Enzymes and methods for fermentation production of monoterpene esters
The development of an alcohol acyltransferase with specific amino acid sequences allows for the efficient esterification of tertiary monoterpene alcohols, overcoming the limitations of existing technologies and enabling high-yield production of linalyl acetate and alpha-terpinyl acetate.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- アイソバイオニクスベーフェー
- Filing Date
- 2021-12-17
- Publication Date
- 2026-07-03
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to an alcohol acyltransferase capable of esterifying a tertiary monoterpene alcohol, preferably within 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes, or 30 minutes, more preferably in a microbial cell, so that at least 30% by mass of the tertiary monoterpene alcohol is esterified. The present invention further relates to nucleic acids comprising a nucleic acid sequence encoding the alcohol acyltransferase of the present invention or a complementary sequence thereof, and vectors or gene constructs comprising the nucleic acids of the present invention. The present invention further provides host cells comprising the vector or gene construct of the present invention, and transgenic non-human organisms comprising the nucleic acids of the present invention, the vector or gene construct of the present invention, or the host cells of the present invention. The present invention also relates to a method for preparing a monoterpene ester, comprising esterifying a monoterpene alcohol to a monoterpene ester in the presence of the alcohol acyltransferase of the present invention. Specifically, this provides a method for preparing linalyl acetate, comprising esterifying linalool to linalyl acetate in the presence of the alcohol acyltransferase of the present invention.The present invention further relates to (i) heterologous reconstitution of terpene biosynthesis pathways; (ii) for producing foaming agents, pesticides, insect repellents or antimicrobial agents for industrial products, preferably flavorings or fragrances, biofuels, fuel compositions, fuel compounds, such as diesel fuel compositions; (iii) for producing aliphatic and / or aromatic monoterpene esters from monoterpene alcohols, preferably tertiary monoterpene alcohols; (iv) for detoxifying monoterpene alcohols in microorganisms, thereby increasing monoterpene production in said microorganisms; and (v) GPP synthase and / or S- or R-linalool synthase The present invention relates to the use of the alcohol acyltransferase of the present invention, nucleic acids of the present invention, vectors or gene constructs of the present invention, host cells of the present invention or transgenic non-human organisms of the present invention in combination with; (vi) to improve the beneficial effect of acetylation in which hydrophobic acetic acid partitioning is more readily transferred to the organic phase compared with monoterpene alcohols; (vii) to express the alcohol acyltransferase of the present invention such that the ratio of monoterpene acetate to monoterpene alcohol is greater than 5:1 or 10:1; or (viii) in microbial production systems for monoterpene esters. The present invention also provides a kit comprising the alcohol acyltransferase of the present invention, nucleic acids of the present invention, vectors or gene constructs of the present invention, host cells of the present invention or transgenic non-human organisms of the present invention and optionally at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol.Ultimately, the present invention relates to a method for producing fuel and / or biolubricant compounds, the method comprising: a) producing one or more monoterpene esters by any one of the methods of the present invention; b) optionally purifying one or more monoterpene esters produced in step a); and c) some or all of the one or more monoterpene esters from step a) or the optionally purified one or more monoterpene esters from step b), preferably: tetrahydrolinalool; 2,6-dimethyloctane (DMO); saturated C20 hydrocarbon dimer; saturated C30 hydrocarbon trimer; hydrogenated methylcyclopentadiene dimer ;converting to one or more fuel and / or biolubricant compounds selected from the group consisting of saturated, high-density polycyclic hydrocarbon compounds suitable for projectile propulsion and hydrogenated C40+ oligomers suitable for producing biolubricant additives;d) optionally combining one or more fuel or biolubricant compounds with further compounds suitable for fuel and / or biolubricants;the fuel and / or biolubricant composition comprises a total of 0.01% (w / w) to 99.99% (w / w) of fuel or biolubricant compounds made from one or more monoterpene esters that can be obtained by one of the methods of the present invention. [Background technology]
[0002] For the past several decades, intensive scientific research has focused on terpenes, the most abundant secondary metabolites in all living organisms. Over 55,000 terpenoid substances are widely distributed among various families of natural products found throughout the entire biological world.
[0003] Many terpenoids are generally secondary metabolites, as they are not primarily essential for the growth, development, or regeneration of any organism. However, this classification does not extend to the broader and further effects of these secondary metabolites in maintaining ecosystem functions. These substances can play important roles and provide plants with evolutionary advantages in terms of their distinct chemosensibility properties, such as odor. Thus, among other things, they can exert insecticidal effects, protecting plants and grains from parasites and pathogens, or act as pollinator attractants in the reproductive process.
[0004] Many terpenoids are well known for their economic importance, being widely used as basic structural components in the production of drugs, flavorings, fragrances, pigments, and disinfectants. For example, linalool, a monoterpene alcohol and a major component of the essential oil of rosewood, Aniba rosaeodora, is one of the most frequently used ingredients, particularly in the production of perfumes. Furthermore, the sesquiterpene lactone artemisinin, extracted from the shrub Artemisia annua, is used in the first-line treatment of malaria. Taxol and its structural analogs, tricyclic diterpenes isolated from the bark of the Pacific yew, Taxus brevifolia, are used as anticancer agents.
[0005] Terpenes are primarily synthesized in plants via common biosynthetic pathways. Regardless of their diverse structures and functions, all terpenes are constructed from isoprene units (5 carbon atoms) according to the isoprene rule. Terpenes are classified according to the number of isoprene units in their structure linked together through head-tail addition, and according to the number of carbon atoms or sesquiterpenoid moieties, respectively: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), or polyterpenes with up to 30,000 linked isoprene units. Similar to terpenes, terpenoids are similarly classified according to the number of isoprene units, and these are further named with the suffix -oids (~like), as in monoterpenoids (C10) or sesquiterpenoids (C15).
[0006] Isopentyl diphosphate (IPP) and its electrophilic isomer, dimethylallyl diphosphate (DMAPP), are universal precursors in terpene biosynthesis. Starting from these two components, linear prenyl diphosphate is synthesized by a group of enzymes belonging to the prenyltransferase group. IPP and DMAPP are condensed under the catalytic effect of prenyltransferase geranyl diphosphate synthase to give C10 geranyl diphosphate (GPP), an intermediate that can be converted into cyclic or linear final products corresponding to the monoterpenes.
[0007] Similarly, sesquiterpenes are produced via the addition of a third isoprene unit to GPP, which forms C15 farnesyl diphosphate (FPP), a biosynthetic precursor of common sesquiterpenes. Further polymerization of IPP and DMAPP produces longer prenyl diphosphates, which form different classes of terpenes named according to the number of isoprene units they contain.
[0008] IPP and DMAPP biosynthesis is completed via two independent pathways: the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although the MVA pathway was considered a universal pathway in terpene synthesis, it has been found in the last decade to be less prominent than the MEP pathway in plant secondary metabolites. MVA is the dominant pathway in the cytoplasm and mitochondria of most eukaryotes, archaea, several bacteria, and plants, producing precursors for multiple analogues such as sesquiterpenes (C15) and triterpenes (C30) within the cytoplasm. On the other hand, the MEP pathway is the dominant pathway in higher plants, cyanobacteria, bacteria, and chloroplasts of algae. Depending on its biosynthetic site in plastids, MEP leads to monoterpenes (C10), diterpenes (C20), and carotenoids (C40).
[0009] The mevalonate pathway (MVA), also known as the mevalonate pathway, isoprenoid pathway, or 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase pathway, was discovered in yeast and animals in the 1950s. The MVA pathway begins with the Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA, catalyzed by the enzyme acetoacetyl-CoA transferase. Acetoacetyl-CoA is converted to HMG-CoA by HMG synthase via an aldol reaction with another acetyl-CoA. In the next two reduction steps, two nicotinamide adenine dinucleotide phosphate molecules are required to convert HMG-CoA to mevalonate (MVA) using HMG-CoA reductase. Subsequent phosphorylation of MVA yields mevalonate 5-bisphosphate (MVAPP) via two reactions catalyzed by mevalonate kinase (MK) and phosphomevalonate kinase (PMK), respectively. Finally, IPP is produced from the decarboxylation of MVAPP by an ATP-coupled decarboxylation reaction catalyzed by mevalonate 5-bisphosphate decarboxylase (MVD). IPP:DMAPP isomerase (IDI) then catalyzes the interconversion between IPP and DMAPP.
[0010] The methylerythritol phosphate pathway (MEP), or MVA-independent pathway, was discovered in the late 1990s and early 2000s by Rohmer, Arigoni, and others in the chloroplasts of bacteria, green algae, and higher plants. This pathway begins with two distinct precursors, namely pyruvate and D-glyceraldehyde 3-phosphate (G3P). Both molecules undergo condensation catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) using thiamine pyrophosphate as a cofactor, yielding 1-deoxy-D-xylulose 5-phosphate (DXP). In the next step, DXP is isomerized to MEP by DXP reductisomerase (DXR). 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) synthase ultimately catalyzes the coupling of MEP and cytidine triphosphate (CTP) to produce methylerythritol cytidyl diphosphate (CDP-ME). In an ATP-dependent reaction, CDP-ME kinase phosphorylates CDP-ME to 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP). Subsequently, the latter undergoes cyclization to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) in a reaction catalyzed by MEcPP synthase, releasing cytidine monophosphate (CMP). This pathway is terminated by ring-opening of the cyclic pyrophosphate and reductive dehydration of MEcPP to 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) catalyzed by HMBPP synthase. HMBPP is ultimately converted by HMBPP reductase into a mixture of IPP and DMAPP.
[0011] In recent years, many terpenes, including monoterpenes, sesquiterpenes, and their alcohols, have been produced in microbial systems to provide alternatives to plant-derived terpenes. Most commercially available terpenes are produced by chemical synthesis or extraction from plant material. Plant-derived terpenes often have drawbacks such as low concentrations, yield dependence, the presence of pesticides, and / or the risk of plant species extinction. Biotechnological production of terpenes can provide sustainable and economically viable alternatives to plant-derived terpenes. Examples of such biotechnological production methods include valencene, artemisinic acid, and sclareol.
[0012] Many terpenes can be produced by microbial biotechnology. Esters such as ethyl acetate can be produced by yeasts such as Kluyveromyces marxianus. These yeasts use EAT proteins from the α / β hydrolase family to convert ethanol and acetyl-CoA to ethyl acetate (e.g., Kruis et al., (2019) Microbial production of short and medium chain esters: Enzymes, pathways, and applications. Biotechnology Advances, 37(7), 107407). The activity of these EAT proteins in more complex molecules has not been studied in much detail.
[0013] The production of monoterpene esters in microorganisms has also been elucidated. When geraniol was produced in E. coli, the chloramphenicol acetyltransferase gene was observed to be involved in the formation of geranyl acetate (Liu et al. Biotechnol Biofuels (2016) 9:58). The use of more specific enzymes has been shown to yield advantages: while monoterpene alcohols such as linalool, as well as geraniol, an acyclic monoterpene found in the floral scents of many plants, are toxic to microorganisms, their esters are often considerably less toxic. The toxicity of monoterpene alcohols often leads to inhibition in growth and / or production, and therefore only very low production titers have been achieved. Chacon et al. showed that RhAAT expression in engineered Escherichia coli, modified to produce geraniol, leads to the formation of geranyl acetate at substantially elevated levels compared to geraniol levels produced without RhAAT (Chacon, MG, et al. Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli. Microb Cell Fact 18, 105 (2019); https: / / doi.org / 10.1186 / s12934-019-1130-0; International Publication No. 2019 / 092388). For this reason, in-situ esterification of monoterpene alcohols such as geraniol has been promoted as a means of detoxifying the product and thus improving terpene production.
[0014] In plants, esters are produced using alcohol acyltransferases (AATs) from the BAHD family of proteins that couple alcohols to acyl-CoA. The BAHD acyltransferase superfamily is named according to the first four biochemically characterized enzymes of this family in plants: benzyl alcohol O-acetyltransferase (BEAT), anthocyanin O-hydroxy-cinnamoyltransferase (AHCT), anthranilate N-hydroxy-cinnamoyl / benzoyltransferase (HCBT), and deacetylvindrin 4-O-acetyltransferase (DAT). Members of the BAHD family share two conserved motifs: the HxxxD motif involved in catalysis and the DFGWG motif, whose role remains unknown (Bayer A et al. (2004) Bioorg Med Chem 12, 2787-2795).
[0015] Well-known examples of alcohol acyltransferases (AATs) generally include those derived from fruits such as strawberries and bananas that produce fruit esters from aliphatic alcohols such as octanol, hexanol, or isoamyl alcohol (Aharoni et al., 2000 Identification of the SAAT gene. Plant Cell. 2000 May;12(5):647-62.doi:10.1105 / tpc.12.5.647.; Beekwilder et al., (2004) Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol. 2004 135(4):1865-78.doi:10.1104 / pp.104.042580).
[0016] Alcohol acyltransferases (AATs) also mediate the esterification of more complex molecules: for example, as part of a series of at least 17 enzymatic steps, saltaridinol 7-O-acetyltransferase mediates a step in morphine biosynthesis using a complex isoquinoline alkaloid as a substrate (Grothe et al., 2001, DOI 10.1074 / jbc.M102688200). Acetyl-CoA:deacetylvindrin 4-O-acetyltransferase catalyzes the final step in the biosynthesis of vindrin, an indole alkaloid.
[0017] Monoterpene alcohols can also be used as substrates for alcohol acyltransferases (AATs). For example, RhAAT from roses (Rosa hybrida) is involved in geranyl acetate formation (Shalit et al., Volatile ester formation in roses. Identification of an acetyl-coenzyme A. Geraniol / Citronellol acetyltransferase in developing rose petals. Plant Physiol. 2003 Apr;131(4):1868-76.doi:10.1104 / pp.102.018572). AAT derived from the genus Lavandula has been described as being involved in the formation of lavandzuryl acetate (Sarker & Mahmoud (2015) Cloning and functional characterization of two monoterpene acetyltransferases from glandular trichomes of Lx intermedia. Planta 242, 709-719).
[0018] Rose AAT (RhAAT) and many other alcohol acyltransferases (AATs) (e.g., strawberry AAT, SAAT) accept a range of alcohol substrates, including phenylethanol, cinnamoyl alcohol, hexanol, isoamyl alcohol, and longer-chain alcohols, but they are not very selective. However, linalool is not an accepted substrate for these enzymes. The exceptional property of linalool that makes it a non-substrate for these AATs may arise from the position of the alcohol group in linalool, which can be considered a tertiary alcohol: the carbon to which the acceptor alcohol group is linked is bonded to three carbon groups. Tertiary alcohols are often more difficult to access the enzyme active site pocket, possibly due to the steric accessibility of the alcohol group.
[0019] Linalyl acetate (CAS number 115-95-7) is an acetyl ester generally formed from linalool. To date, linalyl acetate has been produced chemically, and no large-scale fermentation-based processes for its production have been reported; see, for example, https: / / de.wikipedia.org / wiki / Linalylacetat. Linalyl acetate is an important flavor and aroma molecule. However, no alcohol acyltransferase capable of esterifying linalool or other tertiary monoterpene alcohols in economically significant quantities has yet been identified. Recent studies have reported trace amounts of linalool activity from BAHD acyltransferase from common thyme (Thymus vulgaris) when transiently expressed in Nicotiana benthamiana (Doctoral dissertation "Die Biosynthese von acetylierten Monoterpenen in Thymian (Thymus vulgaris)" by Natalie Wiese, June 2020; https: / / opendata.uni-halle.de / handle / 1981185920 / 34248). However, such trace amounts of activity, accompanied by small amounts of byproduction of linalyl acetate in highly specific experimental settings, may be lost under common conditions in industrial-scale biocatalytic or biotechnological production, or may simply be unsuitable for large-scale production, or may be uneconomical for such use.
[0020] Consequently, the lack of an alcohol acyltransferase capable of esterifying tertiary monoterpene alcohols, such as linalool, in economically significant quantities hinders the production of linalyl acetate by biocatalytic or biotechnological approaches. [Overview of the Initiative] [Problems that the invention aims to solve]
[0021] Therefore, the technical problem underlying the present invention can be considered to be the provision of means and methods that meet the above-mentioned needs. The technical problem is solved by the embodiments characterized in the claims, hereinafter in the description and in the examples.
Means for Solving the Problem
[0022] The present invention relates to an alcohol acyltransferase comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence as shown by any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 16; b) an amino acid sequence having at least 60% sequence identity at the amino acid level with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 16 and having alcohol acyltransferase activity; and c) a fragment of the amino acid sequence of a) or b) having alcohol acyltransferase activity. The alcohol acyltransferase of the present invention can be a natural enzyme. In another embodiment, the alcohol acyltransferase of the present invention is a synthetic (used interchangeably with artificial or non-natural) enzyme.
[0023] In one embodiment, the alcohol acyltransferase of the present invention can esterify a monoterpene alcohol, preferably a tertiary monoterpene alcohol, preferably within 36 hours, more preferably in microbial cells, as defined herein, such that at least 30% by mass of the alcohol is esterified.
[0024] Preferably, the alcohol acyltransferase of the present invention can esterify a monoterpene alcohol, preferably a tertiary monoterpene alcohol, within 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes or 30 minutes in microbial cells, as defined herein, such that at least 30% by mass of the alcohol is esterified.
[0025] In a preferred embodiment, the alcohol acyltransferase of the present invention has 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% or even 100% sequence identity with SEQ ID NO: 2 at the amino acid level, or has at least 65%, at least 70%, at least 75%, at least 80%, 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% or even 100% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 16, and is an alcohol acyltransferase with enzymatic activity, comprising an amino acid sequence having such sequence identity.
[0026] The inventors analyzed the sequence of SEQ ID NO: 2 together with other candidate sequences of alcohol acyltransferase, and further used the experimental results for SEQ ID NO: 6 and 8 (inactive mutants) to determine the types of important amino acids at important positions in SEQ ID NO: 2. For example, it was found that position 371 in SEQ ID NO: 2 is a glycine residue. In contrast, position 371 of its inactive mutant AT9-1-a (SEQ ID NO: 6) is filled with tryptophan. As a result of the substitution of the glycine residue by tryptophan at position 371, although SEQ ID NO: 2 and SEQ ID NO: 6 share 99.77% identity, inactivation of the alcohol acyltransferase occurs. Furthermore, the monoterpene acetyltransferases LiAAT-3 and Li-AAT4 from the glandular protrusions of L. x intermedia described in the publication by Sarker et al. (Planta (2015) 242: p. 709-719) have tryptophan at the position corresponding to position 371 of SEQ ID NO: 2.
[0027] Accordingly, the alcohol acyltransferase of the present invention preferably has an amino acid residue at positions 371 and 372 of SEQ ID NO: 2 that is not tryptophan, tyrosine, or phenylalanine, more preferably not tryptophan. Positions 371 and 372 indicated in SEQ ID NO: 2 are preferably referenced from the sequence list or Figure 1.
[0028] In one embodiment, the alcohol acyltransferase of the present invention further comprises any of the amino acids listed in Table 1 at the position corresponding to the position of SEQ ID NO: 2 shown in Table 1, relative to the position given in the first column of Table 1. For example, but not limited to, the position corresponding to position 62 of SEQ ID NO: 2 is filled with either serine or threonine in the alcohol acyltransferase of the present invention.
[0029] [Table 1]
[0030] To provide an example, the alcohol acyltransferase of the present invention may be an enzymatically active alcohol acyltransferase having an amino acid sequence having 60% sequence identity with SEQ ID NO: 2 at the amino acid level, where the position corresponding to position 62 of SEQ ID NO: 2 is serine, the position corresponding to position 113 of SEQ ID NO: 2 is glycine, the position corresponding to position 158 of SEQ ID NO: 2 is threonine, the position corresponding to position 371 of SEQ ID NO: 2 is glycine, the position corresponding to position 372 of SEQ ID NO: 2 is leucine, and the position corresponding to position 415 of SEQ ID NO: 2 is aspartic acid.
[0031] In a preferred embodiment, the alcohol acyltransferase of the present invention contains the amino acids listed in Table 2 at the position corresponding to the position of SEQ ID NO: 2 shown in Table 2. For example, in the alcohol acyltransferase of the present invention, the position corresponding to amino acid position 197 of SEQ ID NO: 2 is filled with proline.
