGS
FIG. 1 is a pathway showing the oxidation of fatty acids by a P450 enzyme (CYP102) to produce a dicarboxylic acid product.
FIG. 2 is a pathway showing the oxidation of a cyclic hydrocarbon (cyclodecane) to produce a dicarboxylic acid product.
FIG. 3 is a table showing the pairwise percent identity of selected wild type BM3 homologs.
FIG. 4a is a table showing the activities of BM3-wt, YetO-wt and selected mutant enzymes on various fatty acids. Legend: 1: Avg of two different growths; 2: Unit definition: 1U=1 nmole of NADPH consumed per min; 3: NM: not measured; 4: This concentration was not measured in this experiment, however it was shown elsewhere that BM3 and its mutant F87A express about the same. FIG. 4b is a bar chart showing activity comparisons of BM3, YetO, and selected mutant enzymes; bars are, left to right, lauric, myristic, and palmitic.
FIG. 5 provides a bar graph of the relative activities of YetO with different saturated and unsaturated fatty acids compared to lauric acid. Activities in FIG. 5 were calculated by measuring NADPH depletion in cuvette assays using the fatty acids as substrates.
FIG. 6a provides a bar graph of the total yields of dicarboxylic acids (DCAs) vs. fatty acid length. FIG. 6b provides a bar graph of the yields of other hydroxylation products vs. fatty acid length. Bars are, left to right, C15-C20.
FIG. 7 provides a schematic diagram of the oxidation of C15-C20 fatty acids by CYP102 enzymes.
FIGS. 8a-8c provide bar graphs illustrating DCA profiles of CYP102 enzymes with C15-C17 fatty acids. Bars are, left to right, (C15) C11-C14; (C16), C12-C14; (C17), C13-C15.
FIGS. 9a-9b provides bar graphs illustrating DCA profiles of CYP102 enzymes with C18 and C20 fatty acids. Bars are, left to right, (C18), C13-C16; (C20), C13-C18.
FIG. 10 shows HPLC chromatograms for the oxidation of palmitic acid with BM3-YetO. The top chromatogram shows the total ion count and the remaining show peaks corresponding to different masses and putative species.
FIG. 11 shows an HPLC chromatogram for the oxidation of stearic acid (C18) with BM3 enzyme in a lysate.
FIG. 12 shows an HPLC chromatogram for the oxidation of heptadecanoic acid (C17) with purified BM3 enzyme.
FIG. 13: Concentrations of all major products after the oxidation of palmitic acid with BM3 (left bars) and after chemical treatment (right bars). For tridecanediacid (C13 DCA) the yield to product is shown in the left Y-axis (striped bars); for all other compounds due to the lack of authentic standards, the area count from HPLC analysis is shown (right Y-axis).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods of oxidizing fatty acid or hydrocarbon substrates to produce dicarboxylic acid products. The methods are performed through the use of enzymes that have the ability to over-oxidize the fatty acid or hydrocarbon substrates and additionally to cleave a C—C bond of the substrate, resulting in a dicarboxylic acid product. The enzymes can be Cytochrome P450 enzymes that are members of the CYP102 sub-family, and homologs thereof. These enzymes are hemoproteins that catalyze the monooxygenation of various substrates.
The present methods offer several advantages. The present methods can be carried out without the need to utilize toxic chemicals, which are often required in chemical processes for the oxidation of fatty acids or hydrocarbons. The present methods also offer a substantial reduction in the costs of materials involved, as well as higher reaction product yields than have previously been available. Furthermore, the present methods can be performed using a much wider selection of starting materials, such as derivatized, un-derivatized, saturated or unsaturated fatty acids, in addition to hydrocarbons. Yet another advantage offered by the present invention is a much wider class of dicarboxylic acids that can be produced. The invention allows the production not only of dicarboxylic acid products having the same number of carbons as the chain of the starting fatty acid or other hydrocarbons, but also of dicarboxylic acid products having a smaller number of carbons as the chain of the starting fatty acid or hydrocarbon. The present methods can also accomplish the synthesis of dicarboxylic acids using a wider class of petroleum-derived hydrocarbons as substrates, for example, cyclic hydrocarbons (e.g., cyclooctane, cyclodecane, etc.) or aliphatic hydrocarbons. Furthermore, the present methods can be performed using a single enzyme that acts on the hydrocarbon substrate or a product of the reaction of the enzyme on the hydrocarbon substrate. The enzymes of the present invention can perform the initial derivatization of the hydrocarbon as well as the subsequent oxidation and breaking of the C—C bond to arrive at the dicarboxylic acid product of the method. In some embodiments the enzyme acts on a fatty acid substrate and produces a dicarboxylic acid product.
In various embodiments the methods of the invention can further involve one or more steps of converting intermediates or side products of the reactions and/or dicarboxylic acid products of the reaction into other desirable products. For example, various compositions containing diols, vicinal diols, and alcohols (e.g., keto alcohols) can be produced either as intermediates or as reaction side products. The conversion of these intermediates or side products into other desirable products further drives the reaction forward and increases yields compared to methods that do not utilize these one or more steps. Another step that can be included in any of the methods of the invention is the purification of dicarboxylic acids from the reaction products. This purification step can involve the normal processes used to purify dicarboxylic acids, such as by chromatographic methods. Persons of ordinary skill in the art with resort to this disclosure will realize that the specific methods of purifying the dicarboxylic acids will depend on the dicarboxylic acid to be purified or isolated and the contaminants to be eliminated. In some embodiments the purification can involve one or more steps of extracting with diethyl ether (or another organic and/or polar solvent), drying, and fractionally distilling the dicarboxylic acid product. Other methods known to those of skill in the art can also be utilized. In various embodiments the step of purifying the dicarboxylic acid can involve purifying by at least 50% w/w or at least 75% w/w or at least 85% w/w or at least 95% w/w.
In other embodiments the methods of the invention also involve a step of polymerizing the dicarboxylic acid products. The dicarboxylic acids can be polymerized to form polymerized products or intermediates, e.g. for the synthesis of other molecules. In some embodiments the dicarboxylic acids are polymerized to form nylons or precursors for synthesizing nylons. The step of polymerizing the dicarboxylic acid can be polymerizing at least 25% or at least 50% or at least 75% of the dicarboxylic acid product. Polymerization the dicarboxylic acid can mean polymerizing only the dicarboxylic acid product or polymerizing the dicarboxylic acid with another molecule in the mixture. In another embodiment the method can include a step of adding another molecule to the mixture and polymerizing the dicarboxylic acid with the molecule added to the mixture. The method can further involve a step of adding a molecule to initiate or enable polymerization of the dicarboxylic acid product.
The methods of the invention can also involve one or more steps of a) converting dicarboxylic acids to other desirable chemical products, or b) purifying dicarboxylic acids or c) polymerizing dicarboxylic acids, or each of steps a), b), and c), depending on the specific objectives involved.
Reaction Scheme
The methods involve producing a dicarboxylic acid from a hydrocarbon or fatty acid starting material by contacting the hydrocarbon or fatty acid with one or more enzymes that oxidize the hydrocarbon or fatty acid and break a C—C of the hydrocarbon or fatty acid and produce a dicarboxylic acid product. The contacting can be done by combining the reactants in a solution or reaction mixture. The reactants can be incubated or left in contact for an appropriate period of time.
The inventors discovered unexpectedly that certain enzymes have the ability to over-oxidize hydrocarbons and fatty acid substrates and break a C—C bond of the substrate to produce the dicarboxylic acid product. In various embodiments the enzymes are members of the CYP102 subfamily of enzymes. Various forms of the enzyme and mutants or homologs of the enzymes were discovered to have this ability. By over-oxidizing the hydrocarbon or fatty acid substrate it is possible to first oxidize the substrate and then further oxidize to cleave the C—C bond to produce a dicarboxylic acid molecule. By “over-oxidize” is meant that the enzyme reaction performs at least 3 oxidations on the molecule. The three oxidations can be, for example, the oxidations of two carbons to hydroxyls and a third oxidation of either the production of a carbonyl or the cleavage of a C—C bond. The cleavage of a C—C bond can produce a dicarboxylic acid. In some embodiments the enzyme over-oxidizes and performs at least 4 oxidations on the substrate molecule. Thus, oxidation can be the formation of a hydroxyl, or the formation of a ketone, or the breaking of a C—C bond. Each of these oxidations can be performed on atoms or groups of atoms attached to the backbone chain of the hydrocarbon molecule. In one embodiment the methods are carried out using a single enzyme that acts on the substrate to produce the dicarboxylic acid reaction product, thus facilitating large scale production of the dicarboxylic acids. The enzyme can also be a soluble enzyme. In one embodiment the enzyme oxidizes adjacent diols on the hydrocarbon or fatty acid substrate. As an example, FIG. 1 (also below) depicts a reaction scheme of the method of the invention, and the diol-A species exhibits adjacent diols, which are further oxidized in the pathway to form a breakage of the C—C bond and result in the production of a dicarboxylic acid and a fatty acid. Breaking a C—C bond means breaking a covalent bond to separate a molecule into separate molecules thus, for example, causing a double bond to change to a single bond is not considered to break a C—C bond as used herein. But to break a palmitic acid (C16) molecule into a tridecanoic acid (C13) and a three carbon molecule (e.g., propanoic acid) is to break a C—C bond.
Thus, in some embodiments the methods utilize a reaction of the mechanism (I):




