Biosynthesis of γ-lactones

Abstract

Provided herein are methods for preparing gamma lactones comprising reacting a carboxylic acid substrate with a heterologous cytochrome P450 (CYP450) protein having carboxylic acid 4-hydroxylase activity.

Description

[0001] Related patent applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 699,374, filed on July 17, 2018, and U.S. Provisional Application No. 62 / 758,019, filed on November 9, 2018, the disclosure of each of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The field of the invention relates to methods and processes for producing gamma-lactones via recombinant proteins and / or microbial cultures modified to express such recombinant proteins. More specifically, the disclosure relates to the preparation of C4-C20 gamma-lactones from corresponding carboxylic acid substrates via enzymatic conversion. Background Art

[0004] There is a general demand for food, aromatics and cosmetics with pleasant taste and odor. In many cases, these properties of pleasant smell and taste are provided by various lactone compounds (comprising various gamma lactones) with the aromatic characteristics and flavor profile of expectation. The demand for lactone compounds is mainly solved by chemical synthesis or the method for extracting from plants. The production based on plant extraction has obvious shortcomings, such as the influence of the intensity and abundance of the compound of interest, plant diseases and / or the risk of poor harvest, the stability of the compound, the increase in output and trade restrictions on the environment. Industrial production may cause environmental damage, may use dangerous precursors, and itself may run into the problem of cost increase due to the cost increase of key substrates.

[0005] Therefore, there is a need in the art for new methods for economically and reliably preparing gamma lactones without the limitations imposed by plant extraction and chemical synthesis. Summary of the Invention

[0006] According to the present invention, gamma-lactones can be reliably produced in high yields by using genetically modified and fermentative techniques using microorganisms such as bacteria and / or yeast. These microorganisms can synthesize lactones de novo or through biotransformation of fatty acids to provide commercially significant yields. Therefore, novel preparation methods are provided herein to reduce the cost of gamma-lactone production and to minimize the environmental impact of large-scale cultivation and processing of the natural sources from which these lactone compounds can be extracted.

[0007] More specifically, the present disclosure encompasses methods and compositions for producing gamma-lactones by microbial fermentation, wherein the microbial fermentation includes a cellular system expressing a heterologous cytochrome P450 (CYP450) protein.

[0008] The present disclosure is based in part on the discovery that certain CYP450 proteins and their functional variants specifically possess hydroxylase activity at the carbon atom γ to the carbonyl carbon of a carboxylic acid substrate, thereby generating 4-hydroxycarboxylic acids that can spontaneously convert to γ-lactones upon acidification. By overexpressing these CYP450 proteins in modified microbial systems and feeding them with appropriate carboxylic acid substrates, various γ-lactones can be produced in high titers. Thus, the present disclosure provides an economical and reliable method for preparing γ-lactones from carboxylic acids (e.g., various commercially available fatty acids) without the drawbacks associated with chemical synthesis or plant extraction.

[0009] Thus, one aspect of the present disclosure provides a method for producing C4-C20 gamma lactone, comprising: (a) incubating a cell system expressing a heterologous CYP450 protein in a culture medium containing a C4-C20 carboxylic acid substrate to provide 4-hydroxy C4-C20 carboxylic acid, and (b) subjecting the 4-hydroxy C4-C20 carboxylic acid to acidic conditions to produce the C4-C20 gamma lactone. In some embodiments, the 4-hydroxycarboxylic acid can be isolated from the cell system prior to acidification.

[0010] In various embodiments, the cell system can be a transformed host cell comprising a sequence encoding a heterologous CYP450 protein. In some embodiments, the cell system can include bacterial cells, yeast cells, plant cells that do not naturally produce the lactone of interest, algae cells, and / or fungal cells that do not naturally encode the specific fungal CYP450 proteins described herein. In certain embodiments, the cell system may include transformed bacterial cells and / or yeast cells selected from the group consisting of Escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, Methylosinus, Methylomonas, Rhodococcus, Pseudomonas, Rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces In some embodiments, the cell system can include growing cells. In other embodiments, the cell system can include resting cells (e.g., frozen cells that have been resuspended).

[0011] The present disclosure also encompasses methods for preparing C4-C20 gamma lactones, wherein the methods involve providing a reaction mixture comprising a C4-C20 carboxylic acid substrate and a recombinant CYP450 protein as described herein to provide a 4-hydroxy C4-C20 carboxylic acid, and then subjecting the 4-hydroxy C4-C20 carboxylic acid to acidic conditions to produce the C4-C20 gamma lactone. In some embodiments, the recombinant CYP450 protein can be expressed in the reaction mixture by a transformed host cell. In other embodiments, the recombinant CYP450 protein can be isolated and then provided in the reaction mixture.

[0012] In various embodiments of the method for preparing C4-C20γ lactone according to the present disclosure, the CYP450 protein can be a Mucor ambiguous CYP450 protein having carboxylic acid 4-hydroxylase activity or a functional variant thereof. For example, the CYP450 protein can comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. In certain embodiments, the CYP450 protein can comprise the amino acid sequence of SEQ ID NO: 1. In other embodiments, the CYP450 protein can consist of the amino acid sequence of SEQ ID NO: 1. Thus, a transformed host cell transformed to express such a CYP450 protein or a variant thereof can comprise a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 2.

[0013] In some other embodiments, the CYP450 protein can be a Basidiobolus meristosporus CYP450 protein having carboxylic acid 4-hydroxylase activity or a functional variant thereof. For example, the CYP450 protein can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. In certain embodiments, the CYP450 protein can comprise the amino acid sequence of SEQ ID NO: 3. In other embodiments, the CYP450 protein can consist of the amino acid sequence of SEQ ID NO: 3. Thus, a transformed host cell transformed to express such a CYP450 protein or a variant thereof can comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0014] In other embodiments, the CYP450 protein can be an Umbelopsis isabelline CYP450 protein having carboxylic acid 4-hydroxylase activity or a functional variant thereof. For example, the CYP450 protein can comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5. In certain embodiments, the CYP450 protein can comprise the amino acid sequence of SEQ ID NO: 5. In other embodiments, the CYP450 protein can consist of the amino acid sequence of SEQ ID NO: 5. Thus, a transformed host cell transformed to express such a CYP450 protein or a variant thereof can comprise a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 6.

[0015] In some embodiments, the C4-C20 carboxylic acid substrate may include a C4-C20 carboxylic acid, a salt of a C4-C20 carboxylic acid, an ester of a C4-C20 carboxylic acid, a monoglyceride, a diglyceride or a triglyceride of a C4-C20 carboxylic acid, or a combination thereof. In certain embodiments, the C4-C20 carboxylic acid substrate may be a C4-C20 carboxylic acid or a salt thereof. The C4-C20 carboxylic acid may be a straight-chain C4-C20 carboxylic acid, a branched-chain C4-C20 carboxylic acid, a saturated C4-C20 carboxylic acid or an unsaturated C4-C20 carboxylic acid. The C4-C20 carboxylic acid may be optionally substituted with one or more functional groups selected from hydroxyl groups and amino groups, provided that such optional substitution is not on a carbon atom located at the α, β or γ position relative to the carbonyl carbon of the carboxylic acid (see Figure 1 ). In certain embodiments, the C4-C20 carboxylic acid can be a straight-chain fully saturated carboxylic acid. In specific embodiments, the C4-C20 carboxylic acid substrate can be selected from hexanoic acid, hexenoic acid, heptanoic acid, 1-heptenoic acid, 2-heptenoic acid, octanoic acid, 1-octenoic acid, 2-octenoic acid, 3-octenoic acid, nonanoic acid, 1-nonenoic acid, 2-nonenoic acid, 3-nonenoic acid, decanoic acid, 1-decenoic acid, 2-decenoic acid, 3-decenoic acid, undecanoic acid, 1-undecenoic acid, 2-undecenoic acid, 3-undecenoic acid, dodecanoic acid, 1-dodecenoic acid, 2-dodecenoic acid, 3-dodecenoic acid, tridecanoic acid, 1-tridecenoic acid, 2-tridecenoic acid, 3-tridecenoic acid, tetradecanoic acid, 1-tetradecenoic acid, 2-tetradecenoic acid and 3-tetradecenoic acid.

[0016] Depending on the characteristics of the C4-C20 carboxylic acid substrate, the gamma lactone prepared by the method of the present invention may include a C4-C20 gamma lactone selected from the following: gamma-caprolactone, gamma-hexenolactone, gamma-heptenolactone, gamma-heptenolactone, gamma-octanolactone, gamma-octenolactone, gamma-nonenolactone, gamma-nonenolactone, gamma-decenolactone, gamma-decenolactone, gamma-undecalactone, gamma-undecalactone, gamma-dodecenolactone, gamma-tridecalactone, gamma-tridedecenolactone, gamma-tetradecenolactone and gamma-tetradecenolactone.