[0032] [Table 2]
[0033] In a further preferred embodiment, the amino acid sequence of the alcohol acyltransferase of the present invention further includes the conserved amino acid residues shown in Figure 5, i.e., the amino acid residues shown in white letters on a black background.
[0034] Furthermore, Example 4 demonstrates the identification of an alcohol acyltransferase (AAT) homolog from Citrus bergamia in Lavandula angustifolia.
[0035] In one embodiment, the alcohol acyltransferase of the present invention is a synthetic alcohol acyltransferase having enzymatic activity comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity at the amino acid level with SEQ ID NO: 15 or 16. In another embodiment, the amino acid sequence of this synthetic alcohol acyltransferase comprises a "MEIE" motif with consecutive amino acid residues methionine, glutamic acid, isoleucine, and glutamic acid. In a further embodiment, the synthetic amino acid sequence further comprises the conserved amino acid residues shown in Figure 5, i.e., amino acid residues shown in white letters on a black background.
[0036] Another embodiment of the present invention is, a) Amino acid sequences such as those shown in Sequence ID No. 3 (Lavandula AAT-10056) or Sequence ID No. 4 (Lavandula AAT-1461); b) an amino acid sequence having at least 80% sequence identity at the amino acid level with SEQ ID NO: 3 (Lavandula AAT-10056) or SEQ ID NO: 4 (Lavandula AAT-1461); and c) A fragment of the amino acid sequence a) or b) that has enzymatic activity For alcohol acyltransferases containing an amino acid sequence selected from the group consisting of the following.
[0037] A preferred embodiment relates to a linalool acyltransferase, which is an alcohol acyltransferase as defined herein, except for the amino acid sequences of SEQ ID NOs: 3 and 4, and is capable of esterifying linalool to a linalool ester, preferably linalyl acetate.
[0038] Sequence identity as referred to herein is preferably determined over the entire length of the amino acid sequence indicated by the corresponding sequence number.
[0039] Sequence ID 2 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (mutant AAT9-1-c) from Citrus bergamia.
[0040] Sequence ID 3 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (internal name "10056") from Lavandula angustifolia.
[0041] Sequence ID 4 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (internal name "1461") from Lavandula angustifolia.
[0042] Sequence ID 15 corresponds to the artificial amino acid sequence of the alcohol acyltransferase (internal name "10056a") of the present invention.
[0043] Sequence ID 16 corresponds to the artificial amino acid sequence of the alcohol acyltransferase (internal name "1461a") of the present invention.
[0044] The cloning and functional characterization of these enzymes are shown in the following examples.
[0045] Such alcohol acyltransferases of the present invention having the indicated minimum percentage of sequence identity have alcohol acyltransferase activity as defined herein and may be, for example, homologs, mutants, derivatives, or peptide mimes of the alcohol acyltransferase (AAT) of the present invention. Methods for identifying, cloning, and functionally characterizing such alcohol acyltransferases of the present invention having the indicated minimum percentage of sequence identity are known in the art and are described elsewhere herein and in the following examples.
[0046] Preferably, the alcohol acyltransferase (AAT) of the present invention has acyltransferase activity as defined herein.
[0047] "Microbial cells" are defined elsewhere in this specification. Preferably, microbial cells are E. coli cells.
[0048] Preferred tertiary monoterpene alcohols include, but are not limited to, linalool, alpha-terpineol, gamma-terpineol, p-cymen-8-ol, p-ment-3-en-1-ol, p-ment-8-en-1-ol, 4-carbomenthol and / or 4-thujanol; see, for example, Marnett et al. 2014, https: / / onlinelibrary.wiley.com / doi / full / 10.1111 / 1750-3841.12407.
[0049] Preferably, the tertiary monoterpene alcohol includes linalool and alpha-terpineol. In other words, the alcohol acyltransferase of the present invention is advantageous in that it can esterify both linalool and alpha-terpineol compared to alcohol acyltransferases described in the art.
[0050] For example, when linalool is esterified by the alcohol acyltransferase of the present invention, linalyl acetate is produced. When alpha-terpineol is esterified by the alcohol acyltransferase of the present invention, alpha-terpinyl acetate is produced.
[0051] Preferably, the alcohol acyltransferase of the present invention can esterify the tertiary monoterpene alcohol linalool within 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes, or 30 minutes, so that at least 30% by mass of the tertiary monoterpene alcohol linalool is esterified to linalyl acetate.
[0052] Preferably, the alcohol acyltransferase of the present invention can esterify the tertiary monoterpene alcohol alphaterpineol within 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes, or 30 minutes so that at least 30% by mass of the tertiary monoterpene alcohol alphaterpineol is esterified to alphaterpinyl acetate. More preferably, the alcohol acyltransferase of the present invention can esterify the tertiary monoterpene alcohol linalool within 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes, or 30 minutes so that at least 30% by mass of the tertiary monoterpene alcohol linalool is esterified to linalyl acetate, and further can esterify the tertiary monoterpene alcohol alphaterpineol so that at least 30% by mass of the tertiary monoterpene alcohol alphaterpineol is esterified to alphaterpinyl acetate.
[0053] Preferably, the alcohol acyltransferase of the present invention can esterify a tertiary monoterpene alcohol in microbial cells such as bacterial or fungal cells within a specified preferred time, so that at least 30% by mass of the tertiary monoterpene alcohol is esterified. For example, when heterologously expressed in bacteria as shown in the examples, the alcohol acyltransferase of the present invention can esterify a tertiary monoterpene alcohol so that at least 30% by mass of the tertiary monoterpene alcohol is esterified. More preferably, the bacteria are Gram-negative bacteria, and even more preferably, the bacteria are of the genus Escherichia (e.g., E. coli) or the genus Rhodobacter (e.g., Rhodobacter sphaeroides or Rhodobacter capsulatus).
[0054] Preferably, at least 40% by mass, 50% by mass, 60% by mass, 65% by mass, 70% by mass, 75% by mass, 78% by mass, 80% by mass, 90% by mass, or even 100% of the tertiary monoterpene alcohol, such as linalool and / or alpha-terpineol, is esterified by the alcohol acyltransferase of the present invention.
[0055] For determining the content of monoterpene esters produced by the alcohol acyltransferase of the present invention, several useful techniques known in the art may be used, such as gas chromatography-flame ionization detector (GC-FID), gas chromatography-mass selective detector (GC-MSD), high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), high-performance liquid chromatography-differential refractive index detector (HPLC-RID), high-performance liquid chromatography-variable wavelength detector (HPLC-VWD), gel permeation chromatography (GPC), high-performance thin-layer chromatography (HPTLC), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and infrared spectroscopy (IR); see, for example, Jiang et al, Curr Protoc Plant Biol. 2016;1:345-358.doi:10.1002 / cppb.20024. Preferably, GC-FID and GC-MSD are used, along with NMR for further characterization, to determine the content of the monoterpene ester produced by the alcohol acyltransferase of the present invention.
[0056] The alcohol acyltransferase of the present invention may also be capable of esterifying primary and / or secondary monoterpene alcohols in addition to tertiary monoterpene alcohols. Monoterpene alcohols are defined herein as follows: For example, (i) the alcohol acyltransferase of the present invention is capable of esterifying primary, secondary and tertiary monoterpene alcohols, (ii) the alcohol acyltransferase of the present invention is capable of esterifying primary and tertiary monoterpene alcohols, or (iii) the alcohol acyltransferase of the present invention is capable of esterifying secondary and tertiary monoterpene alcohols. Primary alcohols include, for example, geraniol, citronellol, lavandulol, periryl alcohol or thymol; secondary alcohols include, for example, borneol, isoborneol, fencol, verbenol, carveol or menthol.
[0057] Advantageously, the inventors were able to identify a novel alcohol acyltransferase (AAT) isolated from Citrus bergamia that accepts tertiary alcohols, such as those present in the monoterpene alcohol linalool or alpha-terpineol, as substrates. Specifically, when the alcohol acyltransferase of the present invention, as exemplified by mutant AAT9-1-c (SEQ ID NO: 2), is expressed, linalool is converted to linalyl acetate. This is shown in Example 2 and Figure 3. This finding was unpredictable, given that none of the alcohol acyltransferase enzymes described in the prior art are known to use linalool as a substrate, likely because tertiary alcohols are often difficult to access the enzyme active site pocket of alcohol acyltransferases (AATs). To the best of the inventors' knowledge, the only reported exception is an acyltransferase from common thyme (Thymus vulgaris) that, when transiently expressed in Nicotiana benthamiana, exhibited very low catalytic activity for linalool as a side activity (Natalie Wiese's PhD dissertation "Die Biosynthese von acetylierten Monoterpenen in Thymian(Thymus vulgaris)", June 2020; https: / / opendata.uni-halle.de / handle / 1981185920 / 34248; http: / / dx.doi.org / 10.25673 / 34053). This enzyme will be discussed further below. Since the alcohol acyltransferase of the present invention has been established to be active with linalool, a series of monoterpene alcohols and sesquiterpene alcohols were tested in the presence of host cells expressing the alcohol acyltransferase of the present invention or the empty vector pACYCDUET-1 as a control. Surprisingly, the activity of the alcohol acyltransferase of the present invention was also observed with the tertiary monoterpene alcohol alpha-terpineol.Furthermore, the primary monoterpene alcohol geraniol and the secondary monoterpene alcohol verbenol were accepted as substrates, and their corresponding esters were observed. In contrast, the sesquiterpene alcohols nerolidol, pacholol, (-)-alpha-bisabolol, and cedrol were not converted to their corresponding esters.
[0058] In another study, Larkov et al. (Phytochemistry 69 (2008), p.2565-2571) describe entantioselective monoterpene alcohol acetylation in the genera Origanum, Mentha, and Salvia.
[0059] The alcohol acyltransferase of the present invention, as exemplified by AAT9-1-c (SEQ ID NO: 2), appears to be a monoterpene alcohol-specific acyltransferase: it accepts primary, secondary, and tertiary alcohols present in monoterpenes as substrates, but does not accept sesquiterpene alcohols nerolidol, pacholol, (-)-alpha-bisabolol, and cedrol.
[0060] In one embodiment, the monoterpene alcohol is linalool, geraniol, alphaterpineol, gammaterpineol, lavandulol, fencol, periryl alcohol, menthol, or verbenol, preferably linalool, alphaterpineol, or periryl alcohol, more preferably linalool or alphaterpineol.
[0061] Advantageously, the alcohol acyltransferase of the present invention is capable of esterifying tertiary monoterpene alcohols, such as linalool and alpha-terpineol, in economically significant quantities, and thus enabling the production of monoterpene esters, such as linalyl acetate and alpha-terpinyl acetate, on an industrial scale.
[0062] The present invention relates to an alcohol acyltransferase capable of producing at least 50 μg, preferably at least 100 μg, more preferably at least 500 μg, even more preferably at least 1000 μg, even more preferably at least 1500 μg, and most preferably at least 2000 μg of monoterpene esters, preferably tertiary monoterpene alcohols, at 30°C and a pH in the range of 6.0 to 8.5, preferably around pH 7.0, under conditions where the substrate is not limited, and at 30°C and a pH in the range of 6.0 to 8.5, preferably at a pH of around 7.0. The substrates are acyl-CoA and monoterpene alcohols, preferably tertiary monoterpene alcohols, as defined herein.
[0063] The alcohol acyltransferase of the present invention has a Km value for linalool of at least 60 μM, preferably at least 100 μM.
[0064] More advantageously, the alcohol acyltransferase of the present invention is capable of esterifying linalool and / or alphaterpineol more preferably than geraniol, meaning that monoterpene alcohols other than geraniol, and preferably tertiary monoterpene alcohols, are preferred substrates for the enzyme of the present invention. The enzyme of the present invention preferably has a higher affinity for linalool and / or alphaterpineol than for geraniol.
[0065] Therefore, the present invention further relates to an alcohol acyltransferase having a specific activity for at least one tertiary monoterpene alcohol, preferably linalool and / or alpha-terpineol, which is at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times higher than the specific activity of the same enzyme for geraniol. Preferably, the specific activity of the enzyme of the present invention for linalool is at least 0.01 μmol, 0.05 μmol, 0.1 μmol, 0.5 μmol, 1.0 μmol, 2.0 μmol, 3.0 μmol, 4.0 μmol, 5.0 μmol, 10 μmol, 50 μmol, or 100 μmol / nanogram enzyme and / hour.
[0066] This is in comparison to alcohol acyltransferases described in the art, in which, when transiently expressed in Nicotiana benthamiana, it is either completely unable to esterify tertiary monoterpene alcohols due to steric hindrance, or it shows only minimal activity in the tertiary monoterpene alcohol linalool as a BAHD acyltransferase named TvAAT3 from common thyme (Thymus vulgaris) (Natalie Wiese's doctoral dissertation "Die Biosynthese von acetylierten Monoterpenen in Thymian (Thymus vulgaris)", June) 2020; https: / / opendata.uni-halle.de / handle / 1981185920 / 34248). The authors themselves mention the "detectable but very low catalytic activity" of the BAHD acyltransferase TvAAT3 for linalool in this particular transient expression system. Indeed, the conversion of linalool was very low, with less than 10% of linalool being converted. The authors found no conversion of linalool at all when they expressed TvAAT3 in E. coli, so they investigated Nicotiana benthamiana. It is assumed that further enzymatic action is required to produce the estimated linalyl acetate in benthamiana; see page 73, paragraph 2 of the doctoral dissertation mentioned. Furthermore, this very low catalytic activity in linalool in this specific transient expression system has only been confirmed by GC-MS (mass and retention index) and the presence of linalyl acetate has not been confirmed by formal structural assignment. As shown above, BAHD acyltransferase TvAAT3 did not show activity in linalool when expressed in E. coli (section 3.4.2, page 51, last sentence of Natalie Wiese's doctoral dissertation), which is contrary to the alcohol acyltransferase of the present invention; see Example 2.Furthermore, alpha-terpineol could not be esterified by the BAHD acyltransferase TvAAT3 in either E. coli (section 3.4.2, page 51, last sentence of Natalie Wiese's doctoral dissertation) or Nicotiana (section 3.4.3, page 53, last paragraph of Natalie Wiese's doctoral dissertation). The amino acid sequence identity between TvAAT3 (Natalie Wiese, loc.cit's doctoral dissertation) and AAT9-1-c (sequence number 2) of this application is 37.2%.
[0067] In contrast to the reported BAHD acyltransferase TvAAT3, the novel alcohol acyltransferase of the present invention exhibits higher affinity, activity, and improved turnover rate for tertiary monoterpene alcohols in vitro and in vivo, and generates monoterpene esters in host cells or non-human organisms suitable for large-scale industrial production, including but not limited to E. coli and Rhodobacter sphaeroides.
[0068] Preferably, the alcohol acyltransferase (AAT) of the present invention has improved alcohol acyltransferase activity with respect to monoterpene alcohols, preferably tertiary monoterpene alcohols, compared with alcohol acyltransferases (AATs) described in the art.
[0069] Preferably, the alcohol acyltransferase activity with respect to monoterpene alcohols, preferably tertiary monoterpene alcohols, is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or even more than 10 times higher with respect to monoterpene alcohols, preferably tertiary monoterpene alcohols, under the same test conditions.
[0070] More preferably, the alcohol acyltransferase of the present invention has a turnover rate for tertiary monoterpene alcohols that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or even more than 10 times higher than the turnover rate of tertiary monoterpenes by known alcohol acyltransferases under the same test conditions.
[0071] Preferably, the turnover rate is the enzyme turnover rate, or in vivo turnover rate, meaning that kcat is estimated in vivo; see, for example, Heckmann et al., PNAS September 15, 2020 117(37)23182-23190; https: / / doi.org / 10.1073 / pnas.2001562117.
[0072] Preferably, the tertiary monoterpene alcohol is linalool or alphaterpeniol or linalool and alphaterpineol.
[0073] Preferably, the alcohol acyltransferase (AAT) known in the art is the BAHD acyltransferase from common thyme (Thymus vulgaris) named TvAAT3 (Natalie Wiese's PhD dissertation "Die Biosynthese von acetylierten Monoterpenen in Thymian (Thymus vulgaris)", June 2020; https: / / opendata.uni-halle.de / handle / 1981185920 / 34248), and the alcohol acyltransferase activity compared is that for the tertiary monoterpene alcohol linalool under identical test conditions.
[0074] The novel alcohol acyltransferases of the present invention are particularly useful for the production of monoterpene esters, as shown in the examples, especially when the monoterpene ester is derived from a tertiary monoterpene alcohol. For example, they can be used for the production of linalyl acetate or other monoterpene esters as indicated herein.
[0075] BLASTP analysis of the mutant AAT9-1-c sequence (SEQ ID NO: 2) against the SWISSPROT database of characterized proteins revealed that the most closely related characterized protein is a member of the vinolin synthase family; see Example 4. It was not anticipated that this novel alcohol acyltransferase enzyme of the present invention, capable of esterifying tertiary monoterpene alcohols such as linalool, would be most closely related to the family of alcohol acyltransferases (AATs) that act on highly complex phenolic substances, such as saltalidinol. The inventors determined that, in the genus Lavandula, as in bergamot, a member of the vinolin synthase family is a candidate for linalool acetyltransferase. In fact, it is possible to identify alcohol acyltransferase homologs in Lavandula angustifolia, whose amino acid sequences are shown in SEQ ID NO: 3 (Lavandula AAT-10056) and SEQ ID NO: 4 (Lavandula AAT-1461); see Example 4.
[0076] Preferably, the alcohol acyltransferase of the present invention prefers monoterpene alcohols to sesquiterpene alcohols as substrates. For example, the activity of the alcohol acyltransferase of the present invention was found with the monoterpene alcohols geraniol, alpha-terpineol, and verbenol, and the corresponding esters were observed. In contrast, the sesquiterpene alcohols nerolidol, pacholol, (-)-alpha-bisabolol, and cedrol were not observed to be converted to their corresponding esters by the enzyme of the present invention. The alcohol acyltransferase of the present invention is assumed to exhibit activity with other sesquiterpene alcohols, but still prefers monoterpene alcohols to sesquiterpene alcohols as substrates. Similarly, candidates include, for example, nerolidol, pacholol, bisabolol, germacrene D-ol, hedicariol, santalol, and cubebor.
[0077] The alcohol acyltransferase of the present invention can be prepared by chemical synthesis or recombinant molecular biological techniques well known to those skilled in the art, as shown in Example 1. This can be applied with modifications to isolate the alcohol acyltransferase of the present invention from host cells or supernatant; see, for example, Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W.--. 3rd ed.--New York: Cold Spring Harbor Laboratory, 2001; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).
[0078] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural references unless the context explicitly indicates otherwise. For example, “a primary monoterpene alcohol” refers to one or more primary monoterpene alcohols, “a secondary monoterpene alcohol” refers to one or more secondary monoterpene alcohols, “a tertiary monoterpene alcohol” refers to one or more tertiary monoterpene alcohols, or “a cell” refers to one or more cells.
[0079] As used herein, the term “about” means, when quantifying the value of an item, number, percentage, or term mentioned, a range of plus or minus 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the value of the item, number, percentage, or term mentioned. A range of plus or minus 10 percent is preferred.
[0080] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are inclusive or unrestricted, and do not exclude additional, unlisted components, elements, or steps of method. Clearly, the term “comprising” encompasses the term “consisting of.” More specifically, as used herein, the term “comprise” means that a claim encompasses all listed steps of elements or methods, but may also include further unspecified steps of elements or methods. For example, a method comprising steps a), b), and c) encompasses, in the narrowest sense, a method comprising steps a), b), and c). The phrase “consisting of” means that the composition (or apparatus or method) has the elements (or steps) cited, and nothing else. In contrast, the term "comprises" may also encompass methods that include further stages, such as stages d) and e) in addition to stages a), b) and c).
[0081] For the purposes of this specification, the numerical range is used as "concentrations between 1 and 5 micromolar concentrations," and this range includes not only concentrations of 1 and 5 micromolars, but also any values between 1 and 5 micromolar concentrations, such as 2, 3, and 4 micromolar concentrations.
[0082] The term "in vitro," as used herein, refers to the outside or external of an animal or human body. The term "in vitro," as used herein, should be understood to include "ex vivo." The term "ex vivo" generally refers to tissue or cells that have been removed from an animal or human body and are maintained or grown outside the body, for example, in a culture vessel. The term "in vivo," as used herein, refers to the inside or internal of an animal or human body.