where m and n are positive integers. In various embodiments m+n=9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 6-11 or 6-16 or 9-14 or 9-20 or 14-20. In some embodiments m and n are each any of 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 0-9, or 4-7 or 4-10 or 6-16 or 10 or 11 or 12 or 13 or 14 or 0-14 or 12-14. In some embodiments n is 9-14 and m is 0-5; or n can be 12-17 and m is 0.

In some embodiments the reactions of the invention produce mixtures of alcohols and diols. In various embodiments the reactions have greater than 90% selectivity for producing the carbon chain length of the dicarboxylic acid formed from the over-oxidation of the hydrocarbon or fatty acid substrates, meaning that greater than 90% of the dicarboxylic acids produced are of a particular chain length. One or more of the dicarboxylic acids produced by the methods of the invention can have three fewer carbon atoms (in the chain) compared to the hydrocarbon or fatty acid substrate. Thus, in various embodiments the fatty acid palmitic acid (C16) can be used as a substrate to produce tridecanedioic acid (C13) as a major dicarboxylic acid product with at least 90% selectivity or at least 80% selectivity or at least 75% selectivity or at least 60% selectivity or at least 50% selectivity. Selectivity refers to the molar ratio of the product formed. But in other embodiments the fatty acid product can also have two fewer or four fewer or five fewer carbon atoms in its chain compared to the carbon chain of the hydrocarbon or fatty acid substrate, and the product can also be a mixture of fatty acids of two fewer or three fewer or four fewer or five fewer carbon atoms compared to the hydrocarbon or fatty acid substrate. Thus, the reactions may produce a product having 3 or 4 or 5 carbon atoms in its chain, respectively with selectivities of at least 50% or at least 60% or at least 70% or at least 80% or at least 90% molar. In other embodiments the predominant product has three fewer carbon atoms compared to the number of carbon atoms in the chain of the hydrocarbon or fatty acid substrate, and in other embodiments the predominant product has four fewer or five fewer carbon atoms in its chain compared to the carbon chain of the hydrocarbon or fatty acid substrate. Additionally, other mono- and di-hydroxy palmitic acid derivatives may be produced. These additional products may be further processed by chemical or other methods into additional desired products as described herein.
The present methods can also produce the dicarboxylic acids products without the hydrocarbon or fatty acid substrate being bound to or by an acyl carrier protein (ACP) at the time of cleavage by the enzyme, or at any point during the reaction.
Without wishing to be bound by any particular theory it is believed that the reaction of the invention proceeds through a step-wise oxidation of the hydrocarbon or fatty acid substrate to Alcohol-A (FIG. 1) followed by the further oxidation and formation of vicinal diols and eventually to the synthesis of a dicarboxylic acid by the further oxidative cleavage of the C—C bond between the diols. A CYP102 enzyme catalyzes these reactions and NADPH can be a cofactor. A carboxylic acid product can also be produced. Thus, the oxidative cleavage may occur through the over-oxidation of the vicinal diols (FIG. 1). In vicinal diols the hydroxyl groups are attached to adjacent atoms.
In some embodiments in addition to the enzymes an oxidizing agent can be included in the reactions. The oxidizing agent can be contacted with the substrate simultaneously with the enzyme or in a separate subsequent step. The oxidizing agents promote the oxidation of adjacent diols or alcohols to the corresponding carboxylic acids with the simultaneous breaking of the C—C bond. Examples of oxidizing agents that can be included in the reactions include, but are not limited to, H2O2/Na2WO4, tungsten-based poly-acids, ruthenium chloride, potassium permanganate or other strong oxidizing agents. In different embodiments the inclusion of one or more oxidizing agents can increase the reaction yield of di-carboxylic acids in any of the methods by at least 30% or at least 50% or at least 70% or at least 100% or at least 150% or from 30%-200% or from 50% to 150% or from 50% to 200% versus in the absence of the oxidizing agent.
Hydrocarbons and Fatty Acids
The hydrocarbons and fatty acid substrates used in the invention can be any hydrocarbon or fatty acid. As used herein the term “hydrocarbon” indicates molecules consisting entirely of carbon and hydrogen atoms, referring to both straight chain hydrocarbons, branched chain hydrocarbons, and cyclic hydrocarbons (e.g., a cycloalkane). A fatty acid molecule as used herein is a carboxylic acid molecule having an aliphatic tail, which can be saturated or unsaturated. They can be (but are not necessarily) derived from triglycerides or phospholipids. They may also be referred to as free fatty acids when not attached to other molecules. The hydrocarbons and fatty acids used in the invention can be either saturated or unsaturated or partially saturated. A hydrocarbon or fatty acid molecule as used herein comprises a hydrocarbon chain within it consisting only of carbon and hydrogen atoms of at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 11 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or 22 carbon atoms. In other embodiments the hydrocarbon or fatty acid has 12 or fewer, 13 or fewer, 14 or fewer, 15 or fewer, 16 or fewer, 17 or fewer, 18 or fewer, 19 or fewer, 20 or fewer, 21 or fewer, or 22 or fewer carbon atoms, or up to 22, or up to 24, or up to 26, or up to 28, or up to 30 carbon atoms. The carbon atoms can be part of an aliphatic chain. In other embodiments the hydrocarbon or fatty acid can have from 4 to 20 or from 4 to 22 or from 4 to 24 or from 4 to 26 or from 4 to 28 or from 4 to 30 or from 14 to 20 or from 15 to 22 carbon atoms.
In some embodiments the hydrocarbon or fatty acid substrate molecule can also contain other functional groups in the molecule having atoms other than carbon and oxygen. In other embodiments the substrate does not contain a functional group. The functional groups can include carbon, hydrogen, and oxygen atoms, but in other embodiments can include one or more other atoms such as, for example, nitrogen, phosphorus, and halogens, but in other embodiments can be any atoms. In various embodiments the hydrocarbon chain of the hydrocarbon molecule is as described above and can have a number of hydrogen atoms corresponding to the number of carbons, in a saturated or unsaturated hydrocarbon chain within the hydrocarbon molecule. In some embodiments a hydrocarbon molecule according to the invention has only hydrogen and carbon atoms and, when there is a functional group present, not more than 12% or not more than 13% or not more than 14% or not more than 15% oxygen atoms by number.
In embodiments where the substrate is a fatty acid, it can be a fatty acid between 4 and 28 carbons, or from 15 to 20 carbons, or has 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The carbon atoms can be part of an aliphatic chain, and the aliphatic chain can be a straight or branched aliphatic chain. The fatty acid can be saturated or unsaturated or partially unsaturated. Thus, the fatty acid can have 0, 1, 2, 3, or more than 2 or more than 3 double bonds. In some embodiments the hydrocarbon is a saturated straight chain aliphatic hydrocarbon. Examples of suitable hydrocarbons include, but are not limited to, pentadecanoic acid (pentadecyclic acid, C15), hexadecanoic acid (palmitic acid, C16), heptadecanoic acid (margaric acid, C17), octadecanoic acid (stearic acid, C18), nonadecanoic acid (nonadecylic acid, C19), and eicosanoic acid (arachidic acid, C20). In additional embodiments the hydrocarbon can be propanoic acid (C3), butanoic acid (C4), pentanoic acid (C5), hexanoic acid (C6), heptanoic acid (C7), octanoic acid (C8), nonanoic acid (C9), decanoic acid (C10), undecanoic acid (C11), dodecanoic acid (C12), tridecanoic acid (C13), tetradecanoic acid (C14), heneicosanoic acid (C21), docosanoic acid (C22), tricosanoic acid (C23), tetracosanoic acid (C24), pentacosanoic acid (C25), hexacosanoic acid (C26), heptacosanoic acid (C27), octacosanoic acid (C28), nonacosanoic acid (C29), triacontanoic acid (C30), henatriacontanoic acid (C31), dotriacontanoic acid (C32), tritriacontanoic acid (C33), tetratriacontanoic acid (C34), pentatriacontanoic acid (C35), and hexatriacontanoic acid (C36). Persons of ordinary skill with resort to this disclosure will realize additional hydrocarbon or fatty acid molecules that can be used in the methods of the invention. The invention can also be applied to mixtures of hydrocarbons or fatty acids. A mixture of fatty acids or hydrocarbons useful in the invention can contain more than one type or species of any of the fatty acids or hydrocarbons disclosed herein. In some embodiments the mixture can contain 2 or 3 fatty acids or hydrocarbons disclosed herein. The hydrocarbon or fatty acid can also be any that the enzyme of the invention can over-oxidize and break a C—C bond to produce a dicarboxylic acid product.