[0017] Any of the methods described herein may further comprise isolating the gamma lactone from the cell system to provide a crude product. In some embodiments, the crude product obtained from this separation step may comprise a lactone content having a purity of at least 70%. In some embodiments, the method further comprises purifying the crude product comprising the gamma lactone. In some embodiments, the crude product is purified by column chromatography. In some embodiments, the crude product is purified by acid-base extraction. In some embodiments, the crude product is purified by vacuum distillation. In some embodiments, the method further comprises purifying the lactone using semi-preparative HPLC.

[0018] The gamma lactones prepared by the methods described herein can be used alone or mixed with other lactones, flavorings or spices to obtain the desired composition used in a variety of consumer products or food. For example, gamma lactones as described herein can be included in food products (such as beverages, soft drinks, ice cream, dairy products, confectionery, cereal foods, chewing gum, baked goods, etc.), dietary supplements, medical nutrition products and pharmaceutical products to give desired flavor characteristics or aromatic characteristics. In some embodiments, the disclosure provides consumable products, which contain the lactones of the seasoning amount or fragrance amount used in a specific seasoning context, a flavoring formula product or a flavoring formula produced by any of the methods described above or elsewhere herein. In some embodiments, the consumable product can be a food, beverage, spice or beauty product. In some embodiments, the composition can be selected from beverages, confectionery, baked goods, biscuits and chewing gum. In certain embodiments, the composition can be a food, beverage, spice or beauty product with a flavor or aromatic composition designed to taste or smell similar to the following substances: peach, apricot, pear, maple, coconut, vanilla, butterscotch, pomegranate and / or jujube.

[0019] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. However, it should be understood that the drawings and detailed description provided herein are not intended to limit the disclosure to the specific embodiments disclosed, but on the contrary, the intent is to cover all modifications, equivalents, and alternative forms that fall within the spirit and scope of the disclosure as defined by the appended claims.

[0020] Other features and advantages of the present invention will become apparent from the following detailed description of preferred embodiments of the invention when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] Figure 1 The 4-hydroxylase pathway and the β-oxidation pathway for the preparation of γ-lactones from carboxylic acid substrates were compared. Specifically, the preparation of γ-decanoic acid (GC10) was used as an example to demonstrate the two pathways.

[0023] Figure 2 shows GC / MS spectra confirming the production of various gamma-lactones using E. coli cultures transformed to overexpress Mucor ambiguus P450. Figure 2a The production of gamma-heptanoic acid (GC7) from glucosinolate (heptanoic acid, C7) was shown. Figure 2b The production of gamma-octanoic acid (GC8) from caprylic acid (octanoic acid, C8) was shown. Figure 2c The production of gamma-nonalactone (GC9) from pelargonic acid (nonanoic acid, C9) was shown. Figure 2d The production of gamma-decanoic acid (GC10) from caprylic acid (decanoic acid, C10) is shown. Figure 2e The production of gamma-undecalactone (GC11) from undecanoic acid (undecanoic acid, C11) was shown. Figure 2f The production of gamma-dodecalactone (GC12) from lauric acid (dodecanoic acid, C12) was shown. Figure 2g The production of gamma-tridecalactone (GC13) from tridecanoic acid (tridecanoic acid, C13) was shown. Figure 2h The production of gamma-tetradecalactone (GC14) from myristic acid (tetradecanoic acid, C14) was shown.

[0024] Figure 3 Shown are GC / MS spectra confirming the production of γ-lactones using E. coli cultures transformed to overexpress the P450 of Batrachosporon. Specifically, the top panel shows the production of γ-undecalactone (GC11) from undecanoic acid (undecanoic acid, C11). The bottom panel shows the production of γ-undecalactone (GC11′) from undecenoic acid (undecenoic acid, C11:1).

[0025] Figure 4 shows GC / MS spectra confirming the production of various gamma-lactones using E. coli cultures transformed to overexpress Umbelopsis isabellina P450. Figure 4a The production of gamma-caprolactone (GC6) from hexanoic acid (C6) was shown. Figure 4b The production of gamma-heptanoic acid (GC7) from glucosinolate (heptanoic acid, C7) was shown. Figure 4c The production of gamma-decanoic acid (GC10) from caprylic acid (decanoic acid, C10) is shown. Figure 4d The production of gamma-undecalactone (GC11) from undecanoic acid (undecanoic acid, C11) was shown. Figure 4e The production of gamma-dodecalactone (GC12) from lauric acid (dodecanoic acid, C12) was shown. Figure 4f The production of gamma-tridecalactone (GC13) from tridecanoic acid (tridecanoic acid, C13) was shown. Figure 4g The production of gamma-tetradecalactone (GC14) from myristic acid (tetradecanoic acid, C14) was shown. DETAILED DESCRIPTION

[0026] See also Figure 1 , the preparation of gamma lactone by fatty acid biosynthesis can be carried out via at least two different approaches. According to the β-oxidation pathway, medium-chain fatty acids (C6-C12) or long-chain fatty acids (C13 to C21) with hydroxyl groups close to ω carbon (e.g., ω-1, ω-2, ω-3, ω-4, ω-5, ω-6, etc.) can be incubated with yeast to cause multiple β-oxidation cycles (step a1), wherein each β-oxidation cycle removes C2 residues from the hydrocarbon backbone fragment between the hydroxylated carbon atom and the carbonyl carbon atom. Depending on whether there is an odd or even number of carbon atoms in such hydrocarbon backbone fragments, 4-hydroxy (γ-hydroxy) fatty acids or 5-hydroxy (δ-hydroxy) fatty acids can be produced, and when lactonized under acidic conditions (step a2), gamma lactone or δ lactone is produced.

[0027] By comparison, the method of the present invention utilizes a cytochrome P450 protein that has been specifically characterized for its 4-hydroxylase activity. Carboxylic acids or their derivatives can be bioconverted by this cytochrome P450 protein into 4-hydroxycarboxylic acids (step b1), which, upon acidification (step b2), produce the corresponding gamma lactone. Thus, the method of the present invention provides an economical and reliable method for producing gamma lactones suitable for commercial-scale production.

[0028] Method for preparing lactone

[0029] In some embodiments, the methods described herein provide for the preparation of C4-C20 gamma lactones from C4-C20 carboxylic acid substrates. In some embodiments, the methods for preparing such gamma lactones comprise: (a) incubating a cell system expressing a heterologous cytochrome P450 (CYP450) protein with a C4-C20 carboxylic acid substrate to provide a 4-hydroxy C4-C20 carboxylic acid, and (b) contacting or otherwise subjecting the 4-hydroxycarboxylic acid to acidic conditions to prepare the C4-C20 gamma lactone.

[0030] In some embodiments, the method of preparing these lactones comprises incubating a cell pellet harvested from a cell system expressing a heterologous CYP450 protein with a carboxylic acid substrate. In some embodiments, the method of preparing these lactones comprises incubating a resuspended cell pellet harvested from a cell system with a carboxylic acid substrate.

[0031] The method for preparing the gamma lactones described herein includes incubating a cell system expressing a heterologous CYP450 protein in a culture medium comprising a carboxylic acid substrate at any ratio. In some embodiments, the method for preparing the gamma lactones includes incubating a cell system comprising 1 gram of cells per liter of culture medium to 200 grams of cells per liter of culture medium (1g / L to 200g / L). The culture medium may comprise 1 gram of carboxylic acid substrate per liter to 20 grams of fatty acid per liter (1g / L to 20g / L). In some embodiments, the method for preparing the lactones includes incubating a cell system comprising 100 grams of cells per liter (100g / L) in a culture medium comprising 1 gram of carboxylic acid substrate per liter (1g / L).

[0032] In some embodiments, the method for preparing a lactone comprises incubating a cell system with a culture medium containing a carboxylic acid substrate for a suitable period of time sufficient to form a 4-hydroxycarboxylic acid. In some embodiments, the method for preparing a lactone comprises contacting a 4-hydroxycarboxylic acid with a cell culture for a suitable period of time sufficient to form a gamma lactone in the cell culture. In some embodiments, the method for preparing a lactone comprises isolating the 4-hydroxycarboxylic acid prior to contacting the cell culture. In some embodiments, the method for preparing a lactone comprises incubating the 4-hydroxycarboxylic acid in a culture medium with a cell culture.

[0033] The methods for preparing lactones described herein encompass incubating (e.g., incubating a cell system expressing a heterologous CYP450 protein with a culture medium containing an appropriate carboxylic acid substrate) for a certain amount of time. In some embodiments, the methods for preparing lactones comprise incubating for a time between 0.5 hours and 96 hours.

[0034] The methods for producing lactones described herein encompass incubation at a certain temperature (e.g., incubating a cell system expressing a heterologous CYP450 protein with a culture medium containing an appropriate carboxylic acid substrate). In some embodiments, the methods for producing lactones comprise incubation at a temperature between 16°C and 40°C.