[0083] The term "alcohol acyltransferase" (AAT), as used herein, means alcohol O-acetyltransferase. Alcohol O-acetyltransferase catalyzes the reaction acetyl-CoA + alcohol = CoA + acetyl ester. That is, the enzyme catalyzes the esterification step that transfers acyl-CoA to an alcohol (see, e.g., EC2.3.1.84). Therefore, the term "alcohol acyltransferase activity," as used herein, means catalyzing the esterification step that transfers acyl-CoA to an alcohol group. Alcohol acyltransferases (AATs) can combine different alcohols and acyl-CoA groups, resulting in the synthesis of a wide range of esters, including monoterpene esters, released from fruits. Alcohol acyltransferases play a crucial role in ester biosynthesis. The diversity of volatile esters arises from the diversity of genes encoding alcohol acyltransferases with different specific substrate selectivity (Lucchetta et al., (2007) J Agric Food Chem 55, 5213-5220; D'Auria JC (2006), Curr Opin Plant Biol 9, 331-340).
[0084] As is well known to those skilled in the art, enzymes bind to their substrates and convert them into products. A plot of the initial reaction rate against substrate concentration shows a rectangular hyperbola. The reaction rate (v) is equal to (Vmax[A]) / (Km+[A]), as stated by the Michaelis-Menten equation, where Vmax is the maximum rate, [A] is the substrate concentration, and Km is the Michaelis constant or the substrate concentration at the semi-maximal rate. Steady-state enzyme kinetics are used to determine the Km value for the substrate, the Vmax value for the enzyme, and the Ki value for various inhibitors, including drugs.
[0085] The "turnover rate" (kcat or catalytic rate constant) of an enzyme is the maximum number of substrate molecules / active sites / unit time required to convert several different substrates into different products. The kcat / Km values or specificity constants of various substrates can be compared. The substrate with the highest value is the best substrate for the enzyme, explaining the name of the specificity constant. The rate of any reaction is limited by the rate at which reactant molecules collide. The diffusion limiting rate for biomolecular reactions is 10⁻⁶. 8 ~10 9 M -1 s -1 The ratio kcat / Km is the first-order rate constant. The product of kcat / Km and the substrate concentration (at the subsaturated level) gives the rate of the enzyme-catalyzed reaction. This ratio is proportional to the substrate concentration and is therefore specified as first-order. 10 8 ~10 9 M -1 s -1 Enzymes with a kcat / Km ratio near (close to the maximum value possible by diffusion rate) achieve complete catalysis. For example, triose phosphate isomerase (EC 5.3.1.1), an enzyme in the glycolysis pathway, is an enzyme with this property. However, most enzymes have specificity constants of an order below this value. Methods for determining the turnover rate of enzymes are well known in the art; see, for example, https: / / doi.org / 10.1016 / B978-0-12-801238-3.05143-6 or Heckmann et al., PNAS September 15, 2020 117(37)23182-23190; https: / / doi.org / 10.1073 / pnas.2001562117. As used herein, the term "linalool acetyltransferase" means the alcohol O-acetyltransferase that catalyzes the reaction acetyl-CoA + linalool = CoA + linalyl acetate.
[0086] Tests for determining the activity of alcohol acyltransferases as defined herein are well known in the literature (e.g., Chacon, MG, et al. Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli. Microb Cell Fact 18, 105 (2019); https: / / doi.org / 10.1186 / s12934-019-1130-0; International Publication No. 2019 / 092388; Aharoni et al., Identification of the SAAT gene. Plant Cell. 2000 May; 12(5):647-62. doi:10.1105 / tpc.12.5.647.; Beekwilder et al., (2004) Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol. 2004). 135(4):1865-78.doi:10.1104 / pp.104.042580;(Shalit et al.,Volatile ester formation in roses.Identification of an acetyl-coenzyme A.Geraniol / Citronellol acetyltransferase in developing rose petals.Plant Physiol.2003 Apr;131(4):1868-76.doi:10.1104 / pp.102.018572;Sarker & Mahmoud (2015) Cloning and functional characterization of two monoterpene acetyltransferases from glandular trichomes of Lx intermedia.Planta 242,709-719).A suitable test for determining the activity of the alcohol acyltransferase of the present invention is also shown in Example 2.
[0087] By definition, the term "terpene" includes only hydrocarbons composed of carbon and hydrogen. In contrast, the term "terpenoid" refers to terpenes containing further functional groups that give rise to derivatives such as alcohols, aldehydes, ketones, and acids; see, for example, Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability RG Berger; Black et al., Terpenoids and their role in wine flavor: recent advances. Australian Journal of Grape and Wine Research 21, 582-600, 2015; Zhou & Pichersky, More is better: the diversity of terpene metabolism in plants. Current Opinion in Plant Biology 2020, 55:1-10; Degenhardt J, Kollner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70(15):1621-1637). In scientific literature, the term terpene is often used interchangeably with the term terpenoid, although they have different meanings. As used herein, the term "terpene" includes both hydrocarbons and their functionalized derivatives.
[0088] When used herein, "monoterpenes" are the main components of secondary metabolites and essential oils in plants; see, for example, DH Grayson. Monoterpenoids. Nat Prod Rep 1996 Jun;13(3):195-225. doi:10.1039 / np9961300195. Thus, they contribute to the distinctive odor characteristics of plants. Monoterpenes are characterized by their lipophilicity, low molecular weight, and volatility. In plant tissues, they are mainly stored in oil glands, glandular hairs, and leaf trichomes. As mentioned elsewhere herein, C10 geranyl diphosphate (GPP) is a direct precursor in the formation of monoterpenes, involving a series of sequential reactions including hydrolysis, cyclization, and redox reactions.
[0089] Monoterpenes are classified into two main types: acyclic (or linear) and cyclic, which can be monocyclic or bicyclic. Acyclic monoterpenes, such as cis-alpha-ocimene and beta-myrcene, are 2,6-dimethyloctane derivatives. Typical monocyclic monoterpenes, such as limonene and cymene, are mainly cyclohexane derivatives with isopropyl substituents, typically containing a variable double bond moiety. Alpha-pinene and beta-pinene, on the other hand, are common types of bicyclic monoterpenes.
[0090] Oxidation or rearrangement of monoterpene structures is a biochemical modification that produces so-called monoterpenoids. These can retain further functional groups that lead to substances with aldehyde, ketone, ester, or alcohol-based structures.
[0091] Like most secondary metabolites, monoterpenes are crucial to plant evolution. In particular, they play an ecological role in plants in their defense against herbivores, bacteria, and fungi through the release of monoterpenes, which in turn attracts natural enemies of these herbivores. Furthermore, they mediate interactions between plants and their environment when acting as pollinator attractants or allelothermants.
[0092] In aromatic plants, isoprene and especially monoterpenes can provide heat tolerance to those plants. Therefore, plants that secrete these terpenes may have better tolerance to short periods of high temperatures in sunlight than non-terpene-releasing plants. As a result, these releasing plants maintain a higher photosynthetic rate compared to non-releasing plants. Volatile monoterpenes can further protect plants from oxidative damage due to their ability to react with oxidizing agents from the atmosphere. Monoterpenes are also found in oily resins. Together with diterpene resin acids and sesquiterpenes, they act as chemical and physical defense compounds. This complex mixture synthesized by plants repels insects through poisoning and quenching. Furthermore, they heal plant damage by forming a temporary, robust layer.
[0093] Monoterpenes are used industrially as ingredients in flavorings, fragrances, and cosmetics. Fragrances and aromatics are used as important additives to enhance the final quality of food and beverages, as well as body care and other hygiene products. However, recently, there has been a growing demand for naturally derived products. Consequently, the value of natural flavor compounds, which can enhance the sensory appeal of these products, has increased, making them more expensive than their artificial equivalents. Plant-derived essential oils and their monoterpene components are the primary sources of flavorings and fragrances used industrially. One of the first known plants used in cosmetics is the peppermint plant, or Mentha piperita, which contains menthol as a major component of its essential oil. This is primarily applied for its anti-stress, tension-relieving, and cooling effects. Menthol, one of the most well-known monocyclic monoterpenes, is used in mouthwashes, soaps, toothpastes, and other cleansing formulations. Linalool, another well-known acyclic monoterpene alcohol, is a commonly used ingredient, particularly added to perfumes and household cleaning products. Due to its refreshing, sweet citrus scent, it is used as a flavor enhancer in processed foods and beverages. Citral, an acyclic monoterpene aldehyde, is the primary odorant in lemon oil. Due to its citrusy and lemony scent, strongly associated with subjective impressions of freshness and cleanliness, citral is also widely used in household products. Alpha- and beta-pinenes, two bicyclic monoterpenes found in turpentine, are fragrance substances used to enhance the scent of industrial products. Furthermore, they are precursors to several flavor compounds, including citronellol, geraniol, menthol, and verbenol.
[0094] Due to their aromatic properties, essential oils are primarily used as food flavorings, cosmetics, and fragrances. However, research has revealed the high potential of volatile monoterpene components for treating and preventing human diseases. Natural monoterpenes and their synthetic derivatives have been repeatedly described for their various pharmacological effects: studies have shown that monoterpenes possess antibacterial, anti-inflammatory, antispasmodic, analgesic, and anesthetic effects. Furthermore, in recent years, several monoterpenes have been shown to be potential chemotherapeutic agents. Among them, the monocyclic monoterpene limonene has been found to inhibit the development of several types of cancer, including liver, skin, and lung cancer. Geraniol, a monoterpene alcohol found in lemongrass, has been shown to inhibit the proliferation of hepatocellular carcinoma, leukemia, and colon cancer cells. Monoterpenes have also been tested in the prevention and treatment of cardiovascular diseases due to their vasodilatory and blood pressure-lowering effects. Carvacrol, limonene, citronellol, myrtenol, and linalool are among the monoterpenes that exhibit in vivo and in vitro cardiovascular effects in both humans and animals. Separately, other monoterpenoids such as thymol and terpineol have shown potent antioxidant and free radical scavenging activity. Monoterpenes are anti-inflammatory and have also been shown to have beneficial effects on several respiratory disorders. In particular, 1,8-cineole and menthol are among the most effective compounds for alleviating bronchopulmonary injury. Recent studies have shown that both linalool and linalyl acetate, as anti-inflammatory compounds, were able to reduce edema in a rat model of carrageenan-induced limb edema after systemic administration.
[0095] As mentioned elsewhere in this specification, monoterpenes are effective antimicrobial agents. Specifically, some monoterpenoids, such as thymol or menthol, are effective against some Gram-positive bacteria, such as Staphylococcus aureus, and Gram-negative bacteria, such as Escherichia coli. Other monoterpenes, namely geraniol and citral, exhibit antifungal activity against a human fungal pathogen called Cryptococcus neoformans, which typically infects the lungs or central nervous system. Furthermore, citral is not only an antifungal agent but can also reduce the infectivity of herpes simplex virus (HSV), and therefore exhibits significant anti-HSV activity.
[0096] Where used herein, “monoterpene alcohol” means a monoterpene (C10) containing an alcohol group as a functional group. Monoterpene alcohols are described in detail in the Art; see, for example, DH Grayson. Monoterpenoids. Nat Prod Rep 1996 Jun;13(3):195-225. doi:10.1039 / np9961300195. By definition, a primary alcohol is an alcohol in which a hydroxyl group is bonded to a primary carbon atom. This can also be defined as a molecule containing a “-CH2OH” group. In contrast, a secondary alcohol has the formula “-CHROH”, and a tertiary alcohol has the formula “-CR2OH” (where “R” indicates a carbon-containing group).
[0097] Primary alcohols present in monoterpenes are referred to herein as “primary monoterpene alcohols”: the carbon to which the acceptor alcohol group is linked is bonded to a single carbon group. Examples of primary alcohols present in monoterpenes include geraniol, citronellol, and lavandulol.
[0098] Secondary alcohols present in monoterpenes are referred to herein as “secondary monoterpene alcohols”: the carbon to which the acceptor alcohol group is attached is bonded to two carbon groups. Examples of secondary alcohols present in monoterpenes include borneol, isoborneol, fencol, verbenol, carveol, and menthol.
[0099] Tertiary alcohols present in monoterpenes are referred to herein as “tertiary monoterpene alcohols”: the carbon to which the acceptor alcohol group is linked is bonded to three carbon groups. Examples of tertiary alcohols present in monoterpenes include S-linalool, R-linalool, alpha-terpineol, gamma-terpineol, fencol, p-cymen-8-ol, p-ment-3-en-1-ol, p-ment-8-en-1-ol, 4-carbomenthol, and 4-thujanol; see, for example, doi:10.1111 / 1750-3841.12407.
[0100] Acyclic monoterpene alcohols or monoterpenols, occasionally mentioned in the literature, are 2,6-dimethyloctane derivatives containing variable double bond moieties and hydroxyl functional groups. The most important substances in this class are linalool, geraniol, nerol, citronellol, myrcenol, and dihydromyrcenol. These have been used in fragrances since ancient times due to their pleasant olfactory properties.
[0101] By definition, an ester is a chemical compound derived from an acid (organic or inorganic) in which at least one -OH (hydroxyl) group is replaced by an -O-alkyl (alkoxy) group.
[0102] As used herein, “monoterpene ester” means an ester derived from a monoterpene alcohol. This term includes esters derived from primary, secondary, or tertiary monoterpene alcohols, as defined herein. Preferably, as used herein, the term “monoterpene ester” means “monoterpene acetyl ester.” Unless otherwise indicated, these terms are used interchangeably herein.
[0103] As used herein, "sesquiterpene alcohol" means a sesquiterpene (C15) containing an alcohol group as a functional group. Sesquiterpene alcohols are well known in the art; see, for example, Fraga 2012, Natural sesquiterpenoids. Nat. Prod. Rep., 2013, 30, 1226.
[0104] The definitions of primary, secondary, and tertiary alcohols in monoterpenes are applied to sesquiterpene alcohols, with modifications where necessary.
[0105] As used herein, "sesquiterpene ester" means an ester derived from a sesquiterpene alcohol. This term means an ester derived from a primary, secondary, or tertiary sesquiterpene alcohol, as defined herein.
[0106] The terms “protein,” “polypeptide,” “(poly)peptide,” or “peptide” (all terms are used interchangeably unless otherwise indicated) as used herein include isolated and / or purified and / or recombinant (poly)peptides that are essentially free from other host cell polypeptides. When the term "peptide" is used herein, it comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or more amino acid residues, wherein the alpha-carboxyl group of one amino acid residue is bonded to the alpha-amino group of another amino acid residue. Post-translational modifications of proteins or peptides as used and envisioned herein are modifications of newly formed proteins or peptides and may include deletion, substitution or addition of certain amino acids, chemical modifications of certain amino acids, such as amidation, acetylation, phosphorylation, glycosylation, pyroglutamic acid formation, oxidation / reduction of a sulfa group in methionine, or similar addition of small molecules.
[0107] "Homologous" means a bacterial, fungal, plant, or animal homolog of the alcohol acyltransferase of the present invention, preferably a plant homolog, but also includes shortened sequences of coding and non-coding DNA sequences, single-stranded DNA, or RNA.
[0108] As will be shown in Example 4, the inventors can identify an alcohol acyltransferase homolog in Lavandula angustifolia, the amino acid sequences of which are shown in SEQ ID NO: 3 (Lavandula AAT-10056) and SEQ ID NO: 4 (Lavandula AAT-1461).
[0109] Sequence identity, homology, or similarity is defined herein as the relationship between two or more amino acid sequences or two or more nucleic acid sequences as determined by comparing those sequences. Typically, sequence identity or similarity is compared over the entire length of the sequences, but it may also be compared only to a portion of the sequence alignment with one another. Preferably, as described herein, sequence identity or similarity is compared over the entire length of the sequences. In the art, “identity” or “similarity” may also mean, in some cases, the degree of sequence relationship between polypeptide sequences or nucleic acid sequences as determined by such sequence agreements.
[0110] Sequence alignment can be generated using several software tools, including the following: -Needleman and Wunsch Algorithm -Needleman, Saul B. & Wunsch, Christian D. (1970). “A general method applicable to the search for similarities in the amino acid sequence of two proteins”. Journal of Molecular Biology 48(3):443-453.
[0111] This algorithm is incorporated, for example, into the "NEEDLE" program, which performs exhaustive alignment of two sequences. The NEEDLE program is included, for example, in the European Molecular Biology Open Software Suite (EMBOSS). -EMBOSS- A suite of various programs: The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16(6), 276(2000). -BLOSUM (BLOCK'S SUbstitution Matrix)- is generally generated based on the alignment of conserved regions of protein domains, for example (Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the USA. 1992 Nov 15;89(22):10915-9). One of the many BLOSUMs is "BLOSUM62," which is often the "default" setting for many programs when aligning protein sequences. -BLAST (Basic Local Alignment Search Tool)- Consists of several individual programs (BlastP, BlastN) primarily used to search for similar sequences in large sequence databases. The BLAST program also creates local alignments. An improved version ("BLAST2"), the "BLAST" interface provided by NCBI (National Center for Biotechnology Information), is usually used. “Original” BLAST: Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) “Basic local alignment search tool.” J.Mol.Biol.215:403-410;BLAST2: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.
[0112] Sequence identity is the value determined, where used herein, preferably by the EMBOSS pairwise alignment algorithm "Needle". In particular, the NEEDLE program from the EMBOSS package can be used with the NOBRIEF option ('Brief identity and similarity' to NO) to calculate the "longest identity" (version 2.8.0 and later, EMBOSS: The European Molecular Biology Open Software Suite - Rice, P., et al. Trends in Genetics (2000) 16:276-277; http: / / emboss.bioinformatics.nl). Identity, homology, or similarity between two aligned sequences is calculated as follows: the number of corresponding positions in the alignment that show the same amino acid in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. For amino acid sequence alignment, the default parameters are matrix = Blosum62; open gap penalty = 10.0; gap elongation penalty = 0.5. For nucleic acid sequence alignment, the default parameters are matrix=DNAfull; open gap penalty=10.0; gap elongation penalty=0.5.
[0113] For example, enzyme variants may be defined by their sequence identity when compared to a parent enzyme, such as the alcohol acyltransferase of the present invention, with the amino acid sequence shown in SEQ ID NOs: 2, 15, or 16. Sequence identity is typically provided as "% sequence identity" or "% identity". In the first step, to determine the percentage identity between two amino acid sequences, a pairwise sequence alignment is created between these two sequences, and these two sequences are aligned over their entire, whole, or full length (i.e., a pairwise comprehensive alignment). The alignment is generated using a program or software described herein. A preferred alignment for the purposes of the present invention is one in which maximum sequence identity can be determined.
[0114] Any discrepancies between the alcohol acyltransferase following an amino acid sequence or nucleic acid specific to the present invention and the functional homologue of the enzyme or nucleic acid may be the result of modifications made to improve the properties of the enzyme or nucleic acid (e.g., increased enzyme expression or increased enzyme activity) by biological techniques known to those skilled in the art, such as molecular evolution or rational design, or by mutagenesis techniques known in the art and described elsewhere in this specification (e.g., random mutagenesis, site-directed mutagenesis, directional evolution, genetic engineering).
[0115] The sequences of enzymes or nucleic acids may be altered as a result of one or more natural variations. Examples of such natural modifications or variations include differences in glycosylation (more broadly defined as “post-translational modifications”), differences due to alternative splicing, and single nucleic acid polymorphisms (SNPs). Nucleic acids may be modified to encode a polypeptide in which at least one amino acid or two, three, four, five, six or more amino acids differ, so that this polypeptide encodes a polypeptide containing one or more amino acid substitutions, deletions, and / or insertions, and this polypeptide still possesses alcohol acyltransferase activity as defined herein. Furthermore, artificial gene synthesis (synthetic DNA), codon optimization, or codon-pair optimization may be used, for example, based on methods described in International Publication No. 2008 / 000632 or provided by commercial DNA synthesis companies such as DNA2.0, Geneart, and GenScript.