Cyclic Hydrocarbons
Cyclic hydrocarbons comprise one or more rings of carbon atoms. The hydrocarbon chain of a cyclic hydrocarbon that consists entirely of carbon and hydrogen atoms can be a part of one or more of the rings. In some embodiments the methods of the invention can be applied to cyclic hydrocarbons or cycloalkanes in the production of dicarboxylic acids. Cycloalkanes are alkane hydrocarbons and have one or more rings of carbon atoms in the chemical structure of the molecule. In some embodiments the methods can be used to convert cyclic hydrocarbons to produce single hydroxylation products (e.g., alcohols, ketones) or diols and keto alcohols. The cyclic hydrocarbons can contain 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 carbon atoms in the ring. In some particular embodiments the cyclic hydrocarbon can be the cycloalkanes cyclooctane or cyclodecane, or any cyclic hydrocarbon.
In one embodiment cyclodecane is oxidized in the methods to produce a heptanedioic acid (sebacic acid) dicarboxylic acid product, as shown in FIG. 2 and further discussed in Example 3. The enzyme utilized in this embodiment was the CYP102 enzyme BM3-F87A. Other cyclic hydrocarbons can also be utilized in the present invention to form corresponding dicarboxylic acids.
Dicarboxylic Acids
The methods of the invention produce dicarboxylic acid products. The reactions of the invention are able to produce dicarboxylic acids having an even or odd number of carbon atoms in the carbon chain, thus the product can be a dicarboxylic acid having 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or more than 22 carbon atoms. For example the methods of the invention can be applied to palmitic acid (C16) to produce tridecanedioic acid (C13). In another example the methods can be used to convert stearic acid (C18) to pentadecanedioic acid (C15). The methods can also produce a mixture of dicarboxylic acid products, which may be present in varying portions. The dicarboxylic acid product can also be a mono-unsaturated or di-unsaturated or poly-unsaturated dicarboxylic acid.
Persons of ordinary skill with resort to this disclosure will realize that the enzymes used in the methods can be engineered to act on specific substrates and produce specific products. It is possible to also design processes that utilize both chemical and enzymatic methods to produce specific products of interest from specific substrates. By using a combination of chemical and enzymatic methods it is possible to design processes for transforming all or substantially all carbon in the starting substrate or reactant molecules into a desired product. The desired product can be any described herein. For example, with reference to FIG. 1 residual Diol-A that may be present after incomplete oxidation of substrates can be processed to a desired product using one or more additional chemical steps. In the same manner other reactants produced from incomplete reactions or as side products can also be processed to desired products by one or more additional chemical or enzymatic steps. An isolation or purification step can also be added if desirable, which involves the isolation or purification of a desired product from a reaction product mixture. In such manner, by combining chemical and enzymatic steps, product yields can be increased. By substantially all carbon is meant at least 90% of carbon atoms.
In another embodiment the products are not dicarboxylic acids but are precursors to dicarboxylic acids, for example, diols or keto alcohols that can be converted into dicarboxylic acids with one or more additional steps. The precursors may be useful chemicals, or they may be further processed into dicarboxylic acid products, as desired.
Di-carboxylic acids can be purified from reaction media by various methods. Di-carboxylic acids have limited solubility in acidic aqueous media. Therefore, methods can be employed involving one or more steps of the precipitation of di-carboxylic acids by lowering the pH of the reaction solution. They can also be extracted from the reaction mixture at acidic pH (e.g., 1-4) in one or more steps using various organic solvents such as ethers (diethyl, dibutyl, methyl-tert-butyl ether, etc.), esters (ethyl acetate, propyl acetate, butyl acetate, etc.) or other solvents such as, for example, petroleum ether, hexane, toluene etc., or any combination of them.
Additional examples of purification methods that can be employed include one or more steps of precipitation by the addition of aqueous, miscible, organic solvents (e.g., ethanol). Insoluble salts can also be produced, for example by the addition of Ca(OH)2. Another method that can be employed is the esterification of the di-carboxylic acids to form their corresponding diesters using MeOH, EtOH, PrOH, BuOH, iBuOH or other alcohols. Esters can be formed in situ in the aqueous reaction media using enzymes (such as esterases), or by incubating the di-carboxylic acids in these alcohols after they have been precipitated as described above. Esterification of precipitated di-carboxylic acids can be catalyzed by an enzyme (e.g., esterase, lipase) or by a chemical catalyst (e.g., BF3, acid, base etc). Esters can then be distilled under reduced pressure or can be purified by chromatography. Any of the methods described herein can include one or more of the above-described steps to purify or isolate the di-carboxylic acid product.
Enzymes
The enzymes utilized in the methods can be bacterial enzymes, which in some embodiments can be a fatty acid hydroxylase flavocytochrome P450 enzyme. In one embodiment the enzyme is a microbial cytochrome P450 enzyme, which can be a bacterial enzyme. In other embodiments plant, animal, mammal, insect, or fungal cytochrome P450 enzymes can be used. A bacterial cytochrome P450 enzyme can contain a ferredoxin reductase and a ferredoxin, which can be present as a single peptide or as separate proteins, and which transfer electrons to P450. In one embodiment the enzyme is a member of the P450 enzyme sub-family CYP102. It was discovered unexpectedly that the members of this class of enzymes have the ability to over-oxidize hydrocarbon and fatty acid substrates and break a C—C bond in the substrate. Enzymes of this sub-family contain both the heme domain, which performs the oxidation of the substrate, and the reductase domain, which reduces the heme iron in each catalytic cycle. With these domains present in a single protein increased activity and increased catalytic turnovers are available. The P450 heme domain of the enzyme can over-oxidize saturated hydrocarbons, fatty acids, diols, or keto alcohols to produce the dicarboxylic acid with the concomitant breaking of the C—C bond. The enzymes of the invention are able to do this even to free fatty acids, meaning fatty acids that are not bound to ACP. Portions of the enzymes that also have these abilities are referred to as functionally active fragments of the enzymes, and they can also be used in the methods. In various embodiments functionally active fragments can have at least 50% or at least 70% or at least 90% of the described function of the full molecule, and can be of any portion or sub-portion of the sequence of the full molecule having the stated % function compared to the full molecule.
The enzyme(s) can be derived from a bacterium, e.g., a bacterium of the genus Bacillus, or can be derived or synthesized and expressed in E. coli or another appropriate host cell. In one embodiment the particular species of Bacillus is Bacillus megaterium. The enzyme can be BM3, otherwise known as CYP102A1 (EC1.14.14.1), which is a fatty acid hydroxylase from Bacillus megaterium. BM3 contains a P450 heme domain fused to the eukaryotic-like diflavin reductase partner (cytochrome P450 reductase) in a single polypeptide chain, and therefore is catalytically self-sufficient as a monooxygenase. The enzyme can also be a homolog of BM3 having a stated percent sequence identity to BM3. BM3 and other members of the CYP102 sub-family catalyze the NADPH-dependent hydroxylation of long-chain fatty acids at the ω-1, ω-2, and/or ω-3 positions. But in various embodiments many enzymes and homologs of enzymes described herein have the recited characteristics and are useful in the methods of the invention. In other embodiments the enzyme can also be YetO (CYP102A2)(EC1.14.14.1) from a Bacillus or a homolog thereof, or can be yrhJ (CYP102A3) (EC1.14.14.1) or a homolog thereof or any enzyme in the CYP102 sub-family or a homolog thereof. YrhJ can be derived from Bacillus amyloliquifaciens (strain FZB42, among other strains) and has a length of 1053 amino acids. CYP102A2 and CYP102A3 can be derived from Bacillus subtilis and are both homologs of BM3, and are both natural water-soluble fusion proteins. The enzymes used in the methods of the invention can be soluble enzymes, which means in their natural state the enzymes (or the enzyme from which a homolog is derived) are present in the cytosol of the cells from which they are derived. Enzymes that exist primarily in the cell membrane in their natural state (and homologs thereof) are not considered soluble enzymes. Other enzymes finding use in the present invention include Cyp96A and others.