[0035] Cytochrome P450

[0036] Cytochrome P450 (CYP450) proteins are generally enzymes that catalyze substrate oxidation. CYP450 proteins are members of the heme protein superfamily and contain heme as a cofactor. CYP450 enzymes have been identified in many organisms, including but not limited to animals, plants, fungi, bacteria, archaea, and viruses. However, to the inventors' knowledge, prior to the present invention, no CYP450 enzymes with specific carboxylic acid 4-hydroxylase activity had been reported.

[0037] After rigorous high-throughput screening of a large library of candidate genes for fungal cytochrome P450 enzymes with carboxylic acid 4-hydroxylase activity, the inventors identified certain CYP450 genes that showed high carboxylic acid 4-hydroxylase activity.

[0038] The CYP450 protein suitable for use in the methods of the present invention can be an unidentified Mucor CYP450 protein having carboxylic acid 4-hydroxylase activity or a functional variant thereof. Specifically, the CYP450 protein can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In certain embodiments, the CYP450 protein can comprise the amino acid sequence of SEQ ID NO: 1. In other embodiments, the CYP450 protein can consist of the amino acid sequence of SEQ ID NO: 1. Thus, the cell system for expressing such a heterologous CYP450 protein can comprise a host cell having a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0039] In some other embodiments, the CYP450 protein can be a Basidiobolus meristosporus CYP450 protein or a functional variant thereof having carboxylic acid 4-hydroxylase activity. For example, the CYP450 protein can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. In certain embodiments, the CYP450 protein can comprise the amino acid sequence of SEQ ID NO: 3. In other embodiments, the CYP450 protein can consist of the amino acid sequence of SEQ ID NO: 3. Thus, the cell system for expressing such a heterologous CYP450 protein can comprise a host cell having a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0040] In other embodiments, the CYP450 protein can be an Umbelopsis isabelline CYP450 protein having carboxylic acid 4-hydroxylase activity or a functional variant thereof. For example, the CYP450 protein can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5. In certain embodiments, the CYP450 protein can comprise the amino acid sequence of SEQ ID NO: 5. In other embodiments, the CYP450 protein can consist of the amino acid sequence of SEQ ID NO: 5. Thus, the cell system for expressing such a heterologous CYP450 protein can comprise a host cell having a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 6.

[0041] Carboxylic acid substrate

[0042] According to the method described herein, C4-C20 carboxylic acid substrates can be converted into 4-hydroxycarboxylic acids. The carboxylic acid substrate can be a carboxylic acid and / or its derivatives. For example, the C4-C20 carboxylic acid substrate can include a C4-C20 carboxylic acid, a salt of a C4-C20 carboxylic acid, an ester of a C4-C20 carboxylic acid, a monoglyceride, a diglyceride or a triglyceride of a C4-C20 carboxylic acid, or a combination thereof. As used herein, C4-C20 represents an organic compound having 4 to 20 carbon atoms. The range represented by C4-C20 is intended to express any range therein, including but not limited to C5-C20, C6-C20, C7-C20, C8-C20, C9-C20, C10-C20, C6-C14, C7-C14, C8-C14, C9-C14, C10-C14, etc. Thus, the carboxylic acid substrates described herein can include carboxylic acids or derivatives thereof having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, the C4-C20 carboxylic acid substrate can be a C4-C20 carboxylic acid or a salt thereof. Without limitation, the substrate acid can be in the form of a sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or the like.

[0043] The C4-C20 carboxylic acid may be a linear C4-C20 carboxylic acid, a branched C4-C20 carboxylic acid, a saturated C4-C20 carboxylic acid or an unsaturated C4-C20 carboxylic acid. The C4-C20 carboxylic acid may be optionally substituted with one or more functional groups selected from hydroxyl groups and amino groups, provided that such optional substitution is not on a carbon atom located in the alpha, beta or gamma position relative to the carbonyl carbon of the carboxylic acid (see Figure 1 In certain embodiments, the C4-C20 carboxylic acid may be a linear, fully saturated carboxylic acid.

[0044] Examples of C4-C20 carboxylic acids include, but are not limited to, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanic acid, heneicosanoic acid, dotriacontanic acid, trictriacontanic acid, tetratriacontanic acid, pentatriacontanic acid, hexatriacontanic acid, heptacosanoic acid, octatriacontanic acid, nonacosanoic acid, and tetraacontanic acid. Table 1 provides the common names, structural formulas, and lipid numbers of exemplary fatty acids.

[0045] Table 1: Examples of fatty acids .

[0046]

[0047]

[0048] Examples of the unsaturated C4-C20 carboxylic acids include hexenoic acid, 1-heptenoic acid, 2-heptenoic acid, 1-octenoic acid, 2-octenoic acid, 3-octenoic acid, 1-nonenoic acid, 2-nonenoic acid, 3-nonenoic acid, 1-decenoic acid, 2-decenoic acid, 3-decenoic acid, 1-undecenoic acid, 2-undecenoic acid, 3-undecenoic acid, 1-dodecenoic acid, 2-dodecenoic acid, 3-dodecenoic acid, 1-tridecenoic acid, 2-tridecenoic acid, 3-tridecenoic acid, 1-tetradecenoic acid, 2-tetradecenoic acid, and 3-tetradecenoic acid.

[0049] In some embodiments, the culture medium comprising the carboxylic acid substrate comprises a buffer. Examples of buffers include, but are not limited to, phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine buffer.

[0050] In some embodiments, the culture medium containing the carboxylic acid substrate contains a surfactant to aid in the dispersion of the substrate. Non-limiting examples of surfactants include polysorbate 20 ( 20), polyoxyethylene sorbitan monopalmitate ( 40), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (TRITON TM X-100), sodium dodecyl sulfate (SDS), ethyltrimethylammonium bromide (ETMAB), lauryltrimethylammonium bromide (LTAB), and lauryltrimethylammonium chloride (LTAC).

[0051] The substratum comprising the carboxylic acid substrate can comprise the carboxylic acid substrate of any concentration. In some embodiments, the substratum comprising the carboxylic acid substrate can comprise the carboxylic acid substrate of 0.5g / L to 100g / L, for example, the carboxylic acid substrate of 1g / L to 10g / L. In some embodiments, the substratum comprising the carboxylic acid substrate can comprise the carboxylic acid substrate of 0.5g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g / L or 100g / L.

[0052] lactone

[0053] According to the methods described herein, a carboxylic acid substrate is converted into a 4-hydroxycarboxylic acid in the presence of a CYP450 protein as described herein. Upon acidification, the 4-hydroxycarboxylic acid can be converted into the corresponding gamma lactone. Acidification can be carried out with an acid such as hydrochloric acid, acetic acid, etc. According to the present disclosure, the methods provided herein can convert the 4-hydroxycarboxylic acid into various gamma-lactones, including but not limited to gamma-caprolactone, gamma-hexenolactone, gamma-heptenolactone, gamma-heptenolactone, gamma-octalactone, gamma-octenolactone, gamma-nonenolactone, gamma-nonenolactone, gamma-decenolactone, gamma-decenolactone, gamma-undecalactone, gamma-undecalactone, gamma-dodecalactone, gamma-dodecenolactone, gamma-tridecalactone, gamma-tridecenolactone, gamma-tetradecenolactone, and gamma-tetradecenolactone.

[0054] The gamma lactones prepared according to the methods described herein can be isolated or purified from cell cultures using any method known in the art. For example, these gamma lactones can be isolated or purified from cell cultures by solvent extraction, distillation, chromatographic separation, high pressure liquid chromatography, etc. The isolated or purified lactones can be incorporated into products (e.g., foods, beverages, fragrances, or cosmetics).

[0055] Cell system

[0056] As mentioned herein, the cell system according to the methods of the present invention can include any one or more cells that provide expression of the CYP450 proteins described herein. Such cell systems can include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells. In some embodiments, the cell system comprises bacterial cells, yeast cells, or a combination thereof. In some embodiments, the cell system comprises prokaryotic cells, eukaryotic cells, and a combination thereof. In some embodiments, the cell system comprises expressing proteins in vitro based on cellular components (such as ribosomes).

[0057] Bacterial cells of the present disclosure include, but are not limited to, Escherichia spp., Streptomyces spp., Zymomonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp. spp.), Alcaligenes species, Pseudomonas species, Aeromonas species, Azotobacter species, Comamonas species, Mycobacterium species, Rhodococcus species, Gluconobacter species, Ralstonia species, Acidithiobacillus species, Microlunatus species, Geobacter species, Geobacillus species, Arthrobacter species, Flavobacterium species spp.), Serratia spp., Saccharopolyspora spp., Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp.), Pantoea spp., and Vibrio natriegens.

[0058] Yeast cells of the present disclosure include, but are not limited to, engineered Saccharomyces spp., Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Candida boidinii, and Pichia. According to the present disclosure, yeast as claimed herein is a eukaryotic unicellular microorganism classified as a member of the kingdom Fungi. Yeast is a unicellular organism that evolved from a multicellular ancestor, but some species useful in the present disclosure are those that have the ability to develop multicellular properties by forming strings of connected budding cells known as pseudohyphae or pseudohyphae.