[0116] Enzyme sequences or nucleic acid sequences can be modified by gene editing. Gene editing or genome editing can be done by using various techniques such as “gene shuffling” or “directional evolution,” which consists of repeated DNA shuffling and subsequent appropriate screening and / or selection to generate variants of nucleic acids or parts thereof that encode biologically modified proteins (Castle et al., (2004) Science 304(5674):1151-4; U.S. Patent Nos. 5,811,238 and 6,395,547), or by adding “T-DNA activation” tags (Hayashi et al. Science (1992) 1350-1353) (the resulting transgenic organisms exhibit a dominant phenotype due to the modification of gene expression close to the introduced promoter), or by “TILLING” (Targeted Induced Local Lessons In Tilling refers to a type of genetic modification that can be obtained through Genomes, and is a mutagenesis technique useful for creating and / or identifying nucleic acids that encode proteins with altered expression and / or activity. Tilling also allows for the selection of organisms that possess such mutant variants. Methods for tilling are well known in the art (referred to as McCallum et al., (2000) Nat Biotechnol 18:455-457; Stemple (2004) Nat Rev Genet 5(2):145-50). Another technique uses zinc finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems, and modified meganucleases, such as artificially modified nucleases like re-modified homing endonucleases (Esvelt, KM.; Wang, HH. (2013), Mol Syst Biol 9(1):641; Tan, WS. et al. (2012), Adv Genet 80:37-97; Puchta, H.; Fauser, F. (2013), Int. J. Dev. Biol 57:629-637).
[0117] The alcohol acyltransferase derivatives of the present invention include functional, i.e., enzymatically active, mutants that can be obtained by deletion, insertion, or substitution of amino acid residues from / to an amino acid sequence. The modification or mutation may be a substitution, deletion, or insertion of an amino acid residue by a different one. For example, an amino acid residue involved in substrate binding may be modified or mutated. The modified or mutated amino acid sequence preferably exhibits improved, e.g., increased, alcohol acyltransferase activity compared to the wild-type amino acid sequence of SEQ ID NO: 2 or the artificial amino acid sequence of SEQ ID NO: 15 or 16. To provide another example, the modified or mutated amino acid sequence may be an alcohol acyltransferase with altered substrate specificity, for example, that it preferably esterifies specific isomers of monoterpene alcohols, or the alcohol acyltransferase may be capable of esterifying specific sesquiterpene alcohols in addition to esterifying primary, secondary, and / or tertiary monoterpene alcohols. To provide further examples, the modified or mutated amino acid sequence may be an alcohol acyltransferase capable of esterifying other non-terpene compounds, such as phenolic compounds, or producing nor compounds of terpenes, through the acetylation of alcohols, such as coniferyl or 2-phenylethanol.
[0118] DNA and the proteins they encode can be modified using various techniques known in molecular biology for producing mutant proteins or enzymes with new properties or altered properties (see, for example, Sambrook;Ausubel, cited elsewhere in this specification).
[0119] Random PCR mutagenesis is described, for example, in Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471, and combinatorial multiple cassette mutagenesis is described, for example, in Crameri (1995) Biotechniques 18:194-196.
[0120] Alternatively, nucleic acids, such as genes, may be reassembled after random or "stochastic" fragmentation; see, for example, U.S. Patent Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; and 5,605,793.
[0121] Alternatively, modifications, additions, or deletions may be introduced by error-prone PCR, shuffling, site-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis (phage-assisted continuous evolution, in vivo continuous evolution), cassette mutagenesis, recurrent ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, gene reassembly, site-saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recurrent sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch-modified mutagenesis, modified-deletion host-strain mutagenesis, chemical mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer production, and / or combinations thereof and other methods.
[0122] Alternatively, “Site-Saturated Mutagenesis” or “GSSM” includes methods using degenerate oligonucleotide primers to introduce point mutations into polynucleotides, as detailed in U.S. Patent Nos. 6,171,820 and 6,764,835.
[0123] Alternatively, synthetic ligation reassembly (SLR) includes methods for non-stochastically linking oligonucleotide components together, such as disclosed in U.S. Patent No. 6,537,776. Or, tailored multi-site combinatorial assembly ("TMSCA") is a method for producing multiple progeny polynucleotides having different combinations of various multi-site mutations by using at least two mutagenic non-overlapping oligonucleotide primers in a single reaction. Such methods are described, for example, in International Publication No. 2009 / 018449.
[0124] The terms "protein," "polypeptide," or "peptide," as used herein, encompass the peptide mimetics of the alcohol acyltransferase of the present invention. As is known in the art, peptide mimetics are compounds in which essential elements (pharmacophores) mimic natural peptides or proteins in 3D space, retaining the ability to interact with biological targets (such as enzyme substrates) and producing the same biological effect (e.g., alcohol acyltransferase activity). See, for example, the overview by Vagner et al. 2008, Current Opinion in Chemical Biology 12, Pages 292-296. Peptide mimetics are designed to avoid some of the problems associated with natural polypeptides, such as stability against proteolysis (duration of biological activity) and poor bioavailability. Often, certain other properties, such as selectivity for the biological targets described above or the potency of biological activity, such as the biological activity described above, can be substantially improved.
[0125] Preferably, the homologs, mutants, derivatives, or peptide mimes of the alcohol acyltransferase (AAT) of the present invention have at least 50%, 60%, 70%, 80%, 90%, or even 100% of the alcohol acyltransferase activity of the unmodified or unmutated alcohol acyltransferase amino acid sequence of SEQ ID NOs. 2, 15, or 16. To provide an example, the alcohol acyltransferase of the present invention is capable of esterifying a tertiary monoterpene alcohol (e.g., linalool) so that at least 30% by mass of the tertiary monoterpene alcohol is esterified. The homologs, mutants, derivatives, or peptide mimes of the alcohol acyltransferase (AAT) of the present invention have 50% of the alcohol acyltransferase activity of the alcohol acyltransferase of the present invention if they are capable of esterifying the tertiary monoterpene alcohol so that at least 15% by mass of the tertiary monoterpene alcohol is esterified. The homologous, mutant, derivative, or peptide mimetic preferably also maintains the desired substrate specificity and / or substrate preference of the alcohol acyltransferase of SEQ ID NOs: 2, 15, or 16. For example, the homologous, mutant, derivative, or peptide mimetic may esterify primary, secondary, and / or tertiary monoterpene alcohols, preferably tertiary monoterpene alcohols, as defined elsewhere herein.
[0126] To understand the molecular mechanisms involved in ester biosynthesis by the alcohol acyltransferase of the present invention, and to clarify the importance of specific amino acid residues, a structural model of the alcohol acyltransferase can be constructed by comparative modeling. Subsequently, the conformational interactions between the protein and several ligands, alcohol, and acyl-CoA can be explored by molecular docking and molecular dynamics simulations. Such methods are known and described in the art (see, for example, Galaz et al., FEBS Journal 280 (2013) 1344-1357).
[0127] In a preferred embodiment, the alcohol acyltransferase of the present invention is contained in the form of a fusion protein.
[0128] The alcohol acyltransferase of the present invention may also be a fusion protein. As used herein, the term "fusion protein" refers to a chimeric protein (literally, made from parts of different origins) created through the linkage of two or more genes that originally encode separate proteins. Translation of this fusion gene results in a single or multiple polypeptide having functional properties derived from each of the original proteins. For example, a fusion protein as defined herein may include affinity tags for protein purification (His tags, FLAG tags, etc., see, e.g., Kimple et al., 2015, Curr Protoc Protein Sci.; 73:Unit-9.9.doi:10.1002 / 0471140864.ps0909s73) or labels for detection. A "label" as referred herein is a detectable compound or composition that, when directly or indirectly compounded with another molecule, such as the alcohol acyltransferase of the present invention, facilitates the detection of that molecule. Specific non-limiting examples of labels include fluorescent tags, enzyme linkers, and radioactive isotopes, which are well known in the art. In one embodiment, a protease cleavage site and / or linker (i.e., protease cleavage site; or linker; or both protease cleavage site and linker; or linker including protease cleavage site) may be present between the alcohol acyltransferase of the present invention and the labeled or purified tag. For example, the protease cleavage site may be used to cleave the purified tag by treatment with a protease such as enterokinase or thrombin, as needed. For example, the His tag may be used as a tag for expression and purification, while the alcohol acyltransferase of the present invention may be isolated after cleavage with a protease. As is well known to those skilled in the art, beyond its fundamental role in linking functional domains together (in the case of mobile and rigid linkers), linkers can bring about many other advantages for the production of fusion proteins, such as improved biological activity, increased expression levels, and achievement of desired pharmacokinetic profiles.The linker may be a protein / peptide linker, such as a polyglycine linker or other linkers known in the art (see, e.g., Chen et al., Adv Drug Deliv Rev. 2013;65(10):1357-1369). Clearly, the linker may be designed to contain a protease cleavage site. In another embodiment, the fusion protein may harbor a signal peptide for targeting the expressed polypeptide, for example, to a specific organelle, as described elsewhere in this specification.
[0129] Fusion proteins as defined herein can be prepared by chemical synthesis or recombinant molecular biology techniques well known to those skilled in the art. These can be applied, with modifications as appropriate, to the isolation of fusion proteins from host cells or supernatants; see, for example, Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W.--. 3rd ed.--New York: Cold Spring Harbor Laboratory, 2001; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).
[0130] The present invention also relates to an antibody that specifically binds to the alcohol acyltransferase of the present invention.
[0131] As used herein, the term “antibody” refers to a molecule produced by the immune system that is made in response to a particular antigen, specifically binds to that antigen, and includes both native and non-native antibodies. As used herein, “antibody” includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies, dimers or multimers, chimeric antibodies, bispecific antibodies, bispecific single-chain antibodies, polyspecific antibodies, synthetic antibodies, bifunctional antibodies, cell-bound antibodies such as Ig receptors, linear antibodies, diabodies, minibodies, or any fragment of such antibodies that specifically bind to the alcohol acyltransferase of the present invention. The fragments of such antibodies include, for example, Fab, Fv, or scFv fragments or chemically modified derivatives of any of these fragments. Antibodies may be prepared by using the methods described in the Art, see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by techniques first described by Kohler 1975, Nature 256,495 and Galfre 1981, Meth. Enzymol. 73,3. These techniques involve fusing mouse myeloma cells with immunized mammalian spleen cells. The surface plasmon resonance (SPR) optical phenomenon used by the Biacore system enables real-time detection and measurement of protein-protein interactions without the use of labeling. For example, applications of Biacore technology can be used to measure protein-protein interactions using antibodies and their antigens, such as alcohol acyltransferases, fragments thereof, or epitopes of the present invention. The affinity of an antibody for its antigen is determined by measuring the binding dynamics of the interaction. Antibodies can be further improved by techniques well known in the art.Surface plasmon resonance, as used in the Biacore system, can also be used to improve the efficiency of phage antibodies that bind to epitopes; see, for example, Schier 1996, Human Antibodies Hybridomas 7,97; Malmborg 1995, J.Immunol.Methods 183,7.
[0132] The present invention further relates to nucleic acids comprising nucleic acid sequences encoding the alcohol acyltransferase of the present invention or a complementary sequence thereof. Preferably, the nucleic acids of the present invention are suitable for encoding and producing the alcohol acyltransferase having the enzymatic activity of the present invention in microorganisms as defined herein.
[0133] The nucleic acid (or polynucleotide) of the present invention comprises a nucleic acid sequence encoding the alcohol acyltransferase of the present invention. The nucleic acid sequence encoding the alcohol acyltransferase of the present invention is preferably a recombinant and / or isolated and / or purified nucleic acid sequence. The nucleic acid sequence encoding the alcohol acyltransferase of the present invention can be prepared and isolated using known molecular biological standard techniques, sequence information and organisms provided herein.
[0134] For example, homologous sequence stretches or homologous conserved sequence regions can be identified at the DNA or amino acid level in the alcohol acyltransferase sequence of the present invention and the sequences of other vinolin synthase family members or vinolin synthase-like sequences (see Example 3) by comparison algorithms and sequence alignment. The identified sequences can then be used as hybridization probes in standard hybridization techniques, such as those described in Sambrook et al. (cited elsewhere in this specification), for cloning the nucleic acid sequence encoding the alcohol acyltransferase of the present invention. Alternatively, the nucleic acid sequence encoding the alcohol acyltransferase of the present invention can be prepared by polymerase chain reaction, which is well known in the art, using appropriate specific primers. The nucleic acid sequence isolated or produced in a suitable vector, such as those described herein, can then be cloned and characterized by DNA sequence analysis. The alcohol acyltransferase activity of the encoded protein can be determined after expression of the nucleic acid sequence in a suitable host cell, as described elsewhere in this specification and in Example 2.
[0135] For example, the nucleic acid sequence of the present invention can be isolated from Citrus bergamia or Lavandula angustifolia.
[0136] Example 1 shows the cloning of a nucleic acid sequence encoding alcohol acyltransferase from Citrus bergamia. Sequence ID 1 corresponds to the nucleotide sequence of alcohol acyltransferase (AAT) (mutant AAT9-1-c) from Citrus bergamia. Furthermore, Example 4 shows the identification of a homolog of alcohol acyltransferase (AAT) from Citrus bergamia in Lavandula angustifolia.
[0137] The term “nucleic acid,” as used herein, includes references to deoxyribonucleotides or ribonucleotide polymers, i.e., polynucleotides, in either single-stranded or double-stranded form, and unless otherwise limited, includes known analogues that possess the fundamental properties of natural nucleotides in that they hybridize with single-stranded nucleic acids, similar to natural nucleotides (e.g., peptide nucleic acids). Polynucleotides may be native or heterologous structures or full-length or subsequences of regulatory genes. Unless otherwise indicated, the term includes references to the specified sequence and its complementary sequence. Thus, DNA or RNA with a modified backbone for stability or other reasons is a “polynucleotide” as the term intends herein. Furthermore, to give two examples, DNA or RNA containing unusual bases such as inosine or modified bases such as tritylated bases is a “polynucleotide” as the term intends herein. It will be recognized to those skilled in the art that a wide range of modifications have been made to DNA and RNA for many useful purposes known to them. The term “polynucleotide,” as used herein, encompasses the chemical forms of polynucleotides that are chemically, enzymatically, or metabolically modified, as well as the chemical forms of DNA and RNA features of viruses and cells, including, in particular, simple and complex cells. All nucleic acid sequences herein that also encode polypeptides such as alcohol acyltransferases by reference to the genetic code describe all possible silent variations of the nucleic acid. The term “conservatively modified variant” applies to both amino acids and nucleic acid sequences. With respect to a particular nucleic acid sequence, the term “conservatively modified variant” refers to a nucleic acid that encodes a variant of an amino acid sequence that is identical by degeneracy of the genetic code or that is conservatively modified. The term “degeneracy of the genetic code” refers to the fact that a number of functionally identical nucleic acids encode a particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.Therefore, at all positions specified by the codon, alanine can be modified to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and correspond to a type of conservatively modified variation. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The “enzymatic fragment of amino acid sequence” of the alcohol acyltransferase of the present invention means a stretch of at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, or 250 amino acid residues having alcohol acyltransferase activity as defined herein. The terms “polypeptide,” “peptide,” and “protein” also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding natural amino acids, as well as to natural amino acid polymers. The required property of such analogs of natural amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies induced against the same protein but composed entirely of natural amino acids. The terms “polypeptide,” “peptide,” and “protein” include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues. In the context of this application, oligomers (oligonucleotides, oligopeptides, etc.) are considered to be a type of polymer group. Oligomers generally have a relatively small number of monomeric units, typically 2 to 100, and especially 6 to 100, including primer sequences, such as those used for cloning the alcohol acyltransferase of the present invention in the examples.
[0138] The term "heterogeneous," when used in relation to nucleic acids (DNA or RNA) or proteins of the present invention, refers to nucleic acids or proteins that do not occur naturally as part of the organism, cell, genome, or DNA or RNA sequence in which they exist, or that are found at one or more locations (singular or plural) in a cell, genome, or DNA or RNA sequence different from those found naturally. Heterogeneous nucleic acids or proteins of the present invention are not endogenous to the cell into which they are introduced, but are obtained from another cell or produced by synthesis or recombination. Generally, but not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is expressed. Genes that are endogenous to a particular host cell but have been modified from their native form, for example through the use of DNA shuffling, are also called heterogeneous. The term "heterogeneous" also includes non-natural multiple copies of native DNA sequences. Therefore, the term "heterogeneous" may refer to a DNA segment that is foreign or heterogeneous to the cell, or homologous to the cell but is a location and / or number within the host cell nucleic acid in which the segment is not normally found. Foreign DNA segments are expressed to produce foreign polypeptides.
[0139] The “homologous” DNA sequences of the present invention are DNA sequences that are naturally associated with the host cell into which they are introduced. Any nucleic acid or protein that a person skilled in the art would recognize as heterologous or foreign to the cell in which it is expressed is included herein by the term heterologous nucleic acid or protein.
[0140] The terms “modified,” “modification,” “mutated,” or “mutation” are used herein in reference to a protein or polypeptide, with modifications where appropriate, to a nucleotide or nucleic acid sequence, in comparison to another protein or polypeptide (particularly in comparison to the alcohol acyltransferase of the present invention comprising or consisting of the amino acid sequences of SEQ ID NOs. 2, 3, or 4). The terms referred to are used to indicate that a modified nucleotide or nucleic acid sequence encoding a protein or polypeptide having alcohol acyltransferase activity has at least one difference in the nucleotide or nucleic acid sequence compared to, for example, the amino acid sequence of a protein or polypeptide compared to SEQ ID NOs. 2, 15, or 16. The terms are used regardless of whether the modified or mutated protein is actually obtained by mutagenesis of the nucleic acid encoding these amino acids, modification of the polypeptide or protein, or by other means, for example, using artificial gene synthesis methods. Mutagenesis is a well-known method in the art, including, for example, site-directed mutagenesis by PCR or oligonucleotide-mediated mutagenesis, as described in Sambrook, J., and Russell, DWMolecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001). The terms “modified,” “modification,” “mutated,” or “mutation,” as used herein in reference to a gene, are used to indicate that at least one nucleotide in the nucleotide sequence of that gene or its regulatory sequence is different from the nucleotide sequence being compared, for example, the nucleotide sequence encoding the amino acid sequence of SEQ ID NOs. 2, 15, or 16.Modifications or mutations may, in particular, be substitutions, deletions, or insertions of nucleotides by a different nucleotide.
[0141] The present invention also relates to vectors or gene constructs comprising the nucleic acids of the present invention.
[0142] Preferably, the vector or gene construct is suitable for encoding and producing the alcohol acyltransferase of the present invention, which has enzymatic activity in microbial cells as defined herein.
[0143] The nucleic acids of the present invention are operably ligated in a vector or gene construct to an expression regulatory sequence that enables expression in prokaryotic or eukaryotic host cells or isolated fractions thereof. Thus, in one embodiment, the vector is an expression vector. Expression of the nucleic acids of the present invention involves transcription of polynucleotides into translatable mRNA. Regulatory elements that ensure expression in prokaryotic or eukaryotic host cells are well known in the art. In one embodiment, these include a regulatory sequence that ensures transcription initiation and / or a polyA signal that ensures transcription termination and transcription stabilization. Further regulatory elements may include transcriptional and translational enhancers. Possible regulatory elements that enable expression in prokaryotic host cells include, for example, the lac-, trp-, or tac- promoters or the rhodobacter promoter in E. coli (https: / / doi.org / 10.1073 / pnas.2010087117), while examples of regulatory elements that enable expression in eukaryotic host cells include the AOX1- or GAL1- promoter or the CMV-, SV40-, or RSV- promoters (Roussarcoma virus), the CMV-enhancer, the SV40-enhancer, or globin introns in mammalian and other animal cells. Plant promoters are described, for example, in Plant Biotechnology: Principles and Applications, pp. 117-172, 2017. Furthermore, inducible expression regulatory sequences can be used in expression vectors. Such inducible vectors may include tet or lac operator sequences or sequences that are inducible by heat shock or other environmental factors. Suitable expression regulatory sequences are well known in the art. In addition to elements involved in transcription initiation, such regulatory elements may also include transcription termination signals downstream of polynucleotides, such as the SV40-poly-A site or the tk-poly-A site. In this context, suitable expression vectors known in the art include Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (Invitrogen).Viral expression vectors, such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses, can be used for the delivery of polynucleotides or vectors to targeted cell populations.
[0144] Methods well known to those skilled in the art may be used to construct a nucleic acid-containing vector or gene construct of the present invention; see, for example, the techniques described in Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2001), NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).