In various embodiments the enzyme used in the invention can be a cytochrome P450 enzyme of the sub-family CYP102, or a homolog thereof. BM3 can be derived from B. megaterium and techniques are available for producing suitable homologs, for example site-directed mutagenesis. In one embodiment the enzyme is YetO, the nucleic acid and protein sequences of which are disclosed in the accompanying sequence listings as SEQ ID NO: 3 (nucleic acid) and SEQ ID NO: 4 (protein). This enzyme can be isolated and cloned from gDNA, or can be synthesized and optimized for recombinant expression in an appropriate host organism such as, for example, E. coli, Pseudomonas, or Bacillus. Many site-specific mutants can be derived. FIG. 3 illustrates various homologs that have been derived from BM3, and the exemplary sequences of some homologs are disclosed in the accompanying sequence listings as Bac0018 (SEQ ID NO: 5) and Bac2875 (SEQ ID NO: 7), as well as some wild type sequences BM3-wt (SEQ ID NO: 1—DNA and SEQ ID NO: 2—amino acid) and corresponding protein sequences. But persons of ordinary skill with resort to this disclosure will be able to derive a large number of suitable homologs, to BM3 or to other P450 enzymes.
Homologs of any of the enzymes disclosed herein can also be used as the enzyme in the methods. In various embodiments the enzyme utilized in the method can have at least 50% sequence identity to the cytochrome P450 enzyme BM3-wt (CYP102A) from Bacillus megaterium or YetO from Bacillus subtilis (CYP102A2) or yrhJ-wt. In various other embodiments the enzyme of the methods can have at least 55% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity to BM3-wt or YetO-wt or yrhJ-wt. In other embodiments the invention provides an enzyme having 50-99% sequence identity to CYP102A or YetO wild type enzymes or 50-90% or 55-90% or 60-90% or 80-90% or 55-99% or 60-99% or 70-99% or 80-99% or 90-99% or 95-99% or 98-99% sequence identity to CYP102A or YetO wild type enzymes. A number of different search algorithms have been developed, including but not limited to the suite of programs referred to as BLAST programs. There are two implementations of BLAST designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al., Genome Analysis, I: 543, 1997). Such methods involve alignment and comparison of sequences. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Other such software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. In the invention, one or more of these methods can be used to calculate the percent amino acid or nucleotide sequence identity between two homolog molecules.
The methods and/or reactions described herein can be performed in vitro or in vivo in a suitable host cell. In some embodiments any one or more of the nucleic acid or protein/peptide sequences disclosed herein can be comprised in a vector. In some embodiments the vector can contain one or more selection markers. Host cells can be transformed with the vector and the methods and reactions described herein carried out in the organism. The organisms transformed can be an appropriate yeast or bacterial species. Examples include, but are not limited to, bacteria in the Enterobacteriaciae family, such as E. coli, Yersinia, Klebsiella, Shigella, Hafnia (e.g., alvei). When a yeast is used, any suitable yeast can be used (e.g., Saccharomyces, Candida, etc). In one embodiment the invention provides a recombinant organism or cell comprising an exogenous nucleic acid described herein (e.g., any one or more of SEQ IDs 1 or 2 or 3 or 4 or 5 or 6 or 7. “Exogenous nucleic acid” refers to a nucleic acid not found in the organism in its natural environment.
The enzymes utilized in the methods of the invention can have selectivity for a ω-2 or ω-3 fatty acid diol. In various embodiments the enzyme has a selectivity for either an ω-2 or ω-3 fatty acid diol of at least 50% or at least 60% or at least 70% or at least 80% or at least 90% molar selectivity, meaning that the enzyme will cleave the stated percentage of times the ω-2 or ω-3 fatty acid diol.
The invention also provides a composition containing any of the enzymes or stated homologs thereof described herein. In various embodiments the enzymes can be provided in a dried form in a container, or in a liquid form in a container. The enzyme compositions can also be a purified enzyme composition, for example being at least 90% purified w/w or at least 95% pure w/w or at least 98% pure w/w or at least 99% pure w/w. Any of these purified enzyme compositions or any combination thereof can be used in the methods of the invention.
The invention can also provide a kit for conducting methods of the invention. The kits can contain any one or more compositions of the invention provided in a container (either individually or together) and, optionally, instructions for using the composition to conduct a method of the invention and/or a link to a website providing such instructions and/or information about the methods and/or about conducting the methods. The kit can also contain buffers for conducting the methods.
Cell or Cell-Free Systems
The methods, compositions and reactions of the invention can be performed or applied in either a cell culture system or a cell-free system. When cell culture is used it can be either a bacterial cell culture (e.g., using bacteria described herein or other appropriate species) or a yeast cell culture or an insect cell culture. In some embodiments the methods can be applied in a whole cell system utilizing cells that over-express a CYP102 enzyme, which in some embodiments is expressed in the cell as an exogenous enzyme. Any suitable enzyme of the CYP102 sub-family or a homolog thereof can find use in the present invention. In performing the methods cells can be mixed with any appropriate hydrocarbon or fatty acid substrate (or mixture of such substrates) as described herein. Additionally, one or more sugar molecules (e.g., glucose, glycerol, formic acid) can also be present in the mixture for the generation of reducing equivalents. In some embodiments recombinant proteins producing enzymes or other components of the system can be expressed on plasmids or other extra-chromosomal DNA. In one embodiment the enzyme that breaks a C—C bond of a hydrocarbon or fatty acid substrate is expressed on a plasmid comprised in bacteria in a bacterial fermentation.
The methods, compositions and reactions of the invention can also be performed or applied in a cell-free system. The methods can be performed or applied in a crude lysate, or in other embodiments purified enzyme (or a combination of enzymes) can be used in a free soluble enzyme or immobilized on a solid support. A crude lysate is the solution produced when cells are destroyed by lysing or disrupting their cell membranes, and in one embodiment at least 50% of the cells in a medium or solution are lysed or destroyed to produce the crude lysate, but in other embodiments at least 75% or at least 90% or at least 95% or at least 99% f the cells in the medium or solution are destroyed or lysed to produce the crude lysate. Catalytic amounts of NAD and/or NADP and/or another desirable co-factor, as well as a desirable co-factor recycling system can also be present in the reaction mixture, for example a glucose/glucose dehydrogenase or formate/formate dehydrogenase system, or another appropriate system for recycling co-factors. In some embodiments the methods of the invention are performed in a test tube, centrifuge tube, multi-well plate, 96 cell plate, fermenter, flask, cuvettes, glass or plastic vessel, or other vessel that contains the reactants. The methods can involve isolating or obtaining enzyme from a microorganism grown on a nutrient agar or nutrient plate or in a nutrient medium. The methods can also be performed using live or immobilized cells containing or expressing the enzymes.
Any of the methods described herein may involve one or more steps of culturing cells or host cells (either of which examples are provided herein), and/or one or more steps of lysing, disrupting, or destroying the cells or host cells before contacting the hydrocarbon or fatty acid substrate with one or more of the enzymes described herein. The method can also involve one or more steps of producing a dicarboxylic acid product and/or performing a purification step for one or more dicarboxylic acid products from a cell culture or lysate.
Example 1—Enzyme Engineering
This example shows the engineering of wild-type (wt) BM3 and wild-type (wt) YetO to produce a group of mutant enzymes having the desirable activities of sub-terminal over-oxidation of fatty acids and the breaking of the C—C bond. BM3 (or CYP102A1) from B. megaterium and one proprietary homolog from a Bacillus strain (YetO from Bacillus 15_F03) were cloned. Medium homology is shared between BM3 and YetO (60% identity, 75% similarity). The natural gene sequences were used in all plasmids. The YetO gene was PCR-cloned from gDNA, while a synthetic gene using the natural DNA sequence was made for BM3. Various site specific mutants from these sequences were then created as shown in Table 1.