[0059] In some embodiments, a cell pellet is harvested from a cell system expressing CYP450. In some embodiments, the cell pellet can be resuspended at various concentrations. In some embodiments, the cell pellet is resuspended at a concentration of 1 g / L to 250 g / L. In some embodiments, the cell pellet harvested from the cell system is resuspended at a concentration of 1 g / L, 10 g / L, 25 g / L, 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, or 250 g / L.

[0060] Cell culture

[0061] Cell culture refers to any one or more cells in a culture. Cultivation or incubation is a process in which cells are grown under controlled conditions (usually outside their natural environment). For example, cells (such as yeast cells) can be grown as a cell suspension in a liquid nutrient broth. Cell cultures include, but are not limited to, bacterial cell cultures, yeast cell cultures, plant cell cultures, and animal cell cultures. In some embodiments, the cell culture comprises bacterial cells, yeast cells, or a combination thereof.

[0062] The bacterial cell cultures of the present disclosure comprise bacterial cells including, but not limited to, Escherichia species, Streptomyces species, Zymomonas species, Acetobacter species, Citrobacter species, Synechocystis species, Rhizobium species, Clostridium species, Corynebacterium species, Streptococcus species, Xanthomonas species, Lactobacillus species, Lactococcus species, Bacillus species, Alcaligenes species, Pseudomonas species, Aeromonas species, Azotobacter species, Comamonas species, Mycobacterium species, Rhodococcus species, Gluconobacter species, Ralstonia species, Acidithiobacillus species, Lupine species, Geobacter species, Geobacillus species, Arthrobacter species, Flavobacterium species, Serratia species, Saccharopolyspora species, Thermus species, Stenotrophomonas species, Chromobacterium species, Sinorhizobium species, Saccharopolyspora species, Agrobacterium species, Pantoea species, and Vibrio natriuresis.

[0063] Yeast cell cultures of the present disclosure comprise yeast cells, including but not limited to Saccharomyces species, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Candida boidinii, and Pichia.

[0064] In some embodiments, cell culture as described herein can be an aqueous culture medium comprising one or more nutrients as known in the art. This liquid culture medium can comprise one or more carbon sources, nitrogen sources, inorganic salts and / or growth factors. Suitable carbon sources can include glucose, fructose, xylose, sucrose, maltose, lactose, mannitol, sorbitol, glycerol and corn syrup. Examples of suitable nitrogen sources can include organic nitrogen-containing substances and inorganic nitrogen-containing substances, such as peptone, corn steep liquor, average extract, yeast extract, casein, urea, amino acids, ammonium salts, nitrates and mixtures thereof. Examples of inorganic salts can include phosphates, sulfates, magnesium salts, sodium salts, calcium salts and potassium salts. This liquid culture medium can also include one or more vitamins and / or minerals.

[0065] In some embodiments, the cells are cultured at a temperature of 16° C. to 40° C. For example, the cells can be cultured at a temperature of 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40° C.

[0066] In some embodiments, the cells are cultured at a pH in the range of about 3 to about 9, preferably in the range of about 4 to about 8. The pH can be adjusted by adding inorganic or organic acids or bases (such as hydrochloric acid, acetic acid, sodium hydroxide, calcium carbonate, ammonia) or by adding buffers (such as phosphates, phthalates or ) to adjust.

[0067] In some embodiments, the cells are cultured for a period of 0.5 hours to 96 hours or longer. For example, the cells can be cultured for a period of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours. Typically, cells (such as bacterial cells) are cultured for a period of 12 hours to 24 hours. In some embodiments, cells are cultured at a temperature of 37°C for 12 hours to 24 hours. In some embodiments, cells are cultured at a temperature of 16°C for 12 hours to 24 hours.

[0068] In some embodiments, cells are cultured to 1×10 8 (OD 600 <1) viable cells / ml cell culture medium to 2×10 11 (OD is about 200) viable cells / ml cell culture medium. In some embodiments, cells are cultured to a density of 1×10 8 viable cells / ml, 2×10 8 viable cells / ml, 3×10 8 viable cells / ml, 4×10 8 viable cells / ml, 5×10 8 viable cells / ml, 6×10 8 viable cells / ml, 7×10 8 viable cells / ml, 8×10 8 viable cells / ml, 9×10 8 viable cells / ml, 1×10 9 viable cells / ml, 2×10 9 viable cells / ml, 3×10 9 viable cells / ml, 4×10 9 viable cells / ml, 5×10 9 viable cells / ml, 6×10 9 viable cells / ml, 7×10 9 viable cells / ml, 8×10 9 viable cells / ml, 9×10 9 viable cells / ml, 1×10 10 viable cells / ml, 2×10 10 viable cells / ml, 3×10 10 viable cells / ml, 4×10 10 viable cells / ml, 5×10 10 viable cells / ml, 6×10 10 viable cells / ml, 7×10 10 viable cells / ml, 8×1010 viable cells / ml, 9×10 10 viable cells / ml, 1×10 11 viable cells / ml or 2×10 11 The density of viable cells / ml. (Conversion factor: OD 1 = 8 × 10 8 cells / ml).

[0069] synthetic biology

[0070] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, in Sambrook, J., Fritsch, EF, and Maniatis, T., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989 (hereinafter "Maniatis"); and Silhavy, TJ, Bennan, ML, and Enquist, LW, EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1984; and Ausubel, FM, et al., IN CURRENT PROTOCOLS IN MOLECULARBIOLOGY, published by GREENE PUBLISHING and WILEY-INTERSCIENCE in 1987; the entire contents of each of these documents are hereby incorporated herein by reference.

[0071] Bacterial preparation system

[0072] Protein expression in prokaryotes is most often performed in bacterial host cells using vectors containing constitutive or inducible promoters that direct the expression of fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, typically to the amino terminus of the recombinant protein. Such fusion vectors are typically used for three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Typically, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable the recombinant protein to be separated from the fusion moiety after purification of the fusion protein. Such vectors are within the scope of the present disclosure.

[0073] In one embodiment, the expression vector includes those genetic elements for expressing the recombinant polypeptide in bacterial cells. Elements for transcription and translation in bacterial cells may include a promoter, a coding region for the protein complex, and a transcription terminator.

[0074] Those of ordinary skill in the art will be aware of molecular biology techniques that can be used to prepare expression vectors.As described herein, polynucleotides for incorporation into expression vectors of the subject technology can be prepared by conventional techniques such as polymerase chain reaction (PCR).

[0075] A variety of molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive ends. In one embodiment, complementary homopolymer strands can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then linked by hydrogen bonds between the complementary homopolymer tails to form a recombinant DNA molecule.

[0076] In alternative embodiments, the polynucleotide of this theme technology is operably connected to expression vector using the synthetic joint that contains one or more restriction sites.In one embodiment, polynucleotide is generated by restriction endonuclease digestion.In one embodiment, nucleic acid molecules are processed with bacteriophage T4 DNA polymerase or Escherichia coli DNA polymerase I, these enzymes utilize their 3 '-5 '-nuclease exonuclease activity to remove outstanding 3 '-single strand end, and utilize their polymerization activity to fill recessed 3 '-end, thereby generate flat end DNA fragment.Then in the presence of the enzyme (such as bacteriophage T4 DNA ligase) that can catalyze the connection of flat end DNA molecule, flat end fragment is incubated together with the joint molecule of large molar excess.Therefore, reaction product is the polynucleotide that carries polymer linker sequence at its end.Then these polynucleotide are cut and connected to expression vector with suitable restriction enzyme, and this expression vector has been cut with the enzyme of the end compatible with the end of this polynucleotide.

[0077] Alternatively, vectors with ligation-independent cloning (LIC) sites can be used. The desired PCR-amplified polynucleotide can then be cloned into the LIC vector without restriction digestion or ligation (Aslanidis and de Jong, NUCL. ACID. RES. 18, 6069-74, (1990), Haun et al., BIOTECHNIQUES 13, 515-18 (1992), which are incorporated herein by reference to the extent consistent herein).

[0078] In one embodiment, PCR is suitable for use in order to isolate and / or modify the polynucleotide of interest for insertion into a selected plasmid. Appropriate primers for sequential PCR preparation can be designed to isolate the desired coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, and place the coding region in the desired reading frame.

[0079] In one embodiment, polynucleotides for incorporation into expression vectors of the subject technology are prepared using appropriate oligonucleotide primers by using PCR. The coding region is amplified while the primers themselves are incorporated into the amplified sequence products. In one embodiment, the amplification primers comprise restriction endonuclease recognition sites that allow the amplified sequence products to be cloned into suitable vectors.

[0080] Expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Transformation of suitable cells with the expression vectors of the subject technology is accomplished by methods known in the art and generally depends on the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, chemoporation, or electroporation.