[0145] Where used herein, the term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function, such as the nucleic acids of the present invention. Thus, a gene includes coding sequences and / or regulatory sequences required for their expression. For example, a gene refers to a nucleic acid fragment that expresses mRNA or functional RNA or codes for a specific protein, which includes regulatory sequences. A gene also includes non-expressed DNA segments that form, for example, recognition sequences for other proteins. A gene may be obtained from a variety of sources, including cloning from the origin of interest or synthesis from known or expected sequence information, and may include sequences designed to have desired parameters.
[0146] The term “chimeric gene,” as used herein, refers to any gene that contains 1) a DNA sequence comprising regulatory and coding sequences not found together in nature, or 2) a sequence encoding a portion of a protein not adjacent in nature, or 3) a portion of a promoter not adjacent in nature. Thus, a chimeric gene may contain regulatory and coding sequences of different origins, or regulatory and coding sequences of the same origin but organized differently from those found in nature.
[0147] When used herein, the "gene construct" may vary in complexity depending on the insertion of interest. This construct may be designed to be randomly inserted into the genome of an organism, known as gene addition, or to be inserted into the genome at a specific targeted site at a determined chromosomal location, known as gene addition. In both cases, the construct must be complete, possessing structures for controlling gene expression, such as promoters, transcription start sites, polyadenylation sites, and transcription termination sites. That is, the information inserted into the receptor genome has a beginning, middle, and end, thus avoiding the problem of unregulated expression in the host cell or organism.
[0148] The terms “Open Reading Frame” and “ORF,” as used herein, refer to the amino acid sequence encoded between the translation start codon and termination codon of a coding sequence. The terms “start codon” and “termination codon” refer to a unit of three adjacent nucleotides ("codons") in a coding sequence that identify the start of protein synthesis (mRNA translation) and the termination of the chain, respectively.
[0149] Where used herein, “coding sequence” refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes non-coding sequences. This may constitute an “uninterrupted coding sequence,” i.e., one that lacks introns, such as in cDNA, or one or more introns that are joined by appropriate splice junctions. “Intron” is an RNA sequence that is present in the primary transcript but is removed through intracellular RNA cleavage and rejoining to produce mature mRNA that can be translated into a protein.
[0150] When used herein, “regulatory sequence” refers to a nucleotide sequence located upstream (5' non-coding sequence) of a coding sequence, or within or downstream (3' non-coding sequence) of a coding sequence, which affects transcription, RNA processing, or stability or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translational leader sequences, introns, and polyadenylation signal sequences. These include sequences that may be native and synthetic sequences, as well as combinations of synthetic and native sequences. As stated above, the term “appropriate regulatory sequence” is not limited to promoters. Examples of regulatory sequences include promoters (transcriptional promoters, constitutive promoters, inductive promoters), operators, enhancers, mRNA ribosome binding sites, and appropriate sequences that control transcription and translation initiation and termination. Nucleic acid sequences are “operably linked” if the regulatory sequence is functionally related to the DNA or cDNA sequence of the present invention. When used herein, the terms “operably linked” or “operatably linked” refer to proximal relationships that enable the components described in this way to function in the manner they are intended. A regulatory sequence that is "operably ligated" to another regulatory sequence and / or coding sequence is ligated such that transcription and / or expression of the coding sequence is achieved under conditions compatible with the regulatory sequence. Generally, "operably ligated" means that the ligated nucleic acid sequences are close together and, if necessary, in the same reading frame to ligate the two protein coding regions. Each of the regulatory sequences may be independently selected from heterologous and homologous regulatory sequences.
[0151] Where used herein, “promoter” refers to a nucleotide sequence that controls the expression of a coding sequence, typically located upstream (5') of the coding sequence, by providing other factors necessary for recognition by RNA polymerase and proper transcription. A “promoter” includes a minimal promoter, a short DNA sequence consisting of a TATA box and other sequences that work to designate a transcription start site, to which regulatory elements are added for control of expression. A “promoter” also refers to a nucleotide sequence containing a minimal promoter + regulatory elements capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of adjacent and more distal upstream elements, the latter often referred to as enhancers. Thus, an “enhancer” is a DNA sequence that can stimulate promoter activity and may be an inherent element of a heterologous element inserted to enhance the promoter or the promoter's level or tissue specificity. It is manipulable in both directions (normal or flip) and can function whether moved upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived entirely from native genes, or they may consist of different elements derived from different promoters found in nature, or they may even consist of synthetic DNA segments. Promoters may also contain DNA sequences involved in the binding of protein factors that regulate the effectiveness of transcription initiation in response to physiological or developmental conditions.
[0152] Where used herein, “expression cassette” means a DNA sequence capable of directing the expression of a specific nucleotide sequence, such as the alcohol acyltransferase of the present invention, in a suitable host cell as defined herein, including a promoter operably ligated to a nucleotide sequence of interest operably ligated to a termination signal. This also generally includes sequences required for the correct translation of the nucleotide sequence. The coding region typically codes for the protein of interest, but may also code for functional RNA of interest, such as antisense RNA or uncoding RNA, in sense or antisense directions. An expression cassette containing the nucleotide sequence of interest may be a chimeric, meaning that at least one of its components is heterologous to at least one of its other components. An expression cassette may be natural but obtained in a recombinant form useful for heterologous expression. Expression of the nucleotide sequence in an expression cassette may be under the control of a constitutive promoter or an inductive promoter that initiates transcription only when the host cell is exposed to certain specific external stimuli. In multicellular organisms, the promoter may also be specific to a particular tissue or organ or to a developmental stage in, for example, plant development.
[0153] The term “vector,” as used herein, refers to a construct consisting of genetic material designed to direct the transformation of targeted cells. The vector contains multiple genetic elements that are locologically and sequentially oriented, i.e., manipulably linked with other necessary elements, so that nucleic acids in a nucleic acid cassette can be transcribed and, if necessary, translated in the transformed cells. In particular, the vector may be selected from the group of viral vectors, (bacterio)phages, cosmids, or plasmids. The vector may also be a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or an Agrobacterium binary vector. The vector may be self-transmitting or mobile or not, and may be in a double-stranded or single-stranded linear or circular form that can transform host organisms, such as Rhodobacter, either by integration into the cellular genome or by existing outside the chromosome (e.g., an autonomously replicating plasmid with an origin of replication). Specifically, this includes shuttle vectors, which represent DNA vehicles capable of spontaneously or systematically replicating in two different host organisms as defined herein. Preferably, the nucleic acid in the vector is under the control of a promoter or other regulatory element suitable for transcription in host cells as specified herein, and is operably ligated to it. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory element, and in the case of cDNA, it may be under the control of a promoter or other regulatory element suitable for expression in host cells. The nucleic acid-containing vector may be prepared based on methods known in the art. For example, a cDNA sequence encoding the alcohol acyltransferase of the present invention may be used, which is operably ligated to a suitable regulatory element such as a transcriptional or translational regulatory nucleic acid sequence.
[0154] Where used herein, the term “vector” includes references to vectors for standard cloning operations ("cloning vectors"), as well as to more specific types of vectors such as (autosomal) expression vectors and cloning vectors used for integration into the chromosomes of host cells ("integration vectors").
[0155] A "cloning vector" typically contains one or more restriction endonuclease recognition sites into which foreign DNA sequences can be inserted in a manner that can be determined without losing the essential biological function of the vector, as well as marker genes suitable for use in the identification and selection of cells transformed with the cloning vector.
[0156] The term “expression vector,” as used herein, refers to a linear or circular DNA molecule containing a segment encoding a polypeptide of interest under the control (i.e., manipulably ligated) of a further nucleic acid segment that gives rise to its transcription. Such further segments may include a promoter and termination sequence, and optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally originate from plasmids or viral DNA, or may contain elements of both. In particular, expression vectors contain manipulably ligated nucleotide sequences comprising, in the 5' to 3' direction, (a) a transcription and translation initiation region recognized by the host organism, (b) a coding sequence for the polypeptide of interest, and (c) a transcription and translation termination region recognized by the host organism. “Plasmid” refers to autonomously replicating extrachromosomal DNA that is not integrated into the genome of a microbial organism and is usually circular in nature.
[0157] An "integration vector" refers to a linear or circular DNA molecule that can be integrated into, for example, the genome of a microbial organism, such as a bacterial genome, resulting in a stable genetic trait of a gene encoding a polypeptide of interest, such as the alcohol acyltransferase of the present invention. An integration vector generally comprises one or more segments containing a gene sequence encoding the polypeptide of interest, under the control of (i.e., manipulably ligated to) a further nucleic acid segment that results in its transcription.
[0158] Such further segments may include one or more segments that promote the integration of the promoter and termination sequences and the gene of interest into the genome of the target cell, typically through a process of homologous recombination. Generally, the integration vector is one that can be transferred to the target cell but has a non-functional replicon in that organism. Integration of a segment containing the gene of interest may be selected if the segment contains an appropriate marker. One or more nucleic acid sequences encoding an appropriate signal peptide that is not naturally associated with the polypeptide to be expressed in the host cell of the present invention may be incorporated into the (expression) vector. For example, a DNA sequence for a signal peptide reader may be fused in-frame to the nucleic acid of the present invention so that the alcohol acyltransferase of the present invention is first translated as a fusion protein containing the signal peptide. Depending on the nature of the signal peptide, the polypeptide to be expressed is targeted differently. Secretory signal peptides that are functional in the intended host cell promote, for example, extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to certain organelles such as chloroplasts, mitochondria, and peroxisomes. The signal peptide may be cleaved from the polypeptide during transport to or from the intended organelle. It is possible to provide further peptide sequence fusion at the amino or carboxyl terminus of the polypeptide.
[0159] Furthermore, the present invention relates to a host cell comprising the vector or gene construct of the present invention.
[0160] The vector or gene construct of the present invention is used to transform host cells. Those skilled in the art are well aware of the genetic factors that must be present on the gene construct in order to successfully transform, select, and propagate host cells containing the vector or gene construct of the present invention. The host cells of the present invention are capable of expressing polypeptides having alcohol acyltransferase activity, which are included in the vector or gene construct of the present invention.
[0161] Where used herein, "transformation" and "transforming" refer to the introduction of a heterologous nucleotide sequence, such as a nucleotide sequence encoding the alcohol acyltransferase of the present invention, into a host cell, regardless of the method used for insertion, e.g., direct incorporation, transduction, conjugation, f-conjugation, or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, e.g., a plasmid, or may be integrated into the host cell genome.
[0162] The host cells according to the present invention can be prepared based on standard genetic and molecular biology techniques generally known in the art, such as those described in Sambrook, J., and Russell, DW, “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and FMAusubel et al, eds., “Current protocols in molecular biology,” John Wiley and Sons, Inc., New York (1987) and subsequent supplements thereto.
[0163] The host cell may be any cell selected from microbial cells, such as bacterial cells, archaeal cells, fungal cells, such as yeast cells, and protist cells. The host cell may also be an algal cell or cyanobacterial cell, a non-human animal cell or mammalian cell, or a plant cell.
[0164] Specifically, the host cell can be selected from any one of the following organisms:
[0165] Bacteria: The bacterial host cells may be selected from the group consisting of, for example, the genera Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Lactococcus, Ensifer, or Pantoea.
[0166] Gram-positive: Bacillus and Streptomyces genera: Useful Gram-positive bacterial host cells include Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus jautus, Bacillus lentus, and Bacillus richeniformis. Examples include, but are not limited to, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. The most preferred prokaryotes are Bacillus cells, preferably Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus.
[0167] Some other preferred bacteria include species of the order Actinomycetales, preferably the genus Streptomyces, preferably Streptomyces spheroides (ATTC23965), Streptomyces thermoviolaceus (IFO12382), Streptomyces lividans, or Streptomyces murinus, or Strepoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include species belonging to the genus Myxococcus, such as M. virescens.
[0168] Gram-negative: Species of Escherichia, Pseudomonas, Rhodobacter, Paracoccus, Ensifer, or Pantoea: Preferred Gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC15958) or Pseudomonas fluorescens (NRRL B-11) or Pseudomonas denitrificans, Rhodobacter capsulatus, or Rhodobacter sphaeroides. This is Sinorhizobium meliloti, also known as Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens, Pantoea ananatis, or Ensifer meliloti.
[0169] fungi Aspergillus, Fusarium, and Trichoderma genera The host cell may be a fungal cell. As used herein, "fungus" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the phyla Oomycota and Deuteromycotina, and all vegetative spore-forming fungi. Representative groups of Ascomycota include, for example, the genera Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the following yeasts. Examples of Basidiomycota include mushrooms, rust, and smut. Representative groups of Chytridiomycota include, for example, the genera Allomyces, Blastocladilla, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, for example, the Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of vegetative spore-forming fungi include the genera Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of the phylum Zygomycota include, for example, the genera Rhizopus and Mucor.
[0170] Some preferred fungi include species belonging to the subphylum Deuteromycotina, the class Hyphomycetes, such as Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium, or Dreschlera, especially Fusarium oxysporum (DSM2672) and Humicola insolens. Examples include *Trichoderma insolens*, *Trichoderma resii*, *Myrothecium verrucana* (IFO6113), *Verticillum alboatrum*, *Verticillum dahlie*, *Arthromyces ramosus* (FERM P-7754), *Caldariomyces fumago*, *Ulocladium chartarum*, *Embellisia alli*, or *Dreschlera halodes*.Other preferred fungi include species belonging to the subphylum Basidiomycotina, the class Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus, or Trametes, particularly Coprinus cinereus f. microsporus (IFO8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12), or the genus Trametes (formerly called Polyporus), such as T. versicolor (e.g. PR4 28-A). Further preferred fungi include species belonging to the subphylum Zygomycotina, specifically the Mycoraceae class, such as the genera Rhizopus and Mucor, and in particular Mucor hiemalis.
[0171] yeast Pichia genus Saccharomyces genus The fungal host cell may be a yeast cell. As used herein, yeast includes ascosporogenous yeasts (Endomicetales, basidiosporogenous yeasts) and imperfect fungi (Blastomycetes). Ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter consists of four subfamilies: Schizosaccharomycoideae (e.g., the genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., the genera Kluyveromyces, Pichia, and Saccharomyces). Examples of basidiosporogenous yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the imperfect fungi are divided into two families: Sporobolomycetaceae (e.g., the genera Sporobolomyces and Bullera) and Cryptococcaceae (e.g., the genus Candida).
[0172] eukaryotes Eukaryotic host cells may also include, but are not limited to, non-human animal cells, non-human mammalian cells, avian cells, reptile cells, insect cells, or plant cells.
[0173] In a preferred embodiment, the host cell is a host cell selected from the following: a) Bacterial cells of the Gram-negative bacterial group, such as the genera Rhodobacter (e.g., Rhodobacter sphaeroides or Rhodobacter capsulatus), Paracoccus (e.g., P. carotinifaciens, P. zeaxanthinifaciens), Escherichia, or Pseudomonas; b) Bacterial cells selected from the group of Gram-positive bacteria, such as Bacillus, Streptomyces, Corynebacterium, Brevibacterium, and Amycolatopis; c) Fungal cells selected from the genera Aspergillus, Blakeslea, Penicillium, Phaffia (Xanthophyllomyces), Pichia, Saccharamoyces, Kluyveromyces, Yarrowia, and Hansenula; d) Transgenic plant cells or cultures containing transgenic plant cells (these cells are from transgenic plants selected from Arabidopsis spp., Nicotiana spp., Cichorum intybus, Lacuca sativa, Mentha spp., Artemisia annua, tuberous plants, oilseeds, such as Brassica spp. or Brassica napus, fruit-producing flowering plants (angiosperms) and trees); or e) Cultures containing transgenic mushrooms or transgenic mushroom cells (these microorganisms are selected from the genera Schizophyllum, Agaricus, and Pleurotisi).
[0174] More preferred host cells from organisms include microorganisms belonging to the genera Escherichia, Bacillus, Saccharomyces, Pichia, Rhodobacter, Pseudomonas, or Paracoccus (e.g., Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens), and even more preferably E. coli, S. cerevisae, Rhodobacter sphaeroides, and Rhodobacter capsulatus. It is a host cell from a microorganism of *Amycolatopis* sp. (capsulatus).
[0175] Particularly preferred are host cells of the genus Rhodobacter, selected from the groups Rhodobacter capsulatus and Rhodobacter sphaeroides.
[0176] In one preferred embodiment, a host cell is used for the production of the alcohol acyltransferase of the present invention.
[0177] A method for producing the alcohol acyltransferase of the present invention preferably comprises the following steps: (a) culturing host cells containing a nucleic acid sequence encoding the alcohol acyltransferase of the present invention under suitable conditions; and (b) obtaining the alcohol acyltransferase protein of the present invention from the host cells of step (a). In another preferred embodiment, the host cells are suitable for carrying out the method of the present invention.
[0178] For example, a host cell may be used in a method for preparing at least one monoterpene ester, preferably at least one tertiary monoterpene ester, which includes esterifying at least one monoterpene alcohol, preferably at least one tertiary monoterpene alcohol, to at least one monoterpene ester, preferably at least one tertiary monoterpene ester, in the presence of: (i) an alcohol acyltransferase or (ii) an alcohol acyltransferase comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with an amino acid sequence such as that indicated by database acceptance number XP_006493396 (sequence number 13) or UNIPROTKB-A0A2H5PUP1 (sequence number 14), and capable of esterifying monoterpene alcohols to monoterpene esters, or (iii) an alcohol acyltransferase as described in (ii) above, wherein the amino acids corresponding to the amino acids at positions 371 and 372 of SEQ ID NO: 2 are not tryptophan, or (iv) The alcohol acyltransferase of (ii) above, further comprising, at a position corresponding to the position of SEQ ID NO: 2 shown in Table 1, any of the amino acids listed in Table 1 for those positions, or at a position corresponding to the position of SEQ ID NO: 2 shown in Table 2, which is capable of esterifying at least one monoterpene alcohol to at least one monoterpene ester, more preferably at least one tertiary monoterpene alcohol to at least one monoterpene ester.
[0179] Sequence ID 13 corresponds to the amino acid sequence indicated by database access number XP_006493396.
[0180] Sequence ID 14 corresponds to the amino acid sequence indicated by database access number UNIPROTKB-A0A2H5PUP1.
[0181] To prepare at least one monoterpene ester, preferably at least one tertiary monoterpene ester, the host cell preferably heterologously expresses one of the alcohol acyltransferases (i) to (iv) described above.
[0182] The monoterpene alcohol is preferably linalool, geraniol, alpha-terpineol, gamma-terpineol, lavandulol, fencol, periryl alcohol, menthol, or verbenol, and is used as a substrate. The monoterpene alcohol substrate can be produced by the host cell or added to the host cell from outside. The produced monoterpene ester is preferably a monoterpene ester or acetyl ester selected from the group consisting of linalyl acetate, geranyl acetate, alpha-terpineol ester (alpha-terpinyl acetate), gamma-terpineol ester (gamma-terpinyl acetate), lavandulol ester, fencol ester, periryl alcohol ester, menthol ester, and verbenol ester, as shown elsewhere in this specification.
[0183] To provide a concrete example, a host cell may be used in a method for preparing linalyl acetate, which includes esterifying linalool to linalyl acetate in the presence of the alcohol acyltransferases (i) to (iv) described above, which are capable of esterifying linalool to linalyl acetate. In this case, the host cell contains or produces linalool as a substrate.
[0184] In another preferred embodiment, the host cells according to the present invention may be used industrially in the fermentation production of monoterpene esters as illustrated above. Such fermentation production is described elsewhere in this specification.
[0185] Preferably, the alcohol acyltransferases described in (i) to (iv) above may also be used in combination with one or more further enzymes in the host cells of the present invention.
[0186] One example of such additional enzymes that can be used in addition to the alcohol acyltransferases described in (i) to (iv) above is linalool synthase. Linalool synthase is an enzyme that uses geranyl pyrophosphate as a substrate and catalyzes the formation of linalool. For example, S-linalool synthase stereoselectively converts the ubiquitous C10 intermediate geranyl diphosphate (GPP) to S-linalool (Pichersky et al., Arch Biochem Biophys 1995 Feb 1;316(2):803-7.doi:10.1006 / abbi.1995.1107). R-linalool synthase specifically catalyzes the production of R-linalool. Next, S- or R-linalool is esterified by the alcohol acyltransferases described in (i) to (iv) above to linalyl acetate. In this case, the host cell contains or produces geranyl pyrophosphate (GPP) as a substrate for linalool synthase.