TABLE 1







Initial BM3 and homologs cloned











Enzyme (Source)
Source
Mutation
pSGI
Vector





BM3 (P14779)
B. megaterium
—
118
pET24


BM3 (P14779)
B. megaterium
F87A
004
pET24


BM3 (P14779)
B. megaterium
F87A
014
pET28




(w/HisTag)


yetO
Bacillus 15_F03
—
040
pET24


yetO
Bacillus 15_F03
F89A
055
pET24


yetO
Bacillus 15_F03
F89I
056
pET24


yetO
Bacillus 15_F03
F89S
057
pET24


yetO
Bacillus 15_F03
F89V
058
pET24









The sequences are shown herein. For F87 (in BM3) and F89 (in YetO), this phenylalanine was site mutated to alanine in BM3, and to either alanine, isoleucine, serine, or valine to produce the mutants as indicated in Table 1. Two more BM3 homologs were cloned from two Bacillus strains. The native genes of these enzymes were amplified by PCR from genomic DNA and were cloned into pET24a vector. Good functional expression in E. coli was obtained for both enzymes as indicated by their peaks at 450 nm using standard CO binding assays. Their pairwise percent identities with BM3-wt and YetO-wt are listed in FIG. 3b, which shows that the wild-type BM3 does not necessarily possess a very high degree of homology to the two BM3 mutants. Substantial variation is possible while retaining enzymatic activity, is illustrated in FIG. 4. The two functionally expressed enzymes were characterized for both NADPH depletion and overnight reactions on fatty acids. For NADPH depletion assays, 40 μL of lysate was added to 160 μl reaction buffer (4 mM fatty acid, 0.25 mg/mL NADPH) in 96-well microtiter plates. These reaction conditions resulted in lower specific rates than seen in cuvettes but enzymes showed the same relative specific rates when compared to each other. The two modified enzymes showed specific rates between those of the BM3s and YetOs with preference for palmitic and myristic acids, as seen in FIG. 4b. 
FIG. 4a shows the hydroxylation activities of BM3, YetO and selected mutants with various fatty acids and FIG. 4b shows the activity comparison of BM3, YetO, and various enzyme mutants. YetO-wt gives similar oxidation products (hydroxylation at ω-1, 2, 3) as BM3-wt when reacted with lauric acid, but there are differences in the reactivity between these two enzymes. The activity data of FIG. 4b shows that YetO-wt has a preference for lauric (C12) and myristic (C14) compared to palmitic (C16) acid. In contrast, BM3-wt possesses similar activity for lauric and palmitic acids, but its activity is significantly lower for lauric acid compared to YetO-wt. Furthermore, BM3-F87A shows a reversal of fatty acid preference (palmitic being the highest amongst C12/C14/C16) compared to the equivalent mutant(s) of YetO.
Introducing unsaturation to the fatty acid increased the activity of YetO-wt even to fatty acids longer than palmitic, or to fatty acids whose saturated analog was very low or not active at all (FIG. 5). This is shown in the reactivity comparisons between palmitic and palmitoleic, stearic and oleic and even arachidic and erucic. Note that similar reactivities have been reported with BM3-wt.
For quantitative analysis of products from reactions of the enzymes on fatty acids, cell-free reactions with crude lysate were performed. Crude lysate containing each P450 enzyme was mixed with a reaction mixture (100 mM potassium phosphate pH 7.8; 1-5 mL total volume; 8/2, v/v, reaction mix/P450 lysate) containing 2-5 mM fatty acid, 1.0 mM NADPH, 30 mM glucose, and 1 mg/mL glucose dehydrogenase. The glucose/GDH-105 system regenerates NADPH from NADP by the concomitant oxidation of glucose to gluconate. After overnight incubation at 30° C. with good shaking, crude reaction mixtures were mixed with 1-5 drops trifluoroacetic acid (TFA) and equal volume tetrahydrofuran (THF). After centrifugation to precipitate the insoluble protein, samples were analyzed by HPLC-MS.
Reactions were run on lauric (C12), pentadecanoic (C15), palmitic (C16), heptadecanoic (C17), stearic (C18), arachidic (C20), and lignoceric (C24) acids. The reactions on lauric acid produced a mixture of products formed from hydroxylation at the ω-1, ω-2, and ω-3 positions, without any dicarboxylic acids. Reactions on fatty acids with lengths C15-C20 produced several hydroxylation products and dicarboxylic acids (DCAs) resulting from internal C—C bond cleavage. The concentrations of these products were estimated using calibration curves for the available products (single hydroxylation products, dicarboxylic acids, etc.). FIG. 6a shows total yields of all DCAs and hydroxylation products likely in the pathway to DCA formation. BM3-WT and Bac0018 produce the most DCAs, but the YetO enzymes all produce more hydroxylation products than the other enzymes. The concentration of hydroxylation products in these reactions is also higher than the concentration of DCAs in the best reactions. The enzymes produce large amounts of DCAs from C17 fatty acid, but the yields for the C18 fatty acid are actually higher since the solubility of this substrate is lower and thus there was less starting material present. The YetO enzymes produced small amounts of DCAs but high amounts of other hydroxylation products, as shown in FIG. 6b. These enzymes had lower P450 concentrations in the crude lysate (1-2 μM vs. 4-10 μM) and thus DCA yields may be higher if their concentrations were increased.
Both BM3 and the three CYP102 enzyme homologs cloned here perform sub-terminal hydroxylation at the ω-1, ω-2 and ω-3 carbons with lauric acid. The oxidation of longer fatty acids (C15-C20) produces alcohols and diols with the same preferences, and their over oxidation is the source of the observed dicarboxylic acids with three less carbons, as illustrated in FIG. 7. It is believed that the diols and keto alcohols that accumulate during the oxidation of C15 to C20 fatty acids with the CYP102 tested herein are vicinal with ω-2 & 3 selectivity. For a fatty acid of length N, DCAs of length N-7 to N-2 were detected, as illustrated in FIGS. 8-9.