[0081] Successfully transformed cells, i.e., those containing the expression vector, can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce a polypeptide described herein. Cells can be examined for the presence of expression vector DNA by techniques well known in the art.

[0082] The host cell may contain a single copy of the previously described expression vector, or alternatively contain multiple copies of the expression vector,

[0083] In some embodiments, the transformed cell can be a bacterial cell, a yeast cell, an algae cell, a fungal cell, a plant cell, an insect cell, or an animal cell. In some embodiments, the cell is a plant cell selected from the group consisting of a canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.

[0084] Microbial host cell expression systems and expression vectors containing regulatory sequences for directing high-level expression of exogenous proteins are well known to those skilled in the art. Any of these can be used to construct vectors for expressing the recombinant polypeptides of the present invention in microbial host cells. These vectors can then be introduced into appropriate microorganisms via transformation to allow high-level expression of the recombinant polypeptides of the present invention.

[0085] The vectors or boxes used to transform suitable microbial host cells are well known in the art. Typically, the vector or box comprises a sequence for directing the transcription and translation of the relevant polynucleotides, a selective marker, and a sequence that allows autonomous replication or chromosomal integration. Suitable vectors include a 5' region of a polynucleotide with a transcription initiation control and a 3' region of a DNA fragment that controls transcription termination. Preferably, both control regions are derived from genes homologous to the transformed host cell, although it should be understood that such control regions do not need to be derived from the natural genes of the specific species selected as the host.

[0086] The initiation control regions or promoters used to drive expression of recombinant polypeptides in desired microbial host cells are numerous and familiar to those skilled in the art. In fact, any promoter capable of driving these genes is suitable for use in the subject technology, including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (for expression in yeast); AOXI (for expression in Pichia pastoris); and lac, trp, JPL, IPR, T7, tac, and trc (for expression in E. coli).

[0087] Termination control regions can also be derived from various genes native to the microbial host.For the microbial hosts described herein, a termination site can be optionally included.

[0088] In plant cells, the expression vector of the subject technology may include a coding region operably linked to a promoter capable of directing the expression of the recombinant polypeptide of the subject technology in a desired tissue at a desired developmental stage. For convenience, the polynucleotide to be expressed may include a promoter sequence and a translation leader sequence derived from the same polynucleotide. 3' non-coding sequences encoding transcription termination signals should also be present. These expression vectors may also include one or more introns to promote polynucleotide expression.

[0089] For plant host cells, any combination of any promoter and any terminator capable of inducing coding region expression can be used in the vector sequence of the present subject technology. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes. A type of effective plant promoter that can be used is a high-level plant promoter. Such promoters that can be operably connected to the expression vector of the present subject technology should be able to promote the expression of the vector. The high-level plant promoters that can be used in the present subject technology include, for example, promoters (Berry-Lowe et al., J.MOLECULAR AND APP.GEN., 1:483-98 (1982), the document as a whole is hereby incorporated herein by reference in its entirety) and promoters of chlorophyll-binding proteins from the small subunit (s) of ribulose-1,5-bisphosphate carboxylase of soybean. Both promoters are known to be light-inducible in plant cells (see, e.g., GENETIC ENGINEERING OF PLANTS, AN AGRICULTURAL PERSPECTIVE, A. Cashmore, Plenum, NY (1983), pp. 29-38; Coruzzi, G. et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, 258: 1399 (1983), and Dunsmuir, P. et al., JOURNAL OF MOLECULAR AND APPLIED GENETICS, 2: 285 (1983), each of which is hereby incorporated by reference to the extent it is consistent herewith).

[0090] Analyzing sequence similarity using identity scores

[0091] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or peptide sequences are invariant in components (e.g., nucleotides or amino acids) over the entire alignment window. The "identity score" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the segment of the reference sequence, i.e., the entire reference sequence or a smaller, defined portion of the reference sequence.

[0092] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (having appropriate nucleotide insertions, deletions, or gaps totaling less than 20% of those of the reference sequence over the comparison window). Optimal alignment of sequences for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search similarity method of Pearson and Lipman, and preferably by computer implementations of these algorithms, such as those described herein. Wisconsin The "identity score" of the aligned fragment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence fragment, i.e., the entire reference sequence or a smaller, limited portion of the reference sequence. Percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can involve full-length polynucleotide sequences or portions thereof, or longer polynucleotide sequences. For purposes of this disclosure, BLASTX version 2.0 (for translated nucleotide sequences) and BLASTN version 2.0 (for polynucleotide sequences) can also be used to determine "percent identity."

[0093] The percent sequence identity is preferably determined using the Sequence Analysis Software Package TMThe "BestFit" or "Gap" program (Version 10; Genetics Computer Group, Inc., Madison, WI) is used to determine. "Gap" utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, JOURNAL OF MOLECULAR BIOLOGY 48:443-53, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit" uses the local homology algorithm of Smith and Waterman (Smith and Waterman, ADVANCES IN APPLIEDMATHEMATICS, 2:482-489, 1981, Smith et al., NUCLEIC ACIDS RESEARCH 11:2205-2220, 1983) to perform the best alignment of the best segment similarity between the two sequences and insert gaps to maximize the number of matches. Percent identity is most preferably determined using the "BestFit" program.

[0094] Useful methods for determining sequence identity are also disclosed in the Basic Local Alignment Search Tool (BLAST) program, which is publicly available from the National Center Biotechnology Information (NCBI), National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al., J. MOL. BIOL. 215:403-10 (1990); version 2.0 or later of the BLAST program allows gaps (deletions and insertions) to be introduced into the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; and, for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0095] As used herein, the term "significant percentage sequence identity" refers to a percentage sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity. Thus, one embodiment of the present disclosure is a polynucleotide molecule having at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity, to a polynucleotide sequence described herein. Polynucleotide molecules having the activity of a cytochrome P450 gene of the present disclosure are capable of directing the production of a variety of gamma-lactones and delta-lactones, and such polynucleotide molecules may have a significant percentage sequence identity to a polynucleotide sequence provided herein and are encompassed within the scope of the present disclosure.

[0096] Identity and Similarity

[0097] Identity is the fraction of identical amino acids between a pair of sequences after aligning the sequences (which can be done using only sequence information or structural information or some other information, but typically it is based on sequence information alone), while similarity is a score assigned based on an alignment using some similarity matrix. The similarity index can be any of the following BLOSUM62, PAM250, or GONNET, or any matrix used by those skilled in the art for protein sequence alignment.

[0098] Identity is the degree of similarity between two subsequences (without gaps between the sequences). 25% or greater identity indicates functional similarity, whereas 18% to 25% indicates structural or functional similarity. Remember that two completely unrelated or random sequences (which are more than 100 residues) can have greater than 20% identity. Similarity is the degree to which two sequences are similar when compared. This is determined by their identity.

[0099] Explanation of terms used in this article :

[0100] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0101] To the extent that the terms "including," "having," etc. are used in the specification or claims, such terms are intended to be inclusive in a manner similar to the term "comprising" as interpreted when used as a transitional word in a claim.

[0102] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other implementations.

[0103] The term "complementary" will be given its ordinary and customary meaning to those of ordinary skill in the art and is used without limitation to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the subject technology also includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing, as well as those substantially similar nucleic acid sequences.

[0104] The terms "nucleic acid" and "nucleotide" will be given their corresponding ordinary and customary meanings to those of ordinary skill in the art and are used without limitation to refer to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants (e.g., degenerate codon substitutions) and complementary sequences thereof, as well as the sequences explicitly indicated.

[0105] "Coding sequence" will be given its ordinary and customary meaning to those skilled in the art, and is used without limitation to refer to a DNA sequence that encodes a specific amino acid sequence.

[0106] The term "isolated" will be given its ordinary and customary meaning to those of ordinary skill in the art and, when used in the context of an isolated nucleic acid or isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that is artificially removed from its natural environment and, therefore, is not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form, or can exist in a non-natural environment, such as in a transgenic host cell.

[0107] As used herein, the terms "incubating" and "incubation" mean the process of mixing two or more chemical or biological entities (such as chemical compounds and enzymes) and allowing them to interact under conditions favorable to the production of a desired product.

[0108] The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues. A nucleic acid sequence and all its degenerate variants will express the same amino acid or polypeptide.

[0109] The terms "polypeptide," "protein," and "peptide" will be given their corresponding ordinary and customary meanings to those of ordinary skill in the art; these three terms are sometimes used interchangeably and are used without limitation to refer to polymers of amino acids or amino acid analogs, regardless of their size or function. Although "protein" is often used to refer to relatively large polypeptides, and "peptide" is often used to refer to small polypeptides, the use of these terms in the art is overlapping and varied. Unless otherwise indicated, the term "polypeptide" as used herein refers to peptides, polypeptides, and proteins. When referring to polynucleotide products, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing.

[0110] The terms "polypeptide fragment" and "fragment," when used with reference to a polypeptide, will be given their ordinary and customary meanings to those of ordinary skill in the art and are used without limitation to refer to polypeptides that lack amino acid residues compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino terminus or carboxyl terminus, or both, of the reference polypeptide.