[0187] Although linalool synthase uses one catalytic mechanism (exemplified by limonene synthase (LMS)), it possesses sequence motifs that represent both LMS-type synthases and terpene synthases that use different mechanisms (exemplified by copalil diphosphate synthase (CPS)). This suggests that linalool synthase is a composite gene that can evolve from a recombination event between two different types of terpene synthases (Cseke et al., Mol. Biol. Evol. 15(11): 1491-1498. 1998).
[0188] Linalool synthase may be R-linalool synthase or S-linalool synthase.
[0189] R-linalool synthase (EC4.2.3.26) catalyzes the reaction geranyl diphosphate + H2O = (3S)-linalool + diphosphate.
[0190] S-linalool synthase (EC4.2.3.25) catalyzes the reaction geranyl diphosphate + H2O = (3R)-linalool + diphosphate.
[0191] Linalool synthases and their corresponding sequences are well known in the art.
[0192] For example, UniProt Q2XSC5 shows the amino acid sequence of R-linalool synthase from Lavandula angustifolia (lavender). UniProt Q8H2B4 shows the amino acid sequence of R-linalool synthase from Mentha aquatic. UniProt Q84UV0 shows the amino acid sequence of S-linalool synthase from Arabidopsis thaliana.
[0193] Further acceptance numbers, species, and references for linalool synthase are listed below.
[0194] R-linalool synthase: UniProt Q8H2B4 from Mentha aquatica: “Molecular cloning and characterization of a new linalool synthase.” Crowell AL, Williams DC, Davis EM, Wildung MR, Croteau R. Arch. Biochem. Biophys. 405:112-121 (2002).
[0195] UniProt Q2XSC5:“Cloning and functional characterization of three terpene synthases from lavender(Lavandula angustifolia).” [ PubMed ] [ Cross Ref ] Landmann C., Fink B., Festner M., Dregus M., Angel KH, Schwab W. et al. Arch.Biochem.Biophys.465:417–429(2007).
[0196] UniProt Q9SPN0;JW Jia 1,J Crock,S Lu,R Croteau,XY Chen.“(3R)-Linalool synthase from Artemisia annua L.:cDNA isolation, characterization, and wound induction.” Arch Biochem Biophys 1;372(1):143-9.doi:10.1006 / abbi.1999.1466.
[0197] S-State Line: UniProt Q6ZH94;“Jasmonate induction of the monoterpene linalool confers resistance to rice bacterial blight and its biosynthesis is regulated by JAZ protein in rice.” S. Taniguchi, Y. Hosokawa-Shinonaga, D. Tamaoki, S. Yamada, K. Akimitsu, K. Gomi. Plant Cell Environ.37:451–461(2014).
[0198] GenBank AFK09263;“Characterization of S-(+)-linalool synthase from several provenances of Cinnamomum osmophloeum.”Yan-Liang Lin, Yi-Ru Lee, Wen-Ke Huang, Shang-Tzen Chang & Fang-Hua Chu Tree Genetics & Genomes volume 10, pages 75-86(2014).
[0199] UniProt Q96376;N Dudareva,L Cseke,VM Blanc,E Pichersky. “Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C.breweri flower.”The Plant Cell Jul 1996,8(7)1137-1148;DOI:10.1105 / tpc.8.7.1137.
[0200] In another embodiment, the host cell may contain geranyl diphosphate (GPP) synthase in addition to the alcohol acyltransferases and linalool synthases described in (i) to (iv) above. The geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate (GPP). The linalool synthase then converts the ubiquitous C10 intermediate GPP to linalool, which is then esterified to linalyl acetate by the alcohol acyltransferases described in (i) to (iv) above (Burke et al., Proc Natl Acad Sci USA. 1999 Nov 9;96(23):13062-13067). Clearly, in this case, the host cell contains or produces a substrate for geranyl diphosphate synthase.
[0201] The following are examples of non-restrictive acceptance numbers and species for GPP synthase: GenBank CAC16849.1 Geranyl diphosphate synthase (Arabidopsis thaliana). GenBank CAC16851.1 Geranyl diphosphate synthase (Citrus sinensis) GenBank ACA21458.2 Geranyl diphosphate synthase 2 (Picea abies).
[0202] Furthermore, the host cells of the present invention are suitable for carrying out the uses described herein.
[0203] Furthermore, the present invention relates to a transgenic non-human organism containing the nucleic acid of the present invention, a vector or gene construct of the present invention, or a host cell of the present invention.
[0204] The transgenic non-human organism of the present invention comprises nucleic acids, vectors or gene constructs of the present invention, or host cells of the present invention. In preferred embodiments, the transgenic non-human organism of the present invention is used to prepare monoterpene esters, such as linalyl acetate, as detailed elsewhere in this application, as defined herein. The monoterpene esters are prepared by the action of the alcohol acyltransferases of (i) to (iv) above, which esterify the corresponding monoterpene alcohol. The corresponding monoterpene alcohol is linalool in the case of linalyl acetate.
[0205] Preferably, the transgenic non-human organisms of the present invention are bacteria, yeasts, fungi, protists, algae or cyanobacteria, non-human animals or non-human mammals or plants. Specifically, organisms mentioned in relation to the host cells of the present invention may also be used for the creation of the transgenic non-human organisms of the present invention.
[0206] The bacteria are preferably Gram-negative bacteria, preferably belonging to the genera Rhodobacter, Escherichia, Pseudomonas, or Paracoccus.
[0207] With respect to transgenic organisms or transgenic cells, the term “transgenic,” as used herein, means an organism or cell that contains non-natural nucleic acids in the organism or cell, and in which nucleic acids have been introduced into the organism or cell using recombinant DNA techniques known in the art (i.e., introduced in the organism or cell itself or in the ancestor of the organism or cell from which the cell was isolated) (this cell may be the organism itself or the cell of a multicellular organism from which it was isolated). In other words, the nucleic acid is heterogeneous to the transgenic organism or transgenic cell.
[0208] A “transgene” refers to a gene, such as an alcohol acyltransferase gene, that has been introduced into the genome by transformation and is preferably maintained stably. Preferably, the transgene includes a gene that is heterologous to the gene of the specific cell or organism to be transformed. Furthermore, the transgene may include a native gene that is inserted into a non-native organism or a chimeric gene. The term “endogenous gene” refers to a native gene in its natural position in the genome of an organism. A “foreign” gene refers to a gene that is not normally found in the host organism but is introduced by gene transfer.
[0209] Methods for producing transgenic non-human organisms are well known in the art; see, for example, Lee-Yoon Low et al., Transgenic Plants: Gene constructs, vector and transformation method. 2018. DOI.10.5772 / intechopen.79369; Pinkert, CA (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky GM and Robl, JM (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington DC); Sambrook, loc.cit, Ausubel, loc.cit).
[0210] The present invention further relates to a method for preparing monoterpene esters, which comprises esterifying a monoterpene alcohol to a monoterpene ester in the presence of the above-mentioned alcohol acyltransferases (i) to (iv) that are capable of esterifying the monoterpene alcohol to a monoterpene ester.
[0211] In the present invention, the alcohol acyltransferases (i) to (iv) described above, which are capable of esterifying a monoterpene alcohol to a monoterpene ester, are used for the production of monoterpene esters by esterification of the monoterpene alcohol to its corresponding monoterpene ester.
[0212] In one embodiment, a method for preparing the monoterpene ester of the present invention is described in claim 8.
[0213] In principle, the production of monoterpene esters can be carried out based on methods known from prior art; e.g., Jongedijk et al., Appl Microbiol Biotechnol (2016) 100:2927-2938; Yee et al., Metab Eng. 2019 Sep;55:76-84.doi:10.1016 / j.ymben.2019.06.004; Fromighieri et al., Planta. 2018 Oct;248(4):933-946.doi:10.1007 / s00425-018-2948-0; Leferink et al., Sci Rep. 2019 Aug 15;9(1):11936.doi:10.1038 / s41598-019-48452-2; Zhao et al. See al., Microb Cell Fact (2017) 16:17 DOI 10.1186 / s12934-017-0641-9; or refer to International Publication No. 2014 / 014339.
[0214] The following monoterpene alcohols can be used as substrates: Monoterpene alcohols may be primary, secondary, or tertiary monoterpene alcohols, such as linalool, geraniol, alphaterpineol, gammaterpineol, lavandulol, fencol, periryl alcohol, menthol, or verbenol.
[0215] Preferably, the monoterpene alcohol is linalool, geraniol, alphaterpineol, gammaterpineol, lavandulol, fencol, periryl alcohol, menthol, or verbenol.
[0216] The monoterpene alcohol is more preferably a tertiary monoterpene alcohol, such as linalool or alpha-terpineol.
[0217] The monoterpene ester products prepared by the method of the present invention may be linalyl acetate, geranyl acetate, alpha-terpineol ester (alpha-terpinyl acetate), gamma-terpineol ester (gamma-terpinyl acetate), lavandulol ester, fencol ester, periryl alcohol ester, menthol ester, or verbenol ester.
[0218] In a preferred embodiment, a method for preparing a monoterpene ester comprises esterifying a tertiary monoterpene alcohol to a monoterpene ester in microbial cells, preferably as defined herein, within 36 hours, 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes, or 30 minutes, in the presence of an alcohol acyltransferase capable of esterifying at least 30% by mass of the tertiary monoterpene alcohol.
[0219] Preferably, the alcohol acyltransferase used in the method of the present invention to prepare an alcohol ester, preferably a monoterpene ester, is (i) an alcohol acyltransferase or (ii) an alcohol acyltransferase comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, and capable of esterifying monoterpene alcohols to monoterpene esters, or (iii) an alcohol acyltransferase of (i) or (ii) in which the amino acids corresponding to the amino acids at positions 371 and 372 of SEQ ID NO: 2 are not tryptophan or (iv) An alcohol acyltransferase of (i) or (ii) that is capable of esterifying a monoterpene alcohol to a monoterpene ester, further comprising, at a position corresponding to the position of Sequence ID No. 2 shown in Table 1, one of the amino acids listed in Table 1 for that position, and / or, at a position corresponding to the position of Sequence ID No. 2 shown in Table 2, an amino acid listed in Table 2 and / or a conserved amino acid residue shown in Figure 5, i.e., an amino acid residue shown in white letters on a black background.
[0220] In another embodiment, the host cells according to the present invention may be used industrially in the fermentation production of the monoterpene esters described above.
[0221] For example, the host cells or non-human transgenic organisms of the present invention can be used in the fermentation production of monoterpene esters, such as linalyl acetate.
[0222] Preferably, at least one monoterpene ester is produced in a fermentation step, i.e., in a method comprising culturing microbial host cells, such as Rhodobacter host cells, in a culture medium under conditions in which the alcohol acyltransferase of the present invention is expressed. The actual reactions catalyzed by the alcohol acyltransferase of the present invention generally occur intracellularly. It should be noted that the term “fermentation” is used herein in a broad sense to refer to a process that uses the culture of an organism to synthesize a compound from suitable raw materials (e.g., carbohydrates, amino acid sources, fatty acid sources). Thus, a fermentation step as defined herein is not limited to anaerobic conditions but extends to processes under aerobic conditions. Suitable raw materials are generally known for Rhodobacter host cells. Suitable conditions can be based on known methods for Rhodobacter host cells, such as those described in, for example, International Publication No. 2011 / 074954 or International Publication No. 2014 / 014339.
[0223] The prepared monoterpene esters can be isolated or extracted from host cells or non-human transgenic organisms by methods known in the art (for plants, see, for example, Jiang et al., Curr Protoc Plant Biol. 2016;1:345-358.doi:10.1002 / cppb.20024; for the genus Rhodobacter, see, for example, International Publication No. 2014 / 014339).
[0224] Generally, this method comprises the following steps: 1) disrupting cells to release their chemical components; 2) extracting the sample using a suitable solvent (or through distillation or compound trapping); 3) separating the desired monoterpene ester from other undesirable contents of the extract that would interfere with the analysis and quantification; and 4) using a suitable analytical method (e.g., thin-layer chromatography (TLC), gas chromatography (GC), or liquid chromatography (LC), or another method as described herein).
[0225] Preferably, the alcohol acyltransferases described in (i) to (iv) above are used in combination with linalool synthase and / or geranyl diphosphate (GPP) synthase in these methods of the present invention. Linalool synthase and geranyl diphosphate (GPP) synthase have already been described herein in relation to host cells. The definitions, sequences and descriptions relating to the latter enzyme are applied to the methods of the present invention, with modifications where appropriate.
[0226] The monoterpene esters produced according to these methods of the present invention may be used, for example, as flavorings or fragrances, as insect repellents, as pesticides or antimicrobial agents; they may also be used to produce biofuels, fuel compositions or fuel compounds—such as but not limited to blowing agents for diesel fuel compositions—or as starting materials for other compounds, such as other flavorings or fragrances or fuel compounds.
[0227] The present invention also relates to a method for preparing linalyl acetate, comprising esterifying linalool to linalyl acetate in the presence of the alcohol acyltransferases (i) to (iv) described above, which are capable of esterifying linalool to linalyl acetate.
[0228] In this method, the alcohol acyltransferases (i) to (iv) described above, which are capable of esterifying linalool to linalyl acetate, are used for the production of linalyl acetate by esterifying linalool to its corresponding monoterpene ester, linalyl acetate.
[0229] In one embodiment, a method for preparing linalyl acetate according to the present invention is described in claim 10.
[0230] In a preferred embodiment, a method for preparing linalyl acetate comprises esterifying linalool to linalyl acetate in microbial cells, preferably within 36 hours, 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 45 minutes, or 30 minutes, in the presence of an alcohol acyltransferase capable of esterifying linalool to at least 30% by mass.
[0231] Preferably, the alcohol acyltransferase used in the method for preparing linalyl acetate of the present invention is as follows: (i) an alcohol acyltransferase of the present invention as defined elsewhere herein, or (ii) an alcohol acyltransferase comprising an amino acid sequence such as that indicated by database acceptance number XP_006493396 (sequence number 13) or UNIPROTKB-A0A2H5PUP1 (sequence number 14), or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence indicated by database acceptance number XP_006493396 (sequence number 13) or UNIPROTKB-A0A2H5PUP1 (sequence number 14), which is capable of esterifying linalool to linalyl acetate, or (iii) an alcohol acyltransferase of (i) or (ii) in which the amino acids corresponding to the amino acids at positions 371 and 372 of SEQ ID NO: 2 are not tryptophan, (iv) An alcohol acyltransferase of (i) or (ii) that is capable of esterifying linalool to linalyl acetate, further comprising, at the position corresponding to the position of Sequence ID No. 2 shown in Table 1, any of the amino acids listed in Table 1 for that position, or at the position corresponding to the position of Sequence ID No. 2 shown in Table 2, an amino acid listed in Table 2, or a conserved amino acid residue shown in Figure 5, i.e., an amino acid residue shown in white letters on a black background.
[0232] In a further preferred embodiment, a method for preparing linalyl acetate comprises esterifying linalool to linalyl acetate in the presence of the alcohol acyltransferases (i) to (iv) above, which are capable of esterifying linalool to linalyl acetate. In one embodiment, a method for preparing linalyl acetate uses an alcohol acyltransferase of the present invention other than those of SEQ ID NOs: 3 and 4.
[0233] In another preferred embodiment of this method of the present invention, linalyl acetate is prepared in a host cell or non-human transgenic organism expressing the alcohol acyltransferases (i) to (iv) described above, which are capable of esterifying linalool to linalyl acetate.
[0234] Linalool, or 3,7-dimethyl-1,6-octadien-3-ol, is an acyclic, unsaturated, tertiary monoterpene alcohol containing two enantiomers found in plants: (3S)-(+)-linalool and (3R)-(-)-linalool. The latter is therefore more common in nature. It is one of the main components of essential oils in over 200 plant species, belonging to more than 50% of the plant family. Among them, the most well-known plants rich in linalool are rosewood (Aniba rosaeodora Ducke, Lauraceae), ho leaf (Cinnamomum camphora Nees, Lauraceae), coriander fruit (Coriandrumsativum L., Apiaceae), and lavender (the flower tips of Lavendula officinalis Chaix sin., L. angustifolia Mill., Lamiaceae).
[0235] As explained in the introduction, linalyl acetate, a monoterpene ester of linalool, is an important flavor and aroma molecule. Linalyl acetate is used in many flavors, including amber, bubblegum, ginger, lavender, orange, rose, sandalwood, sherry, night-blooming lilac, and ylang-ylang. In particular, it is an important component of the flavoring ingredients lavender oil and bergamot oil. In flavoring agents, it is used for tomato, tropical, and Earl Grey tea flavors.
[0236] Linalyl acetate occurs in two optical forms: R-type (CAS No. 115-95-7) and S-type (CAS No. 51685-40-6).
[0237] (R)-Linalyl acetate is found in bergamot, lavender, Mentha citrata (bergamot mint), and various other plants.
[0238] In cardamom oil, (S)-linalyl acetate is present.
[0239] Natural linalyl acetate, which is mostly R-type, is described as having sweet, green, citrusy, spicy scents and floral, green, terpy, waxy, citrusy, and woody notes.
[0240] Most linalyl acetate currently on the market is produced through synthetic chemistry. This linalyl acetate is a mixture of both S- and R-isomers.
[0241] Enantiopure R-linalyl acetate can be extracted from many sources, including bergamot oil, petitgrain (a steam distillate from bitter orange plant material including leaves and twigs), neroli bigarade oil, and bergamot mint oil. It is also known to be available from ho wood oil (Cinnamomum camphora) from China, but this is difficult due to the presence of impurities.
[0242] Preferably, the linalyl acetate prepared by the method of the present invention is (i) R-type (Cas No. 115-95-7), (ii) S-type (Cas No. 51685-40-6), or (iii) R-type (Cas No. 115-95-7) and S-type (Cas No. 51685-40-6). The latter embodiment (iii) includes a mixture of the R-type and S-type linalyl acetate.
[0243] Preferably, the alcohol acyltransferases (i) to (iv) above, which are capable of esterifying linalool to linalyl acetate, are used in combination with linalool synthase and / or geranyl diphosphate (GPP) synthase in this method of the present invention. Linalool synthase and geranyl diphosphate (GPP) synthase have already been described herein in relation to host cells. The definitions, sequences and descriptions relating to the latter enzyme are applied to the method of the present invention, with modifications where appropriate.
[0244] The present invention (i) For heterologous reconstruction of the terpene biosynthesis pathway; (ii) for producing industrial products, preferably flavorings or fragrances, biofuels, fuel compositions, fuel compounds, such as foaming agents, pesticides, insect repellents or antimicrobial agents for diesel fuel compositions; (iii) For producing aliphatic and / or aromatic monoterpene esters from monoterpene alcohols, preferably tertiary monoterpene alcohols; (iv) For detoxifying monoterpene alcohols in microorganisms such as bacteria or fungi (e.g., yeast), thereby increasing monoterpene production in said microorganisms; (v) Combined with GPP synthase and / or S- or R-linalool synthase; (vi) To improve the beneficial effect of acetylation in that hydrophobic acetic acid partitioning proceeds more easily to the organic phase compared to monoterpene alcohols; (vii) To express the alcohol acyltransferase of the present invention such that the ratio of monoterpene acetate to monoterpene alcohol is greater than 5:1 or 10:1; (viii) In microbial production systems for monoterpene esters, The above also relates to the use of alcohol acyltransferases, nucleic acids of the present invention, vectors or gene constructs of the present invention, host cells of the present invention, or transgenic non-human organisms of the present invention.
[0245] In one embodiment, an alcohol acyltransferase other than the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4 is used.
[0246] The use of the present invention to enhance the beneficial effect of acetylation in that the hydrophobic acetic acid portion more readily enters the organic phase (see item vi above) can be tested, for example, by comparing the logP values of monoterpene alcohols and monoterpene esters, compared with monoterpene alcohols.
[0247] For example, the logP value for linalool is 2.970 (see http: / / www.thegoodscentscompany.com / data / rw1007872.html#tophyp), and for linalyl acetate it is 3.930 (see http: / / www.thegoodscentscompany.com / data / rw1007892.html#tophyp).
[0248] For example, a two-phase system can be used to produce the monoterpene esters described herein using fermentation. The monoterpene ester product is extracted into the oil layer, while the microorganisms remain in the aqueous layer. More hydrophobic molecules result in a higher percentage (%) distribution to this second layer. This percentage may be important if it makes the difference between exceeding or not exceeding the toxicity threshold in the aqueous layer. A two-phase fermentation process for the production of organic compounds is described, for example, in International Publication No. 2015 / 002528.