Example 2—Oxidation of Palmitic Acid with BM3-YetO
Strain BL21DE3/pSGI-040 (pET24a-BM3-yetO-WT) was grown on 150 mL of M9TV/TB (3/1 v/v)+glucose (2 g/L)+FeCl3 (0.05 mM) in a baffled flask (500 mL). Cells grew at 30° C. for 3-4 h until OD600 ̃0.5 was reached. At this point 0.5 mM δ-aminolevulinic acid and 0.25 mM IPTG were added and the flask was moved into a shaker at RT ( ̃23° C.) where it was allowed to shake for an additional 16 h at 120 rpm.
Cells were harvested by centrifugation and were frozen at −80° C. for at least 1 h. Frozen cells were re-suspended in 15 mL of lysis buffer (100 mM potassium phosphate, 10% glycerol (v/v), 1 mM DTT and 1 mg/mL lysozyme). After thawing the pellet it was incubated on ice for 15 min before cells were sonicated twice. Lysed cells were centrifuged at 12,000 rpm to precipitate insoluble cell debris. Clarified lysate was used without any further purification in enzymatic reactions. Approximately 8-10 uM (or nmole/mL) of P450 was calculated in the lysate.
Enzymatic reactions were prepared by mixing 0.25 mL of the above enzyme lysate with 0.75 mL of reaction buffer. The reaction buffer contained 100 mM potassium phosphate pH=7.8, 50 mM glucose, 1 mM NADP, 1.5 mg/mL glucose dehydrogenase and 2.5 mM lauric acid. The reaction was moved in a 10 mL borosilicate test tube and was thoroughly shaken at 30° C. for 12 h. Sample preparation: in the 1 mL of the reaction add 50 uL of HCl (3M) and 1 mL MeOH. The sample was centrifuged to precipitate proteins and submit for HPLC analysis.
The HPLC analysis shows peaks corresponding to various hydroxylation and over-oxidation products, as illustrated in FIG. 10.
Example 3—Oxidation of Cyclic Hydrocarbons
This example illustrates that a mutant CYP102 enzyme (BM3-F87A) oxidizes cyclic hydrocarbons, including cyclooctane and cyclodecane and also produces C—C bond cleavage of cyclodecane. It was found that when the enzyme is placed in the presence of cyclodecane, sebacic acid (decanedioic acid) is produced after over-oxidation.
By performing the reaction in the presence of deuterated lauric acid, we were able to trace the carbons of the produced sebacic acid to the cyclodecane and not the lauric acid. The reaction was thus performed using a mixture of cyclodecane and fully deuterated lauric acid (C12D23CO2H).
Cells of BL21(DE3)/pSGI-004 (pET24a-BM3-F87A) were grown and lysed as described above. A concentration of  ̃14-20 μM of P450 is obtained under these conditions. Reactions were prepared by mixing 1.5 mL reaction buffer and 0.5 mL of enzyme lysate. Reaction buffer was prepared in 100 mM potassium phosphate (pH 7.8) by adding 4 mM deuterated lauric acid (C12D23CO2H), 1 mM NADP, 1 mg/mL GDH-105 and 50 mM glucose. In this solution cyclodecane dissolved in acetonitrile was added giving a final concentration of 2 mM for cyclodecane and 2% (v/v) for acetonitrile co-solvent. The mixture (1.5 ml) was sonicated for 30 sec to create a milky solution before it was mixed with the enzyme lysate (0.5).
The enzymatic reaction was incubated at 30° C. in a glass test tube with good shaking. After 12 h of incubation 1 mL of the reaction mixture was mixed with 50 uL HCl (3M) and 1 mL of MeOH, centrifuged to remove insoluble proteins and was analyzed by HPLC-MS.
Product analysis revealed various hydroxylated products of the deuterated lauric acid (C12), and a peak that correlates with the same retention time and molecular mass as non-deuterated sebacic acid (C10) (data not shown). Therefore, the carbons of the non-deuterated sebacic acid produced by the reaction had their origin in the cyclodecane, and not the lauric acid (C12). Therefore, the BM3-F87A mutant was able to break the C—C bond of the cyclodecane.
Example 4—Oxidation of Stearic Acid (C18) with a CYP102 Enzyme in a Lysate
Overnight cultures were initiated in 5 mL LB broth from glycerol stocks stored at −80° C. These were grown at 30° C. overnight and 0.5 mL was used to inoculate 50 mL TB broth cultures that were grown at 30° C. to an OD600=0.5-1 (3-4 hr) before having 0.25 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 0.250 mM 8-aminolevulinic acid (dALA), and 0.50 mM ferric ammonium citrate added for induction. These cultures were grown at 25° C. for 20 h before being centrifuged at 4750×g for 10 min. The supernatant was decanted and the pellets stored at −80° C.
Pellets were resuspended in 5 mL (10% v/v of original culture volume) of 100 mM potassium phosphate, pH 7.8, 10% glycerol, 1 mg/mL lysozyme, 2 U/mL DNase, 1 mM Dithiothreitol (DTT), 5 mM magnesium chloride, and 0.5 mM calcium chloride by vortexing and then shaken at 600 rpm for 1 hr at 37° C. Lysates containing BM3 were then sonicated, centrifuged at 14000×g for 10 min, and decanted. P450 concentrations were calculated using CO binding difference spectra by adding 100 μL lysate to 100 μL 140 mM Na2S2O4 and prereading before a 15 min incubation under 10 psi CO. Plates were then read and P450 concentration calculated. Concentrations ranged from 13 nM for BM3 to 2 nM for YetO.