[0111] The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of a full-length polypeptide or protein and has substantially the same biological activity as the full-length polypeptide or protein, or performs substantially the same function as the full-length polypeptide or protein (e.g., performs the same enzymatic reaction).

[0112] The terms "variant polypeptide," "modified amino acid sequence," or "modified polypeptide," used interchangeably, refer to an amino acid sequence that differs from a reference polypeptide by one or more amino acids (e.g., by one or more amino acid substitutions, deletions, and / or additions). In one aspect, the variant is a "functional variant" that retains some or all of the capabilities of the reference polypeptide.

[0113] The term "functional variant" also includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions and retains some or all of the activities of the reference peptide. "Conservative amino acid substitution" is the replacement of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine replacing another; one charged or polar (hydrophilic) residue replacing another, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; one basic residue such as lysine or arginine replacing another; or one acidic residue such as aspartic acid or glutamic acid replacing another; or one aromatic residue such as phenylalanine, tyrosine or tryptophan replacing another. It is expected that such substitutions have little effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "conservatively substituted variant" also includes peptides in which residues are replaced by chemically derivatized residues, provided that the resulting peptide retains some or all of the activities of the reference peptide as described herein.

[0114] The term "variant" in connection with the polypeptides of the subject technology also includes functionally active polypeptides having an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical to the amino acid sequence of the reference polypeptide.

[0115] "Percent (%) amino acid sequence identity" with respect to variant polypeptide sequences of the subject technology refers to the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide, after aligning the candidate sequence and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0116] The comparison for the purpose of measuring amino acid sequence identity percentage can be realized in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required for maximum comparison over the full length of the compared sequence. For example, the sequence comparison program NCBI-BLAST2 can be used to measure amino acid sequence identity%. The NCBI-BLAST2 sequence comparison program can be downloaded from ncbi.nlm.nih.gov. NCBI BLAST2 uses some search parameters, wherein all those search parameters are set to default values, including for example unmasking "yes", chain=all, expected occurrence rate 10, minimum low complexity length=15 / 5, multiple e-value=0.01, multiple constant=25, the drop value of the final gap comparison=25, and scoring matrix=BLOSUM62. In the case of amino acid sequence comparisons using NCBI-BLAST2, the % amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a determined % amino acid sequence identity relative to, with, or to a given amino acid sequence B), to, or to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A.

[0117] In this sense, the technology for determining the "similarity" of amino acid sequences is well known in the art. Generally speaking, "similarity" refers to the accurate comparison of amino acids and amino acids at appropriate positions of two or more polypeptides, wherein the amino acids are identical or have similar chemical properties and / or physical properties, such as charge or hydrophobicity. The so-called "similarity percentage" between the compared polypeptide sequences can then be determined. The technology for determining the identity of nucleic acids and amino acid sequences is also well known in the art, including determining the nucleotide sequence of the mRNA of the gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded therein, which is then compared with a second amino acid sequence. Generally speaking, "identity" refers to the precise nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by measuring their "identity percentage", as can two or more amino acid sequences. The programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) (e.g., the GAP program) can calculate the identity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known to those skilled in the art.

[0118] An amino acid position that "corresponds to" a reference position refers to a position that is aligned with a reference sequence, as identified by aligning the amino acid sequences. Such alignment can be performed manually or by using well-known sequence alignment programs (such as ClustalW2, Blast 2, etc.).

[0119] Unless otherwise indicated, percent identity between two polypeptide or polynucleotide sequences refers to the percentage of identical amino acid residues or nucleotides over the entire length of the shorter of the two sequences.

[0120] The term "homologous" in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or polypeptides having a "common evolutionary origin", including polynucleotides or polypeptides from superfamilies and homologous polynucleotides or proteins from different species (Reeck et al., CELL 50:667, 1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous polypeptides can have amino acid sequences that are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical.

[0121] "Suitable regulatory sequences" will be given their ordinary and customary meaning to those of ordinary skill in the art and are used, without limitation, to refer to nucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0122] "Promoter" will be given its common and customary meaning for those of ordinary skill in the art, and is used, without limitation, to refer to a DNA sequence that can control the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. Promoters can all be derived from natural genes, or consist of different elements derived from different promoters found in nature, or even comprise synthetic DNA fragments. It will be understood by those skilled in the art that different promoters can direct the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. Promoters that cause genes to be expressed in most cell types at most times are generally referred to as "constitutive promoters." It is also recognized that, since the precise boundaries of regulatory sequences have not been fully determined in most cases, DNA fragments of different lengths may have the same promoter activity.

[0123] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either sense or antisense orientation.

[0124] As used herein, the term "expression" will be given its ordinary and customary meaning to those of ordinary skill in the art and is used, without limitation, to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA of nucleic acid fragments derived from the subject technology. "Overexpression" refers to the production of a gene product in a transgenic or recombinant organism that exceeds the level of production in a normal or non-transformed organism.

[0125] "Transformation" will be given its ordinary and customary meaning to those skilled in the art and is used without limitation to refer to the transfer of a polynucleotide into a target cell for further expression by the cell. The transferred polynucleotide may be incorporated into the genomic or chromosomal DNA of the target cell, thereby producing genetically stable inheritance, or it may replicate independently of the host chromosome. A host organism containing the transformed nucleic acid fragment is referred to as a "transgenic," "recombinant," or "transformed" organism.

[0126] When used herein in conjunction with a host cell, the terms "transformed," "transgenic," and "recombinant" will be given their corresponding ordinary and customary meanings to those of ordinary skill in the art and are used without limitation to refer to cells of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate themselves. A transformed cell, tissue, or subject should be understood to encompass not only the end product of the transformation process, but also its transgenic progeny.

[0127] When used herein in conjunction with polynucleotides, the terms "recombinant," "heterologous," and "exogenous" will be given their ordinary and customary meanings to those of ordinary skill in the art and are used without limitation to refer to polynucleotides (e.g., DNA sequences or genes) that are derived from a source that is foreign to a particular host cell or that is modified relative to its original form if derived from the same source. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example, by the use of site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA fragment that is foreign or heterologous to a cell or homologous to a cell but in a location or form in which the element is not normally found within the host cell.

[0128] Similarly, the terms "recombinant," "heterologous," and "exogenous," when used herein in conjunction with a polypeptide or amino acid sequence, refer to a polypeptide or amino acid sequence that is derived from a source foreign to the particular host cell or, if derived from the same source, is modified relative to its original form. Thus, a recombinant DNA segment can be expressed in a host cell to produce a recombinant polypeptide.

[0129] The terms "plasmid," "vector," and "cassette" will be given their corresponding ordinary and customary meanings to those of ordinary skill in the art and are used without limitation to refer to an extrachromosomal element that typically carries genes that are not part of the central metabolism of the cell and is typically in the form of a circular double-stranded DNA molecule. Such elements can be linear or circular self-replicating sequences, genome integrating sequences, phages, or nucleotide sequences derived from single-stranded or double-stranded DNA or RNA from any source, many of which have been linked or recombined into a unique construct capable of introducing a promoter fragment and DNA sequence for a selected gene product, as well as appropriate 3' non-translated sequences, into a cell. A "transformation cassette" refers to a specific vector that contains an exogenous gene and has, in addition to the exogenous gene, elements that promote transformation of a specific host cell. An "expression cassette" refers to a specific vector that contains an exogenous gene and has, in addition to the exogenous gene, elements that allow enhanced expression of the gene in an exogenous host.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described below.

[0131] The present disclosure will be more fully understood by considering the following non-limiting examples. It should be understood that these examples, although indicating preferred embodiments of the subject technology, are given by way of illustration only. Based on the above discussion and these examples, those skilled in the art can determine the essential characteristics of the subject technology and, without departing from its spirit and scope, can make various changes and modifications to the subject technology to adapt it to various uses and techniques.

[0132] Example

[0133] Example 1: Identification of candidate genes for fungal cytochrome P450 enzymes with fatty acid 4-hydroxylase activity

[0134] Cytochrome P450 (CYP) enzymes can catalyze the oxidation of a wide variety of organic substrates, including fatty acids, to produce a variety of hydroxylated compounds, including hydroxy fatty acids. However, the position of the carbon atom along the hydrocarbon backbone at which the hydroxylation occurs can be crucial. For example, among the various P450s, the bacterial and fungal P450 BM3 enzyme has been reported to catalyze the formation of ω-1 to ω-3 hydroxy fatty acids. See alsoFor example, Miura, Y. and Fulco, AJ, “ω-1,ω-2andω-3Hydroxylation of Long-Chain Fatty Acids, Amides and Alcohols by a Soluble Enzyme System from Bacillus megatyerium,” Biochimica et Biophysica Acta-Lipids and LipidMetabolism, 388(3):305-317(1975); and Kitazume, T. et al., "Fusarium oxysporumFatty-acid Subterminal Hydroxylase(CYP505)Is a Membrane-bound EukaryoticCounterpart of Bacillus megaterium Cytochrome P450BM3," J. Bio. Chem., 275: 39734-39740 (2000).