[0249] The present invention also relates to a kit comprising the alcohol acyltransferases of (i) to (iv) above, preferably the alcohol acyltransferase of the present invention, the nucleic acids of the present invention, the vectors and / or gene constructs of the present invention, the host cells and / or transgenic non-human organisms of the present invention, and optionally at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol. Preferably, the kit is for carrying out the method of the present invention to prepare at least one monoterpene ester, preferably at least one tertiary monoterpene ester, such as linalyl acetate.
[0250] Where used herein, the term “Kit” refers to a group of means, including the alcohol acyltransferases of (i) to (iv) above, nucleic acids of the present invention, vectors or gene constructs of the present invention, host cells of the present invention, and / or transgenic non-human organisms of the present invention, provided in ready-to-use individual or common vials for carrying out the methods of the present invention as defined herein. The Kit may optionally include, for example, at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol, as a substrate for the alcohol acyltransferase of the present invention. In one embodiment, the Kit includes further means for carrying out the methods, such as further enzymes, e.g., S- or L-linalool synthase and / or GPP synthase, as defined herein. Furthermore, in one embodiment, the Kit includes instructions for carrying out the methods of the present invention. These instructions may be provided as a manual. Furthermore, the present invention relates to monoterpene esters, preferably produced from tertiary monoterpene alcohols, produced by methods for preparing the alcohol acyltransferases of (i) to (iv) above, or the monoterpene esters of the present invention, and compositions comprising the monoterpene esters. Optionally, the composition comprises the alcohol acyltransferases described in (i) to (iv) above, and further optionally, at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol. Preferably, the composition is cell-free and / or not found in nature.
[0251] The present invention also relates to a method for preparing linalyl acetate produced by the alcohol acyltransferases (i) to (iv) described above, preferably the alcohol acyltransferases of the present invention, or linalyl acetate produced from linalool, preferably linalool, and to a composition comprising the linalyl acetate. Optionally, the composition comprises the alcohol acyltransferases (i) to (iv) described above and, further optionally, linalool. Preferably, the composition is cell-free and / or not found in nature.
[0252] Furthermore, the present invention relates to a composition comprising the alcohol acyltransferases described in (i) to (iv) above and at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol. The alcohol acyltransferase may be in an active state, or its activity may be activated by, for example, a change in pH or the removal of an inhibitor.
[0253] The present invention also relates to fermentation broth, preferably fermentation broth in a biphasic fermentation system, the fermentation broth comprising the alcohol acyltransferases of (i) to (iv) above, the nucleic acids of the present invention, the vector or gene construct of the present invention, the host cells of the present invention, and / or the non-human transgenic organism of the present invention, and one or more monoterpene alcohols and / or one or more monoterpene esters produced by the alcohol acyltransferases of (i) to (iv) above or by the method of the present invention.
[0254] Monoterpene esters produced by any one of the methods of the present invention can be used on their own. For example, linalyl acetate can be used as an additive or as a blowing agent in diesel fuel compositions, as shown elsewhere herein.
[0255] In other embodiments, the monoterpene ester produced by any one of the methods of the present invention may be used as a starting material for another compound, such as a fuel and / or biolubricant compound or composition. For example, the monoterpene ester may be converted into a fuel and / or biolubricant compound.
[0256] Accordingly, the present invention further relates to a method for producing fuel and / or biolubricant compounds, the method comprising the following steps: a) To produce one or more monoterpene esters by any one of the methods of the present invention; b) optionally purifying one or more monoterpene esters produced in step a); and c) Optionally converting some or all of one or more monoterpene esters from step a) or one or more purified monoterpene esters from step b) into one or more fuel and / or biolubricant compounds that are not the one or more monoterpene esters produced in step a).
[0257] Preferably, the fuel and / or biolubricant compound is selected from the group consisting of the following: i. Tetrahydrolinalool; ii. 2,6-Dimethyloctane (DMO); iii. Saturated C20 hydrocarbon dimers; iv. Saturated C30 hydrocarbon trimers; and v. Hydrogenated methylcyclopentadiene dimer
[0258] The present invention further provides a method for producing fuel and / or biolubricant compositions, the method comprising the following steps: a) Producing one or more monoterpene esters by any one of the methods of the present invention; b) Optionally purifying one or more monoterpene esters produced in step a); and c) converting some or all of the one or more monoterpene esters from step a) into one or more fuel and / or biolubricant compounds, or converting some or all of the optionally purified monoterpene esters from step b); and d) To form a fuel and / or biolubricant composition by combining one or more fuel and / or biolubricant compounds with further compounds suitable for a fuel and / or biolubricant composition, and optionally one or more monoterpene esters produced in step a).
[0259] Further such compounds suitable for fuel and / or biolubricant compositions are known in the art; see, for example, U.S. Patent No. 9,816,043 or U.S. Patent No. 9,802,873.
[0260] Preferably, at least one fuel and / or biolubricant compound is selected from the group consisting of the following: i. Tetrahydrolinalool; ii. 2,6-Dimethyloctane (DMO); iii. Saturated C20 hydrocarbon dimers; iv. Saturated C30 hydrocarbon trimers; and v. Hydrogenated methylcyclopentadiene dimer
[0261] Preferably, the fuel and / or biolubricant composition comprises one or more fuel and / or biolubricant compounds of one or more monoterpene esters, which are prepared from one or more monoterpene esters in total at a concentration of 0.001% (w / w) to 99.99% (w / w), preferably 0.01% (w / w) to 99.9% (w / w), and which can be obtained by any one of the methods of the present invention.
[0262] The present invention also relates to a method for producing a fuel composition, and this method is a) To produce linalyl acetate by any one of the methods of the present invention; b) Optionally purifying the linalyl acetate produced in step a); and c) generating a fuel composition by combining the linalyl acetate of step a) or the optionally purified linalyl acetate of step b) with further compounds suitable for the fuel composition comprising the step of; This fuel composition contains 0.001% (w / w) to 99.99% (w / w), preferably 0.01% (w / w) to 99.9% (w / w) of linalyl acetate.
[0263] Preferably, the fuel composition is a diesel fuel composition, and linalyl acetate is used as a foaming agent as described elsewhere herein. Generally, at least about 0.5% (w / w) of linalyl acetate is included in the fuel composition prepared by this method of the present invention.
[0264] A method for preparing a monoterpene ester, which comprises esterifying a monoterpene alcohol to a monoterpene ester in the presence of the alcohol acyltransferase of (i)-(iv) above, is described elsewhere herein. This applies to a method for preparing linalyl acetate, which comprises esterifying linalool to linalyl acetate in the presence of the alcohol acyltransferase of (i)-(iv) above, with such modifications as may be appropriate. This definition and embodiment applies, with such modifications as may be appropriate, to the method of the present invention for the production of fuels and / or biolubricant compounds or compositions.
[0265] Preferably, one or more monoterpene esters are made from tertiary monoterpene alcohols, more preferably from linalool, perillyl alcohol and / or alpha terpineol, and most preferably from linalool and / or alpha terpineol.
[0266] Step a) of these methods of the present invention is preferably carried out in a fermentation system, preferably a two-phase system, using a bio-based substrate for the provision of one or more monoterpene alcohols. A bio-based substrate is a substrate intentionally made from substances derived from living (or formerly living) organisms.
[0267] Preferably, optional step b) of these methods of the present invention is used, if necessary, for further purification of the monoterpene esters produced by fermentation prior to conversion to one or more fuel or biolubricant compounds preferably selected from the group consisting of: i. Tetrahydrolinalool; ii. 2,6-Dimethyloctane (DMO); iii. Saturated C20 hydrocarbon dimer; iv. Saturated C30 hydrocarbon trimer; v. Hydrogenated methylcyclopentadiene dimer; vi. Saturated high-density polycyclic hydrocarbon compounds suitable for propellant propulsion; and vii. Hydrogenated C40+ oligomers suitable for making biolubricant additives.
[0268] If a bio-based substrate is used in fermentation and the monoterpene esters produced by fermentation are then used for the production of said fuel and / or biolubricant compounds, the resulting fuel and / or lubricant composition comprising said fuel and / or biolubricant compounds is at least in part a bio-based fuel and / or biolubricant composition, which is advantageous with respect to the carbon footprint of transport, traffic and aviation.
[0269] As an example of a hydrogenated methylcyclopentadiene dimer, RJ-4 is shown in FIG. 6.
[0270] U.S. Patent No. 9,816,043 describes methods for manufacturing fuel and biolubricant additives. U.S. Patent No. 9,802,873 describes methods for the conversion of linalool to a drop-in high-density fuel suitable for ramjet or propellant propulsion. Biolubricants are described, for example, at https: / / www.sciencedirect.com / topics / agricultural-and-biological-sciences / biolubricants.
[0271] This is further referenced in compilations and summaries of biolubricants, for example, K. Belafi-Bako, in Handbook of Waste Management and Co-Product Recovery in Food Processing, Volume 1, 2007; Ionic Liquids in the Production of Biodiesel and Other Oleochemicals by Bethala Lakshmi Anu Prabhavathi Devi, et al., in Ionic Liquids in Lipid Processing and Analysis, 2016; and Production of fine chemicals from food wastes, V. Godvin Sharmila, et al., in Food Waste to Valuable Resources, 2020.
[0272] The fuel and / or biolubricant compositions produced by the method of the present invention can be used as gasoline, kerosene fuel, jet fuel, projectile fuel, heavy diesel fuel, marine fuel, and / or lubricants. The advantages of the fuel compositions produced by the method of the present invention are improved carbon dioxide footprint, reduced sulfur content, reduced particulate emissions from engines, and lower NOx emissions in both diesel and turbine applications.
[0273] The present invention relates to a fuel composition which is gasoline, kerosene fuel, jet fuel, projectile fuel, heavy diesel fuel, or marine fuel, largely based on a monoterpene ester produced by the method of the present invention.
[0274] Preferably, the fuel composition is a biofuel composition; see, for example, https: / / doi.org / 10.1016 / B978-0-12-815162-4.00011-2;Second and Third Generation of Feedstocks;The Evolution of Biofuels;2019,Pages 291-320;Chapter 11-Physical properties and chemical composition of biofuels;Mohd Hafizil, MatYasin,Mohd Affandi Ali,Rizalman Mamat,Ahmad Fitri Yusop,Mohd HafizAli.
[0275] In another aspect of the present invention, linalyl acetate produced by the method of the present invention may be used as a blowing agent in diesel fuel compositions, for example, as disclosed in International Publication No. 2019 / 201630. The use of the fermentation method described herein for the production of linalyl acetate has the advantage of providing bio-based linalyl acetate with a predetermined fermentation production and minimal impact on natural resources, without the need to isolate it from high-value plants such as lavender or to use chemical synthesis which involves a harmful carbon footprint.
[0276] Furthermore, the present invention relates to industrial products, preferably flavorings or fragrances, biofuels, fuel compositions, fuel compounds, such as foaming agents, pesticides, insect repellents or antimicrobial agents for diesel fuel compositions, for example, by using the alcohol acyltransferases described in (i) to (iv) above or by the method of the present invention, preferably from tertiary monoterpene alcohols. Such industrial products can be considered based on renewable resources if at least part of them use monoterpene esters produced by the alcohol acyltransferases described in (i) to (iv) above or by the method of the present invention. Moreover, natural or naturally occurring substances are considered more attractive than petroleum-based synthetic substitutes in many markets.
[0277] The present invention further relates to a microbial production system for one or more monoterpene esters, the microbial production system comprising: a. One or more microbial cells; b. One or more alcohol acyltransferases from (i) to (iv) above; c. At least one monoterpene alcohol, preferably a tertiary monoterpene alcohol; d.(b) An appropriate amount of acetyl-CoA for the alcohol acyltransferase to be active; e.(b) A suitable aqueous medium for one or more alcohol acyltransferases to be active; and f. A non-aqueous solvent in which at least one monoterpene ester produced by the alcohol acyltransferase of (b) is miscible, optionally.
[0278] At least one monoterpene alcohol, preferably a tertiary monoterpene alcohol, and / or acetyl-CoA may be added and / or produced by the cells of one or more microorganisms.
[0279] Preferably, the microorganism is a bacterium or fungus as shown elsewhere in this specification, and more preferably, a bacterium of the genus Rhodobacter.
[0280] Preferably, the microbial production system of the present invention is carried out in a fermentation system, preferably a biphasic system, as described elsewhere in this specification.
[0281] Advantageously, the microbial production system according to the present invention can be used industrially in the fermentation production of monoterpene esters as defined herein.
[0282] The definitions and embodiments relating to the fermentation production of monoterpene esters in host cells or non-human transgenic organisms of the present invention are applicable to the microbial production system of the present invention, with modifications as necessary.
[0283] In one embodiment, the microbial production system may include microbial cells that are inducible with respect to the production of at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol, and / or acetyl-CoA and / or one or more of the alcohol acyltransferases of (i)-(iv).
[0284] Preferably, the microbial production system includes means for inactivating or destroying the microorganisms when a sufficient amount of at least one monoterpene alcohol, preferably a tertiary monoterpene alcohol, and / or acetyl-CoA and / or one or more of the alcohol acyltransferases of (i)-(iv) and / or one or more monoterpene esters has been produced.
[0285] Microbial production systems for monoterpenes are known in the art; see, for example, Zhang et al., Biotechnol Adv. 2017 Dec;35(8):1022-1031.doi:10.1016 / j.biotechadv.2017.09.002.Epub 2017 Sep 6; Cao et al., Appl Microbiol Biotechnol. 2018 Feb;102(4):1535-1544.doi:10.1007 / s00253-017-8695-5.Epub 2017 Dec 20). <s
[0286] Suitable fermentation conditions can be based on known methods for Rhodobacter, for example, as described in WO 2011 / 074954 or WO 2014 / 014339.
[0287] sequence SEQ ID NO: 1 corresponds to the nucleotide sequence of an alcohol acyltransferase (AAT) (AAT9-1-c) from Citrus bergamia.
[0288] Sequence ID 2 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (AAT9-1-c) from Citrus bergamia.
[0289] Sequence ID 3 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (10056) from Lavandula angustifolia.
[0290] Sequence ID 4 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (1461) from Lavandula angustifolia.
[0291] Sequence ID 5 corresponds to the nucleotide sequence of alcohol acyltransferase (AAT) (AAT9-1-a) from Citrus bergamia.
[0292] Sequence ID 6 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (AAT9-1-a) from Citrus bergamia.
[0293] Sequence ID 7 corresponds to the nucleotide sequence of alcohol acyltransferase (AAT) (AAT9-2-a) from Citrus bergamia.
[0294] Sequence ID 8 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (AAT9-2-a) from Citrus bergamia.
[0295] Sequence ID 9 corresponds to the nucleotide sequence of primer AAT9-1fw.
[0296] Sequence ID 10 corresponds to the nucleotide sequence of primer AAT9-2fw.
[0297] Sequence ID 11 corresponds to the nucleotide sequence of primer AAT9-1re.
[0298] Sequence ID 12 corresponds to the nucleotide sequence of primer AAT9-2re.
[0299] Sequence ID 13 corresponds to the amino acid sequence indicated by database access number XP_006493396.
[0300] Sequence ID 14 corresponds to the amino acid sequence indicated by database access number UNIPROTKB-A0A2H5PUP1.
[0301] Sequence ID 15 corresponds to the artificial amino acid sequence of the alcohol acyltransferase of the present invention (internal name "10056a").
[0302] Sequence ID 16 corresponds to the artificial amino acid sequence of the alcohol acyltransferase of the present invention (internal name "1461a"). [Brief explanation of the drawing]
[0303] [Figure 1] Figure 1 shows the multiple sequence alignment of the amino acid sequences of the alcohol acyltransferase (AAT) mutants AAT9-1-c (SEQ ID NO: 2), AAT9-1-a (SEQ ID NO: 6), and AAT9-2-a (SEQ ID NO: 8) from Citrus bergamia. These sequences are indicated by a combination of bold and italicized fonts at positions deemed important by the inventors. [Figure 2] Figure 2 shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the alcohol acyltransferase (AAT) mutant AAT9-1-c from Citrus bergamia. [Figure 3]The GC FID chromatogram shows the conversion of linalool to linalyl acetate in the alcohol acyltransferase (AAT) mutant AAT9-1-c (SEQ ID NO: 2) from Citrus bergamia, which is not observed in the empty vector pACYCDUET-1 (control). [Figure 4-1] Figure 4 shows the alignment (using the NEEDLE algorithm with standard settings) of the amino acid sequence of alcohol acyltransferase (AAT) from Lavandula angustifolia (referred to as 10056) (SEQ ID NO: 3) and another amino acid sequence of alcohol acyltransferase (AAT) from Lavandula angustifolia (referred to as 1461) (SEQ ID NO: 4). [Figure 4-2] Continuation of Figure 4-1. [Figure 5] Figure 5 shows the multiple sequence alignments of the amino acid sequence of the mutant AAT9-1-c from Citrus bergamia (SEQ ID NO: 2), the amino acid sequence of alcohol acyltransferase (AAT) (10056) from Lavandula angustifolia (SEQ ID NO: 3), and the amino acid sequence of alcohol acyltransferase (AAT) (1461) from Lavandula angustifolia (SEQ ID NO: 4). The MEIE motif may be found at the beginning of SEQ ID NOs. 3 and 4. Conserved amino acid residues are shown with a black background. The position numbering for SEQ ID NO: 2 in this figure differs from the position numbering for SEQ ID NO: 2 in Figure 1 or in the sequence list. The alignment of SEQ ID NOs. 3 and 4 requires insertions in the alignment, resulting in an apparently larger number for the position relative to the end of the protein sequence of SEQ ID NO: 2 in this figure. [Figure 6] RJ-4 is shown as an example of a hydrogenated methylcyclopentadiene dimer. [Modes for carrying out the invention]
[0304] The present invention is illustrated by the following embodiments, but this should not be interpreted as limiting the scope of the invention.
[0305] Examples Example 1: Cloning of alcohol acyltransferase from Citrus bergamia Fruits of Citrus bergamia, approximately 5 cm in diameter and not yet fully mature, were obtained from an orchard in Calabria, Italy. The outer peel (flavedo) of the fruit was collected using a zester. The weight of 0.5 g of plant material was measured in a pre-cooled glass tube, and 2 mL of dichloromethane was added. The suspension was vortexed for 1 minute, sonicated in an ultrasonic bath for 5 minutes, and centrifuged at 1500 g for 5 minutes at room temperature. The supernatant was collected and filtered through a 1 g sodium sulfate column. Approximately 2 μL was analyzed by GC / MS using a gas chromatograph, as described in detail by Cankar et al. (Biotechnol J. 2015 Jan; 10(1): 180-9. doi: 10.1002 / biot. 201400288. Epub 2014 Sep 18). Linalyl acetate was identified by comparing its retention time and mass spectrum with that of the original standard of racemic linalyl acetate (Sigma-Aldrich). Further tissue samples were taken for RNA extraction.
[0306] RNA from the root material of Citrus bergamia was isolated as follows: Approximately 15 mL of extraction buffer (2% hexadecyl-trimethylammonium bromide, 2% polyvinylpyrrolidone K30, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, 0.5 g / L spermidine, and 2% β-mercaptoethanol) was heated to 65°C, and then 3 g of lysated tissue was added and mixed. The mixture was extracted twice with equal volumes of chloroform:isoamyl alcohol (1:24), and one-quarter volume of 10 M LiCl was added to the supernatant and mixed. The RNA was precipitated overnight at 4°C and recovered by centrifugation at 10000 g for 20 minutes. The pellet was dissolved in 500 μL of SSTE [1.0 M NaCl, 0.5% SDS, 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA (pH 8.0)] and extracted once with equal volumes of chloroform:isoamyl alcohol. Two volumes of ethanol were added to the supernatant, and the mixture was incubated at -20°C for at least 2 hours. The mixture was then centrifuged at 13000 g, and the supernatant was removed. The pellet was air-dried and resuspended in water. Total RNA (60 μg) was sent to Vertis Biotechnology AG (Freising, Germany). PolyA+ RNA was isolated, and randomly primed cDNA was synthesized using randomized N6 adapter primers and M-MLV H-reverse transcriptase. The cDNA was sheared and fragmented, and 500 bp fragments were used for further analysis. The cDNA possessed adapter sequences A and B ligated to its 5'- and 3'-terminuses, as defined by Illumina. Next, the substance was analyzed on an Illumina HiSeq sequencing instrument. A total of 93,001,205 sequences were read by HiSeq. Sequences from the Illumina sequencing adapter were prepared using Trimmomatic-0.32, paired end sequences were duplicated using Seqprep, and phiX contamination was removed using bowtie2 (version 2.2.1) (phiX DNA is used as a spike-in control, which is typically present in <1%). Trinity assembly (trinityrnaseq-2.0.2) was used, with both paired end readings and single reads.A total of 191,426 contigs were constructed using Trinity.