Crude lysates (200 μL) containing each P450 enzyme were mixed with a reaction mixture (800 μL) containing 2 mM stearic acid (C18), 1.0 mM NADPH, 30 mM glucose, and 1 mg/mL glucose dehydrogenase in 100 mM potassium phosphate pH 7.8. After overnight incubation at 30° C. with good shaking, crude reaction mixtures were mixed with 1-5 drops trifluoroacetic acid (TFA) and equal volume tetrahydrofuran (THF). After centrifugation to precipitate the insoluble protein, samples were analyzed by HPLC-MS. A sample chromatogram of a BM3 lysate reaction on stearic acid is shown in FIG. 11. Peaks corresponding to hydroxylation products, including hydroxy stearic acid and dihydroxy stearic acid are clearly visible. Additional peaks for products of the cleavage of a C—C bond are clearly visible, including a C15 (C18-3) dicarboxylic acid and a C17 dicarboxylic acid, are also clearly visible.
Example 5—Oxidation of Heptadecanoic Acid (C17) Using Purified BM3
Overnight cultures were started in 5 mL LB broth from glycerol stocks stored at −80° C. These were grown at 30° C. overnight and used to inoculate 500 mL TB broth cultures that were grown at 30° C. to an OD600=0.5-1 (3-4 hr) before having 0.250 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 0.250 mM δ-aminolevulinic acid (dALA), and 0.50 mM ferric ammonium citrate added for induction. These cultures were grown at 25° C. for 20 h before being centrifuged at 4750×g for 10 min. The supernatant was decanted and the pellets stored at −80° C.
Pellets were resuspended in 50 mL (10% v/v of original culture volume) of 100 mM potassium phosphate, pH 7.8, 10% glycerol, 1 mg/mL lysozyme, 2 U/mL DNase, 1 mM Dithiothreitol (DTT), 5 mM magnesium chloride, 0.5 mM calcium chloride, 500 mM NaCl and 5 mM imidazole by vortexing and then shaken at 600 rpm for 1 hr at 37° C. Lysates were then sonicated, centrifuged at 14000×g for 10 min, and decanted. Ni-columns were brought to room temperature and centrifuged at 700×g for 2 minutes to remove storage buffer. The 3 mL bed volume columns were then equilibrated with 6 mL of 20 mM Tris HCl, 0.5 M NaCl, 5 mM imidazole and centrifuged at 700×g for 2 minutes. The columns were then filled with lysate and shaken at 160 rpm for 30 min before being centrifuged at 700×g for 2 minutes. This step was repeated until all the lysate had been passed through the columns. The columns were then washed with 6 mL of 20 mM Tris HCl, 0.5 M NaCl, 50 mM imidazole and centrifuged at 700×g for 2 minutes. Three separate elutions were performed with 3 mL of 20 mM Tris HCl, 0.5 M NaCl, 500 mM imidazole and centrifuging at 700×g for 2 minutes. The eluate was then desalted using 10DG desalting columns into 100 mM potassium phosphate pH 7.8. P450 concentrations were calculated using CO binding difference spectra by adding 100 μL purified protein to 100 μL 140 mM Na2S2O4 and prereading before a 15 min incubation under 10 PSI CO. Plates were then read and P450 concentration calculated. The concentration of BM3 obtained was 3 nM.
Purified protein (600 μL) was mixed with a reaction mixture (1400 μL) containing 2 mM heptadecanoic acid (C17), 1.0 mM NADPH, 30 mM glucose, and 1 mg/mL glucose dehydrogenase (GDH-105) in 100 mM potassium phosphate pH 7.8. After overnight incubation at 30° C. with good shaking, crude reaction mixtures were mixed with 1-5 drops trifluoroacetic acid (TFA) and equal volume tetrahydrofuran (THF). After centrifugation to precipitate the insoluble protein, samples were analyzed by HPLC-MS. A sample chromatogram of a BM3 lysate reaction on heptadecanoic acid is shown in FIG. 12. Hydroxylation products of the fatty acid are clearly visible, including hydroxyl heptadecanoic acid and dihydroxy heptadecanoic acid, as well as products of the cleavage of a C—C bond, including C14 dicarboxylic acid (C17-3), C15 dicarboxylic acid, and C16 dicarboxylic acid.
Example 6—Chemical Oxidation of Enzymatic Products with Palmitic Acid
Diols and keto-alcohols and dicarboxylic acids constitute the majority of products from the oxidation of C15-C20 fatty acids with CYP102 enzymes. This example shows that in palmitic acid these diols and keto alcohols are located at the C13 and C14 carbons (□-2 and □-3), and can be converted to C13 di-carboxylic acid upon chemical oxidation as shown in FIG. 7 except that instead of Cyp102 the second step is chemical.
In 1.6 ml of KPi, buffer (50 mM pH 8.0) that contained 2 mM palmitic acid, 50 mM glucose, 1 mg/mL GDH-105, 0.5 mM NADP), 0.4 mL of 5 nM BM3 was added. BM3 enzyme was prepared by 0-60% ammonium sulfate precipitation of crude lysate, and re-dissolving in KPi buffer (50 mM, pH 8.0). After shaking for 16 h at 30° C., 0.2 mL were mixed with 2 drops of TFA, 0.2 mL THF, centrifuged to remove insoluble proteins, and were analyzed by HPLC for product formation (blue bars FIG. 13). Separate samples, 0.4 mL, were removed from the reaction, and were mixed with 8 uL of 30% H2O2 (w 200 mM final concentration), 1.3 mg of sodium tungstate dihydrate (Na2WO4.2H2O, 10 mM final) phosphoric acid to 1 mM to pH-2. This method has been reported to selectively oxidize adjacent diols to the corresponding carboxylic acid by C—C bond breaking (Venturello, C; Ricci, M. J Org. Chem. 1986, 51, 1599). After overnight (16 h) incubation at 90° C. the sample was filtered to remove precipitated proteins and was analyzed by HPLC. The results are shown in FIG. 13 (blue bars).
As shown in FIG. 13, the enzymatically produced diols and keto alcohols disappeared after chemical treatment with H2O2/Na2WO4. At the same time, the concentration (and associated yield) of C13 di-carboxylic acid increased in the same sample without the accumulation of other HPLC-detectable byproducts. Therefore, the oxidized derivatives are located on C13 and C14 carbons of palmitic acid. BM3 and other enzymes of the CYP102 family stereoselectively produce diols and keto alcohols from saturated fatty acids at ω-2 & ω-3 carbons as shown in FIG. 1. Chemical oxidation of these intermediates produces a single dicarboxylic acid and various chemical conditions and reagents are available for the chemical oxidation step.