[0135] See also Figure 1 The efficient synthetic pathway from various fatty acids to gamma-lactones involves hydroxylating the carbon atom at the gamma position to the carbonyl carbon of the fatty acid substrate, thereby generating 4-hydroxy fatty acids (4-hydroxylase pathway). Subsequently, 4-hydroxy fatty acids can be converted to gamma-lactones under acidic conditions.

[0136] It is reported that some filamentous fungi such as Mucor circinelloides, Aspergillus oryzae and Mortierella isabellina can prepare various γ-lactones or δ-lactones from corresponding carboxylic acids. Referring to U.S. Patent No. 5,032,513 and U.S. Patent No. 7,863,023. However, the molecular mechanism of their biotransformation activity is largely unknown. In addition, the complex morphology of such filamentous fungi makes the amplification of these processes a huge challenge.

[0137] To the best of the inventors' knowledge, prior to the present invention, no specific fatty acid 4-hydroxylase genes and enzymes had been identified. After rigorous high-throughput screening of a large library of candidate genes for fungal cytochrome P450 (CYP, P450, or CYP450) enzymes with fatty acid 4-hydroxylase activity, the inventors identified the following candidate genes: a cytochrome P450 monooxygenase gene from an unidentified species of Mucor (GenBank: GAN03094.1), the amino acid sequence of which is provided as SEQ ID NO: 1; a cytochrome P450 monooxygenase gene from Batrachoderma schizosporum (GenBank: ORX85448.1), the amino acid sequence of which is provided as SEQ ID NO: 3; and a cytochrome P450 monooxygenase gene from Agaricales fusca (Agaricales fusca CYP), the amino acid sequence of which is provided as SEQ ID NO: 5.

[0138] Example 2: Generation of high-yielding strains and overexpression of cytochrome P450 for the production of γ-lactones To facilitate protein expression in E. coli, each candidate gene was codon-optimized for the E. coli genome and synthesized by Gene Universal Inc. (Newark, DE).

[0139] The codon-optimized unidentified Mucor P450 gene was cloned into the pET17b vector (AMP+, Novagen) via the HindIII and XhoI sites. This construct was transformed into BL21 (DE3) cells for expression. In a typical experiment, an overnight culture was inoculated with a 2% LB medium containing 100 mg / L carbenicillin. The culture was first grown at 37°C to an OD600 of 0.6 and then cooled to 25°C. 1 mM IPTG was then added to induce protein expression. After incubation at 25°C for 3 hours, a fatty acid substrate was added at 1 g / L to prepare γ-lactone.

[0140] Each of the codon-optimized P450 gene of Batrachoma schizosporum and the P450 gene of Agaricus fuscae is cloned into pET-32a-(+) carrier (AMP+, Novagen) by HindIII site and XhoI site. This construct is transformed into BL21 (DE3) cells and expressed. In a typical experiment, the liquid LB medium (2%) containing 100mg / L carbenicillin is inoculated with overnight culture. The culture is first grown to OD600 of 0.8 at 37°C and then cooled to 16°C. Then 1mM IPTG is added to induce protein expression. After incubation at 16°C for 5 hours, the temperature is raised to 30°C, and fatty acid substrates are added to prepare gamma-lactone with 1g / L.

[0141] Example 3: Analysis of γ-lactone production

[0142] To analyze the production of γ-lactones, 0.5 ml of the E. coli culture described in Example 2 was acidified with 10 μl of 2N HCl and then extracted with 0.5 ml of ethyl acetate at room temperature with shaking for 60 minutes. After centrifugation at 14,000 rpm for 15 minutes, the ethyl acetate phase was used for GC / MS or GC / FID analysis.

[0143] GC / MS analysis was performed on a Shimadzu GC-2010 system coupled to a GCMS-QP2010S detector. The analytical column was a SHRXI-5MS (thickness 0.25 μm; length 30 m; diameter 0.25 mm), and the injection temperature was 265°C in split mode. The temperature gradient was: 80°C from 0 min to 3 min; 120°C to 263°C from 3 min to 8.7 min, with a gradient of 25°C; and 263°C from 8.7 min to 10.7 min.

[0144] GC / FID analysis was performed on a Shimadzu GC-2014 system. The analytical column was a Restek RXi-5ms (thickness 0.25 μm; length 30 m; diameter 0.25 mm), and the injection temperature was 240°C in split mode. The temperature gradient was: 100°C from 0 min to 3 min; 100°C to 280°C from 3 min to 9 min, with a gradient of 30°C; and 280°C from 9 min to 12 min.

[0145] Example 4: Preparation of γ-lactone using a high-yielding strain overexpressing an unidentified Mucor P450

[0146] The procedure described in Example 2 was performed using an E. coli culture overexpressing an unidentified Mucor P450. Specifically, samples of the E. coli culture were taken and acidified either 2 days or 16 hours after addition of the fatty acid substrate. Figure 2 shows the production of various γ-lactones from the corresponding carboxylic acids: (a) γ-heptanoic acid (GC7) is produced from gluconic acid (heptanoic acid, C7), (b) γ-octanoic acid (GC8) is produced from caprylic acid (octanoic acid, C8), (c) γ-nonanoic acid (GC9) is produced from pelargonic acid (nonanoic acid, C9), (d) γ-decanoic acid (GC10) is produced from caprylic acid (decanoic acid, C10), (e) γ-undecalactone (GC11) is produced from undecanoic acid (undecanoic acid, C11), (f) γ-dodecalactone (GC12) is produced from lauric acid (dodecanoic acid, C12), (g) γ-tridecalactone (GC13) is produced from tridecanoic acid (tridecanoic acid, C13), and (h) γ-tetradecalactone (GC14) is produced from myristic acid (tetradecanoic acid, C14).

[0147] like Figures 2a to 2hAs shown, GC7-GC14 γ-lactones are effectively produced by corresponding C7-C14 carboxylic acids in a heterologous system overexpressing an unknown mucor cytochrome P450 (SEQ ID NO: 1). It is noteworthy that the GC7 to GC12 peaks match the corresponding standards from Sigma-Aldrich (St Louis, MO) in terms of retention time and mass spectrum. The standards of γ-tridecalactone (GC13) and γ-tetradecalactone (GC14) are not commercially available. However, it is noted that the putative GC13 and GC14 peaks have a retention time longer than that of GC12.

[0148] Example 5: Preparation of γ-lactone using a high-yielding strain overexpressing Batrachoderma P450

[0149] The procedure described in Example 2 was performed using an E. coli culture overexpressing the P450 of R. schizosporum. Specifically, a sample of the E. coli culture was taken and acidified 17 hours after the addition of the fatty acid substrate. Figure 3 The production of various γ-lactones from the corresponding carboxylic acids is shown: (a) γ-undecalactone (GC11) from undecanoic acid (undecanoic acid, C11), and (b) γ-undecalactone (GC11′) from undecenoic acid (undecenoic acid, C11:1).

[0150] like Figure 3 As shown, GC11γ-lactone and GC11'γ-lactone are efficiently produced from the corresponding C11 carboxylic acid and C11:1 carboxylic acid in a heterologous system overexpressing Batrachodactylum cytochrome P450 (SEQ ID NO:3). Notably, the C11 peak, GC11 peak, and C11:1 peak match the corresponding standards from Sigma-Aldrich (St Louis, MO) in both retention time and mass spectrum. The standard for GC11' is not commercially available. However, it is noted that the putative GC11' peak shows a retention time similar to GC11.

[0151] Under the reaction conditions, about 69 mg / L of GC11 and about 18 mg / L of GC11′ were produced 17 hours after 1 g / L of C11 or C11:1, respectively, was added to the E. coli growing cell culture.

[0152] In addition to growing cells, resting cells were also studied for their potential use in bioconversion of fatty acids into gamma-lactones. In this experiment, liquid SOC culture medium (3%) containing 100 mg / L carbenicillin was inoculated with overnight cultures. Culture was first grown at 37°C to an OD600 of 0.8, then cooled to 16°C. 1 mM IPTG was then added to induce protein expression. After incubation at 16°C for 16 hours, cultures were harvested and cell pellets were collected by centrifugation, then stored at -80°C until use.

[0153] In a typical bioconversion experiment, frozen cells were resuspended in 100 mM K-Pi buffer (pH 7) at a cell concentration of 100 g / L fresh weight. Then, 1 g / L of C11 or C11:1 was added along with 0.1% Tween 40 and 10 mM NADPH. The mixture was then incubated at 30°C while shaking at 250 rpm. Samples were taken after 3 hours. The titers of GC11 and GC11' were 419 mg / L and 185 mg / L, respectively.