[0307] To identify alcohol acyltransferases, a database of cDNA sequences was created using Citrus bergamia contigs. In this database, the TBLASTN program was deployed to identify cDNA sequences encoding proteins identical to the alcohol acyltransferase (AAT) (AAW31948.1) protein sequence, particularly from Rosa hybrida. A total of 14 contigs were identified in C. bergamia that exhibited significant homology to alcohol acyltransferase (AAT) and encoded full-length proteins. These 14 contigs were further characterized by analyzing them using the BLASTX program to align them with protein sequences present in the UniProt database (downloaded August 28, 2019).
[0308] Full-length open reading frames were amplified from cDNA of Citrus bergamia. Forward and reverse primers, as shown in Table 3, were designed and used to amplify the entire translational region in plasmid pACYC-DUET-1 (Novagen) so that the reading frame was fused to the C-terminus of the His-6 tag. Cloned mutants were analyzed by sequencing of alcohol acyltransferase (AAT) inserts. A total of 37 different alcohol acyltransferase (AAT) open reading frames (ORFs) were cloned. Three different closely related cDNAs were obtained using primers AAT9-1fw (SEQ ID NO: 9) and AAT9-1re (SEQ ID NO: 11), and primers AAT9-2fw (SEQ ID NO: 10) and AAT9-2re (SEQ ID NO: 12) (see Table 1).
[0309] [Table 3]
[0310] Three different alcohol acyltransferase (AAT) mutants obtained using primer pairs AAT9-1fw (SEQ ID NO: 9) and AAT9-1re (SEQ ID NO: 11) from Citrus bergamia show the following sequences:
[0311] Sequence ID 1 corresponds to the nucleotide sequence of alcohol acyltransferase (AAT) (mutant AAT9-1-c) from Citrus bergamia. Sequence ID 2 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (mutant AAT9-1-c AAT9-1-c) from Citrus bergamia.
[0312] Sequence ID 5 corresponds to the nucleotide sequence of alcohol acyltransferase (AAT) (mutant AAT9-1-a) from Citrus bergamia. Sequence ID 6 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (mutant AAT9-1-a) from Citrus bergamia.
[0313] Sequence ID 7 corresponds to the nucleotide sequence of alcohol acyltransferase (AAT) (mutant AAT9-2-a) from Citrus bergamia. Sequence ID 8 corresponds to the amino acid sequence of alcohol acyltransferase (AAT) (mutant AAT9-2-a) from Citrus bergamia.
[0314] Figure 1 shows the multiple sequence alignment of the amino acid sequences of the alcohol acyltransferase (AAT) mutants AAT9-1-c (SEQ ID NO: 2), AAT9-1-a (SEQ ID NO: 6), and AAT9-2-a (SEQ ID NO: 8) from Citrus bergamia. At these positions, indicated in bold and italics, amino acids such as those found in SEQ ID NO: 2 are preferred in the AAT enzyme of the present invention.
[0315] Figure 2 shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the alcohol acyltransferase (AAT) mutant AAT9-1-c from Citrus bergamia.
[0316] Example 2: Activity of alcohol acyltransferase mutant AAT9-1-c (SEQ ID NO: 2) from Citrus bergamia Subsequently, three different alcohol acyltransferase (AAT) mutants obtained using primer pairs AAT9-1fw (SEQ ID NO: 9) and AAT9-1re (SEQ ID NO: 11) from Citrus bergamia, possessing SEQ ID NOs: 2, 6, and 8, were tested for their ability to convert linalool to linalyl acetate. For this purpose, the three different mutants and empty pACYC-DUET-1 were introduced into chemically competent E. coli BL21-RIL (Stratagene) by heat shock transformation and selected on LB agar containing 1% glucose and 50 ul / ml chloramphenicol. The transformants were transferred to 5 ml of LB liquid medium containing 1% glucose and 50 ul / ml chloramphenicol and grown overnight at 37°C at 250 rpm.
[0317] 200 μL of these cultures were transferred to 20 mL of LB medium containing the appropriate antibiotic in a 100 mL Ehrenmeier flask, and incubated at 37°C at 250 rpm until the A600 was 0.4–0.6. Subsequently, 1 mM IPTG supplemented with 5 mM linalool (racemic) and 2 mL of dodecane were added, and the cultures were incubated overnight at 18°C at 250 rpm. The following day, the dodecane layer was collected by centrifugation (8000 x g for 10 minutes), diluted in ethyl acetate, and analyzed by GC FID.
[0318] Interestingly, the alcohol acyltransferase (AAT) mutant AAT9-1-c (SEQ ID NO: 2) showed a significant decrease in linalool (Rt = 11.15 min) and the appearance of a peak corresponding to linalyl acetate (15.45 min) (Figure 3). Clearly, linalool was converted to linalyl acetate when AAT9-1-c was expressed. In one experiment, approximately 65% by mass of linalool was esterified, and in another experiment, according to the inventors' preliminary estimate, approximately 78% by mass of linalool was esterified.
[0319] Such transformations were not observed when an empty vector was expressed or when AAT9-1-a (SEQ ID NO: 6) or AAT9-2-a (SEQ ID NO: 8) was expressed, revealing that changes at key positions in the AAT enzyme can impair its function. Preferred amino acid types at key positions in the AAT enzyme of the present invention are given in Tables 1 and 2.
[0320] Since it has been established that AAT9-1-c (SEQ ID NO: 2) is active against linalool, a set of monoterpene and sesquiterpene alcohols was tested in the presence of E. coli cells expressing AAT9-1-c or empty pACYCDUET-1. Activity of AAT9-1-c (SEQ ID NO: 2) was observed against the monoterpene alcohols geraniol, alpha-terpineol, and verbenol, and the corresponding esters could be observed.
[0321] No conversion was observed for the sesquiterpene alcohols nerolidol, pacholol, (-)-alpha-bisabolol, and cedrol to their corresponding esters.
[0322] Therefore, AAT9-1-c (SEQ ID NO: 2) appears to be a monoterpene alcohol-specific acyltransferase that accepts primary, secondary, and tertiary alcohols. This result is particularly surprising, for example, since linalool is not a commonly recognized substrate for AAT enzymes described in the art. The exceptional properties of linalool that define it as a non-substrate for these AATs may arise from the position of the alcohol group in linalool, which can be considered a tertiary alcohol: the carbon to which the acceptor alcohol group is linked is bonded to three carbon groups. Tertiary alcohols are often more difficult to access from the enzyme active site pocket, possibly due to the steric accessibility of the alcohol group. Advantageously, the alcohol acyltransferase mutant AAT9-1-c (SEQ ID NO: 2) from Citrus bergamia is capable of esterifying linalool to linalyl acetate, as shown in Figure 3.
[0323] Example 3: Sequence comparison with alcohol acyltransferase mutant AAT9-1-c (SEQ ID NO: 2) from Citrus bergamia. Protein sequence alignment was performed between the alcohol acyltransferase mutant AAT9-1-c (SEQ ID NO: 2) from Citrus bergamia and its inactive mutants AAT9-1-a (SEQ ID NO: 6) and AAT9-2-a (SEQ ID NO: 8) (Figure 1). AAT9-1-c (SEQ ID NO: 2) and AT9-1-a (SEQ ID NO: 6) differ by only one amino acid at position 371, and therefore share 99.77% identity. Position 371 of AT9-1-a (SEQ ID NO: 6) is filled with tryptophan. This is interesting because other candidates for the transferase also have tryptophan at the position corresponding to position 371 or 372 in SEQ ID NO: 2. The DFGWG motif has been described in the art for the BAHD enzyme superfamily (Ma et al. (2005), Journal of Biological Chemistry, Vol 280, pages 13576-13583), and the region around position 371 in AAT9-1-c (SEQ ID NO: 2) and AT9-1-a (SEQ ID NO: 6) is the expected region for this DFGWG motif. AAT9-1c (SEQ ID NO: 2) is 81.60% identical to AAT9-2-a (SEQ ID NO: 8).
[0324] BLASTP analysis of the AAT9-1-c sequence (SEQ ID NO: 2) against the SWISSPROT database of characterized proteins (updated 2020 / 09 / 15) revealed that the most closely related proteins are stemadenine O-acetyltransferase (CrSAT) from Catharanthus roseus (A0A2P1GIW7.1; 34.3% identical), vinolin synthase from Rauvolfia serpentine (Q70PR7.2; 35.7%), minovincinin 19-hydroxy-O-acetyltransferase from Catharanthus roseus (Q8GZU0.1; 35.4%), and Papaver somniferum. It is revealed that the enzyme encodes saltaridinol 7-O-acetyltransferase (salAT) (Q94FT4.1; 34.5%) from somniferum. Each of these enzymes is involved in the acetylation of highly complex alkaloid molecules (compared to linalool), and the inventors define them as part of the vinolin synthase family. This finding was unexpected, as the novel alcohol acyltransferase enzymes of the present invention are most closely related to the family of alcohol acyltransferases (AATs) that act on highly complex phenolic substances such as saltaridinol.
[0325] BLASTP analysis of the AAT9-1-c sequence (SEQ ID NO: 2) against the GenBank database (updated 2020 / 09 / 14) revealed that the most closely related sequences are a vinolin synthase-like sequence from Citrus sinensis (XP_006493396.1; 88.5%), a hypothetical protein CUMW_168950 from Citrus unshiu (GAY56063.1; 88.3%), a hypothetical protein CISIN_1g044243mg from Citrus sinensis (KDO41287.1; 82.16%), and Citrus clementina (Citrus It has become clear that these proteins encode proteins from citrus species, including one annotated as vinolin synthase (XP_006423966.1; 81.9%) from clementina. Clearly, the function of these proteins has not yet been identified, other than by their homology to alcohol acyltransferases.
[0326] Example 4: Identification of alcohol acyltransferase in Lavandula angustifolia The inventors determined that, similar to bergamot, members of the vinolin synthase family were candidates for alcohol acyltransferase in the genus Lavandula. Therefore, using the RhAAT protein sequence (AAW31948.1), they searched the TBLASTN for sequence data present in the Transcriptome Shotgun Assembly of BioProject PRJNA391145 (https: / / www.ncbi.nlm.nih.gov / bioproject / ?term=txid1196215[Organism:noexp]), revealing a set of 16 proteins. Among these, two protein sequences showed significant homology to AAT9-1-c (SEQ ID NO: 2), encoded by ctg1461 (41.9% identical) and ctg10056 (35.3%). As expected, manual editing of the incomplete protein sequences yielded a sequence considered to be a candidate linalool alcohol acyltransferase. Sequence ID 3 shows the amino acid sequence of alcohol acyltransferase (AAT) (10056) from Lavandula angustifolia. Sequence ID 4 shows the amino acid sequence of alcohol acyltransferase (AAT) (1461) from Lavandula angustifolia. The sequences mentioned are shown in Figure 4. However, initial tests showed that the two alcohol acyltransferases of Sequence IDs 3 and 4 do not prefer linalool as a substrate, but can act with other alcohols.
[0327] Figure 5 shows the multiple sequence alignment of the amino acid sequence of the linalool acetyltransferase mutant AAT9-1-c from Citrus bergamia (SEQ ID NO: 2), the amino acid sequence of alcohol acyltransferase (AAT) (10056) from Lavandula angustifolia (SEQ ID NO: 3), and the amino acid sequence of alcohol acyltransferase (AAT) (1461) from Lavandula angustifolia (SEQ ID NO: 4). Synthetic sequences with mutations were constructed compared to SEQ ID NOs. 3 and 4, respectively (SEQ ID NOs. 15 and 16, respectively). Based on alignment with linalool acetyltransferase AAT9-1-c (SEQ ID NOs. 2) and other sequences of SEQ ID NOs. 13 and 14, the inventors intentionally substituted the tryptophan in SEQ ID NOs. 3 and 4 with the amino acid found at the corresponding position in SEQ ID NOs. 2. Sequence IDs 15 and 16 will be tested for the conversion of linalool to linalyl acetate and for the activity of other alcohol acyltransferases.
Claims
1. a) The amino acid sequence shown in Sequence ID No. 2; and b) An amino acid sequence having alcohol acyltransferase activity that has at least 90% sequence identity at the amino acid level with SEQ ID NO: 2; An alcohol acyltransferase comprising an amino acid sequence selected from the group consisting of, An alcohol acyltransferase capable of esterifying a tertiary monoterpene alcohol so that at least 30% by mass of the tertiary monoterpene alcohol is esterified.
2. The alcohol acyltransferase according to claim 1, which is capable of esterifying a tertiary monoterpene alcohol within 36 hours so that at least 30% by mass of the tertiary monoterpene alcohol is esterified.
3. The alcohol acyltransferase according to claim 1, which is capable of esterifying a tertiary monoterpene alcohol in microbial cells so that at least 30% by mass of the tertiary monoterpene alcohol is esterified.
4. The alcohol acyltransferase according to any one of claims 1 to 3, which is capable of esterifying a tertiary monoterpene alcohol at 30°C and a pH in the range of 6.0 to 8.5 so that at least 50 μg of monoterpene ester is produced per minute per gram of alcohol acyltransferase.
5. A nucleic acid comprising a nucleic acid sequence encoding an alcohol acyltransferase according to any one of claims 1 to 4, or a complementary sequence thereof.
6. A vector or gene construct comprising the nucleic acid described in claim 5.
7. A host cell comprising the vector or gene construct according to claim 6, which is a bacterial cell, yeast cell, fungal cell, algal cell or cyanobacterial cell, non-human animal cell or non-human mammalian cell or plant cell.
8. The host cell according to claim 7, which is a bacterial cell, a yeast cell, or a fungal cell.
9. A transgenic non-human organism comprising the nucleic acid described in claim 5, the vector or gene construct described in claim 6, or the host cell described in claim 7 or 8.
10. A method for preparing monoterpene esters, the following: (i) a) Having the amino acid sequence shown in Sequence ID No. 2; or b) Having an amino acid sequence that has alcohol acyltransferase activity and has at least 90% sequence identity with SEQ ID NO: 2 at the amino acid level, Alcohol acyltransferase; Or, (ii) an alcohol acyltransferase of (i) in which the amino acids corresponding to the amino acids at positions 371 and 372 of SEQ ID NO: 2 are not tryptophan, or (iii) an alcohol acyltransferase of (i) that further comprises any of the amino acids listed in Table 1 at the position corresponding to the position of SEQ ID NO: 2 shown in Table 1, or an amino acid listed in Table 2 at the position corresponding to the position of SEQ ID NO: 2 shown in Table 2, and is capable of esterifying a monoterpene alcohol to a monoterpene ester; (iv) any of the alcohol acyltransferases (i) to (iii) capable of esterifying a monoterpene alcohol so that at least 30% by mass of the monoterpene alcohol is esterified; or (v) Any of the alcohol acyltransferases (i) to (iv) capable of esterifying a monoterpene alcohol at 30°C and a pH in the range of 6.0 to 8.5, so that at least 50 μg of monoterpene ester is produced per minute per gram of alcohol acyltransferase. A method comprising esterifying a monoterpene alcohol to a monoterpene ester in the presence of [a specific substance].
11. The method according to claim 10, wherein the alcohol acyltransferase (iv) can esterify the monoterpene alcohol within 36 hours so that at least 30% by mass of the monoterpene alcohol is esterified.
12. The method according to claim 10, wherein the alcohol acyltransferase of (iv) can esterify a monoterpene alcohol in a microbial cell so that at least 30% by mass of the monoterpene alcohol is esterified.
13. The method according to any one of claims 10 to 12, wherein the monoterpene alcohol is a primary, secondary, or tertiary monoterpene alcohol.
14. The method according to claim 13, wherein the monoterpene alcohol is a tertiary monoterpene alcohol.
15. A method for preparing linalyl acetate, the following: (i) a) Having the amino acid sequence shown in Sequence ID No. 2; or b) Having linalool acyltransferase activity and an amino acid sequence having at least 90% sequence identity at the amino acid level with SEQ ID NO: 2, Alcohol acyltransferase; or (ii) The alcohol acyltransferase of (i) in which the amino acids corresponding to the amino acids at positions 371 and 372 of SEQ ID NO: 2 are not tryptophan, (iii) an alcohol acyltransferase of (i) that further comprises any of the amino acids listed in Table 1 for the positions corresponding to the positions of SEQ ID NO: 2 shown in Table 1, or an amino acid listed in Table 2 for the positions corresponding to the positions of SEQ ID NO: 2 shown in Table 2, and is capable of esterifying linalool to linalyl acetate; or (iv) any of (i) to (iii) alcohol acyltransferases capable of esterifying linalool so that at least 30% by mass of linalool is esterified; or (v) Any of the alcohol acyltransferases (i) to (iv) capable of esterifying linalool at 30°C and a pH in the range of 6.0 to 8.5, so that at least 50 μg of linalyl acetate is produced per minute per gram of alcohol acyltransferase. A method comprising esterifying linalool to linalyl acetate in the presence of [a specific substance].
16. The method according to claim 15, wherein the alcohol acyltransferase (iv) can esterify linalool within 36 hours so that at least 30% by mass of linalool is esterified.
17. The method according to claim 15, wherein the alcohol acyltransferase (iv) can esterify linalool in microbial cells so that at least 30% by mass of linalool is esterified.
18. The method according to any one of claims 10 to 17, wherein the alcohol acyltransferase is used in combination with GPP synthase and / or S- or R-linalool synthase.
19. The method according to any one of claims 10 to 17, wherein the monoterpene ester according to claim 10 or linalyl acetate according to claim 15 is prepared in a host cell according to claim 7 or 8 or a transgenic non-human organism according to claim 9 that expresses the alcohol acyltransferase.
20. (i) For heterologous reconstruction of the terpene biosynthesis pathway; (ii) For the manufacture of industrial products; (iii) For the preparation of aliphatic and / or aromatic monoterpene esters from monoterpene alcohols; (iv) For detoxifying monoterpene alcohols in microorganisms and thereby increasing monoterpene production in said microorganisms; (v) Combined with GPP synthase and / or S- or R-linalool synthase; (vi) To enhance the beneficial effect of acetylation in that the hydrophobic acetic acid partitioning proceeds more easily to the organic phase compared to the monoterpene alcohols; (vii) for expressing an alcohol acyltransferase according to any one of claims 1 to 4 such that the ratio of monoterpene acetate to monoterpene alcohol is greater than 5:1 or 10:1; or (viiii) In a microbial production system for producing monoterpene esters, Use of an alcohol acyltransferase according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector or gene construct according to claim 6, a host cell according to claim 7 or 8, or a transgenic non-human organism according to claim 9.
21. Industrial products are selected from flavorings or fragrances, biofuels, fuel compositions, fuel compounds, pesticides, insect repellents or antimicrobial agents; or A monoterpene alcohol is a tertiary monoterpene alcohol; or The microorganism is either a bacterium or a fungus. The use described in claim 20.
22. A kit comprising an alcohol acyltransferase according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector and / or gene construct according to claim 6, a host cell according to claim 7 or 8 and / or a transgenic non-human organism according to claim 9.
23. The kit according to claim 22, further comprising at least one monoterpene alcohol.
24. The kit according to claim 23, wherein the monoterpene alcohol is a tertiary monoterpene alcohol.
25. A method for producing fuel and / or biolubricant compounds, a) Prepare one or more monoterpene esters by any one of the methods described in any one of claims 10 to 19; b) Optionally, purify one or more monoterpene esters prepared in step a); c) Using some or all of one or more monoterpene esters from step a) or one or more optionally purified monoterpene esters from step b) as a fuel and / or biolubricant compound; and / or converting some or all of one or more monoterpene esters from step a) or one or more optionally purified monoterpene esters from step b) into one or more fuel and / or biolubricant compounds. A method that includes steps.