## Claims

1. A method of producing a dicarboxylic acid comprising:
contacting a saturated fatty acid substrate having 15-20 carbon atoms and not bound to or by an acyl carrier protein with a CYP102 sub-family enzyme that comprises a ferredoxin reductase and a ferredoxin and that oxidizes the fatty acid substrate and breaks a C—C bond of the fatty acid substrate to produce a dicarboxylic acid product, wherein the dicarboxylic acid product has 3 less carbon atoms than the substrate and the enzyme has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NOs: 2, 4, 6 and 8; and
detecting the dicarboxylic acid product.

2. The method of claim 1 further comprising that hydroxylated fatty acid products are produced in addition to the dicarboxylic acid product when the enzyme is incubated with the fatty acid.

3. The method of claim 1 wherein the enzyme overoxidizes the fatty acid substrate.

4. The method of claim 3 wherein the reaction comprises the over-oxidation of adjacent diols.

5. The method of claim 1 wherein oxidizing the fatty acid substrate and breaking the C—C bond comprises the reaction mechanism:



wherein m+n=9-14.

6. The method of claim 5 further comprising contacting the substrate with an oxidizing agent.

7. The method of claim 1 wherein
the substrate is tridecanedioic acid (C19) and the dicarboxylic acid is palmitic acid (C16).

8. The method of claim 1 wherein the enzyme has at least one amino acid residue different from CYP102A1 wt (BM3) and/or CYP102A2 wt (YetO) and/or CYP102A3 wt (yrhJ) enzymes.

9. The method of claim 1 wherein the enzyme is selected from the group consisting of: BM3 (F87A), YetO (F89A), YetO (F89I), YetO (F89S), YetO (F89V).

10. The method of claim 9 wherein the substrate is palmitic acid or heptadecanoic acid.

11. The method of claim 9 wherein the enzyme is BM3-F87A.

12. The method of claim 1 wherein the enzyme has at least one amino acid residue different from CYP102A1 wt (BM3) or CYP102A2 wt (YetO).

13. The method of claim 1 wherein the substrate is palmitic acid or heptadecanoic acid.

14. The method of claim 13 wherein the substrate is palmitic acid.

15. The method of claim 1 wherein the enzyme has the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.