[0154] Example 6: Preparation of γ-lactone using a high-yielding strain overexpressing Agaricales flavus P450

[0155] The procedure described in Example 2 was performed using an E. coli culture overexpressing the Agaricus luteus P450. Specifically, a sample of the E. coli culture was taken and acidified 25 hours after addition of the fatty acid substrate. Figure 4 shows the production of various γ-lactones from the corresponding carboxylic acids: (a) γ-undecalactone (GC11) from undecanoic acid (undecanoic acid, C11), and (b) γ-undecalactone (GC11') from undecenoic acid (undecenoic acid, C11:1), with retention times (min).

[0156] Figure 4 shows that various γ-lactones are produced from the corresponding carboxylic acids: (a) γ-caprolactone (GC6) is produced from hexanoic acid (C6), (b) γ-heptanoic acid (GC7) is produced from gluconic acid (heptanoic acid, C7), (c) γ-decanoic acid (GC10) is produced from capric acid (decanoic acid, C10), (d) γ-undecalactone (GC11) is produced from undecanoic acid (undecanoic acid, C11), (e) γ-dodecalactone (GC12) is produced from lauric acid (dodecanoic acid, C12), (f) γ-tridecalactone (GC13) is produced from tridecanoic acid (tridecanoic acid, C13), and (g) γ-tetradecanoic acid (GC14) is produced from myristic acid (tetradecanoic acid, C14).

[0157] like Figures 4a to 4gAs shown, GC6γ-lactone, GC7γ-lactone, GC10γ-lactone, GC11γ-lactone, GC12γ-lactone, GC13γ-lactone and GC14γ-lactone are effectively produced by corresponding C6 carboxylic acid, C7 carboxylic acid, C10 carboxylic acid, C11 carboxylic acid, C12 carboxylic acid, C13 carboxylic acid and C14 carboxylic acid in the heterologous system of overexpressing Agaricus flavoceae cytochrome P450 (SEQ ID NO:5).It should be noted that GC6 peak, GC7 peak, GC10 peak, GC11 peak and GC12 peak are matched with the corresponding standard substance from Sigma-Aldrich (St Louis, MO) in terms of retention time and mass spectrum.The standard substance of γ-tridecaprolide (GC13) and γ-tetradecaprolide (GC14) is not commercially available.However, it is noted that the GC13 peak and GC14 peak of inference have a retention time longer than the retention time of GC12.

[0158] Under the reaction conditions, about 42 mg / L of GC6 was produced 25 hours after 1 g / L of C6 was added to the E. coli growing cell culture.

[0159] In addition to growing cells, resting cells were also studied for their potential use in bioconversion of fatty acids into gamma-lactones. In this experiment, liquid SOC culture medium (3%) containing 100 mg / L carbenicillin was inoculated with overnight cultures. Culture was first grown at 37°C to an OD600 of 0.8, then cooled to 16°C. 1 mM IPTG was then added to induce protein expression. After incubation at 16°C for 16 hours, cultures were harvested and cell pellets were collected by centrifugation, then stored at -80°C until use.

[0160] In a typical bioconversion experiment, frozen cells were resuspended in 100 mM K-Pi buffer (pH 7) at a cell concentration of 100 g / L fresh weight. 1 g / L of C6 was then added, and the mixture was incubated at 30°C while shaking at 250 rpm. Samples were taken after 3 hours. The titer of GC6 was 198 mg / L.

[0161] Sequence of interest :

[0162] Unidentified Mucor cytochrome P450

[0163] Amino acid sequence of GAN03094.1 gene (SEQ ID NO: 1) :

[0164]

[0165] The nucleotide sequence of GAN03094.1 was codon-optimized for the E. coli genome (SEQ ID NO: 2) ：

[0166]

[0167] Cytochrome P450 of Batrachoderma spp

[0168] Amino acid sequence of ORX85448.1 gene (SEQ ID NO: 3) ：

[0169]

[0170] The nucleotide sequence of ORX85448.1 was codon-optimized for the E. coli genome (SEQ ID NO: 4) ：

[0171]

[0172] Agaricus cytochrome P450

[0173] Amino acid sequence (SEQ ID NO: 5)

[0174]

[0175] Nucleotide sequence codon-optimized for the E. coli genome (SEQ ID NO: 6) ：

[0176]

Claims

1. A method for preparing gamma lactone, comprising: providing a reaction mixture comprising a recombinant CYP450 protein having carboxylic acid 4-hydroxylase activity and a C4-C20 carboxylic acid substrate to provide a 4-hydroxy C4-C20 carboxylic acid; and subjecting the 4-hydroxy C4-C20 carboxylic acid to acidic conditions to produce the gamma lactone; wherein the gamma lactone is selected from the group consisting of gamma-heptanolactone, gamma-octanolactone, gamma-nonanolactone, gamma-decanolactone, gamma-undecalactone, gamma-dodecalactone, gamma-tridecalactone, and gamma-tetradecalactone; the C4-C20 carboxylic acid substrate is a fatty acid selected from the group consisting of heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid; and the recombinant CYP450 protein comprises the amino acid sequence of SEQ ID NO: 1; or wherein the gamma lactone is selected from gamma-undecalactone and gamma-undecenolactone, the C4-C20 carboxylic acid substrate is a fatty acid selected from undecanoic acid and undecenoic acid; and the recombinant CYP450 protein comprises the amino acid sequence of SEQ ID NO: 3; or wherein the gamma lactone is selected from the group consisting of gamma-caprolactone, gamma-heptanolactone, gamma-decanolactone, gamma-undecalactone, gamma-dodecalactone, gamma-tridecalactone, and gamma-tetradecalactone, the C4-C20 carboxylic acid substrate is a fatty acid selected from the group consisting of hexanoic acid, heptanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid, and the recombinant CYP450 protein comprises the amino acid sequence of SEQ ID NO:

5. 2 . The method of claim 1 , wherein the recombinant CYP450 protein is expressed by a transformed host cell in the reaction mixture.

3. The method according to claim 2, wherein the transformed host cell is selected from the group consisting of a bacterial cell and a yeast cell.

4. The method according to any one of claims 1 to 3, wherein the C4-C20 carboxylic acid substrate comprises the fatty acid, a salt of the fatty acid, an ester of the fatty acid, a monoglyceride, a diglyceride or a triglyceride of the fatty acid, or a combination thereof.

5. A method for preparing gamma lactone, comprising: incubating a transformed host cell comprising a sequence encoding a CYP450 protein having carboxylic acid 4-hydroxylase activity with a C4-C20 carboxylic acid substrate to provide a 4-hydroxy C4-C20 carboxylic acid; and subjecting the 4-hydroxy C4-C20 carboxylic acid to acidic conditions to produce the gamma lactone; wherein the gamma lactone is selected from the group consisting of gamma-heptanolactone, gamma-octanolactone, gamma-nonanolactone, gamma-decanolactone, gamma-undecalactone, gamma-dodecalactone, gamma-tridecalactone, and gamma-tetradecalactone; the C4-C20 carboxylic acid substrate is a fatty acid selected from the group consisting of heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid; and the sequence of the CYP450 protein comprises the nucleotide sequence of SEQ ID NO: 2; or wherein the gamma lactone is selected from gamma-undecalactone and gamma-undecenolactone, the C4-C20 carboxylic acid substrate is a fatty acid selected from undecanoic acid and undecenoic acid; and the sequence of the CYP450 protein comprises the nucleotide sequence of SEQ ID NO: 4; or wherein the gamma lactone is selected from the group consisting of gamma-caprolactone, gamma-heptanolactone, gamma-decanolactone, gamma-undecalactone, gamma-dodecalactone, gamma-tridecalactone and gamma-tetradecalactone, the C4-C20 carboxylic acid substrate is a fatty acid selected from the group consisting of hexanoic acid, heptanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid and tetradecanoic acid, and the sequence of the CYP450 protein comprises the nucleotide sequence of SEQ ID NO:

6. The method according to claim 5 , wherein the transformed host cell is selected from a bacterial cell or a yeast cell. 7 . The method of claim 6 , wherein the nucleotide sequence is SEQ ID NO: 2, and the CYP450 protein comprises the amino acid sequence of SEQ ID NO:

1.

8. The method of claim 6, wherein the nucleotide sequence is SEQ ID NO: 4, and the CYP450 protein comprises the amino acid sequence of SEQ ID NO:

3. 9 . The method of claim 6 , wherein the nucleotide sequence is SEQ ID NO: 6, and the CYP450 protein comprises the amino acid sequence of SEQ ID NO:

5.

10. The method of any one of claims 5 to 9, wherein the C4-C20 carboxylic acid substrate comprises the fatty acid, a salt of the fatty acid, an ester of the fatty acid, a monoglyceride, a diglyceride, or a triglyceride of the fatty acid, or a combination thereof.

11. The method according to any one of claims 5 to 9, wherein the transformed host cell is a growing cell.

12. The method according to any one of claims 5 to 9, wherein the transformed host cell is a quiescent cell.

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