Recombinant host systems for the production of aleuritic acid and methods therefor

Abstract

The present disclosure provides a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid), wherein the host system is transformed with: (i) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxide hydrolase enzyme and the monooxygenase enzyme, or (ii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the bifunctional epoxygenase hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme. The present disclosure further provides methods for producing aleuritic acid using the recombinant host system.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates primarily to the field of synthetic biology. In particular, the present disclosure provides materials and methods for the synthesis of aleuritic acid. BACKGROUND OF THE INVENTION

[0002] The primary acid component of lac resin produced by the Indian lac bug Kerria lacca is aleuritic acid (9,10,16-trihydroxyhexadecanoic acid). Aleuritic acid is a unique acid with three hydroxyl groups, two of which are adjacent carbon atoms. Aleuritic acid is a valuable molecule because it is used as a starting material for the synthesis of macrocyclic perfume compounds such as ambrettolide, iso-ambrettolide, civetone, dehydrocivetone, exaltone, glucose manoaleuritate and related lactones, insect sex pheromones, pharmaceutical chemicals, cosmetics, esters, metal salts and stabilizers, plant growth regulators, and biodegradable polymers such as poly(aleuritic acid), polyhydroxyalkanoic acid (PHA), etc. (Kun Li et al. 2019 Mater. Res. Express 6 075328).The current approach of purifying aleuritic acid to the required quantity by alkaline hydrolysis of lacquer resin is complex and time-consuming (Characterization of Different Shellac Types and Development of Shellac-Coated Dosage Forms, Dissertation, Hamburg 2010). Therefore, there is still a need for an alternative route for the bioconversion or de novo biosynthesis of aleuritic acid.

[0003] The production of an intermediate of the aleuritic acid pathway, 9,10-dihydroxyhexadecanoic acid, was demonstrated by the in vitro reconstitution of three different enzymes using an E. coli-based expression system (Kaprakkaden A, Srivastava P, Bisaria VS. In vitro synthesis of 9, 10-dihydroxyhexadecanoic acid using recombinant Escherichia coli. Microb Cell Fact. 2017 May 18; 16(1):85. doi: 10.1186 / s12934-017-0696-7).

[0004] Patent document WO2019217226A1 discloses recombinant microorganisms expressing a heterologous biochemical pathway comprising: (i) a delta-12 fatty acid epoxygenase and an epoxide hydrolase, (ii) a heterologous FatA thioesterase, (iii) an acyl-CoA synthetase, (iv) an ester synthase, and (v) a cypl53A cohydroxylase from Marinobacter aquaeolei for the production of 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid).

[0005] However, the known methods do not provide an efficient and time-saving method for producing aleuritic acid for large-scale commercial applications. Therefore, there is an urgent need to accurately characterize the biosynthetic pathway of aleuritic acid in a recombinant host system to increase the efficiency of aleuritic acid production. SUMMARY OF THE INVENTION

[0006] In one aspect of the 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid), the host system is transformed with: (i) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxide hydrolase enzyme and the monooxygenase enzyme, or (ii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the bifunctional epoxygenase hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme.

[0007] In one aspect of the present disclosure, there is provided a method for producing 9,10,16-trihydroxyhexadecanoic acid, comprising: growing the recombinant host system as disclosed herein in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, monooxygenase enzyme, or combinations thereof.

[0008] In one aspect of the present disclosure, there is provided a method for producing 9,10,16-trihydroxyhexadecanoic acid, comprising: growing the recombinant host system as disclosed herein in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, bifunctional epoxygenase hydrolase enzyme, monooxygenase enzyme, or combinations thereof.

[0009] These and other features, aspects, and advantages of the present subject matter will be better understood by reference to the following description and the appended claims. This summary is intended to present a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE CHARACTERS

[0010] The following figures are part of this specification and serve to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the figures in combination with the detailed description of the specific embodiments presented herein. Fig.Figure 1 illustrates the identified biosynthetic pathway (1) for aleuritic acid, involving at least four types of enzymes: 1. fatty acid desaturase, 2. epoxygenase, 3. epoxyhydrolase, and 4. cytochrome P450 monooxygenase (CYPs), in accordance with one embodiment of the present disclosure. Fig. Figure 2 illustrates the identified biosynthetic pathway (2) for aleuritic acid involving three types of enzymes: 1. fatty acid desaturase, 2. bifunctional epoxygenase / hydrolase, and 3. cytochrome P450 monooxygenase, in accordance with one embodiment of the present disclosure. Fig. 3A-E illustrate the strategy for producing aleuritic acid using biosynthetic pathways 1 and 2 according to one embodiment of the disclosure. Fig.Figures 5A-G show the gas chromatography-mass spectrometry (GC-MS) analysis of intermediates and final products of the different aleuritic acid preparation processes, where A) represents the elution peaks of 1) aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, and 4) 9,10-dihydroxyhexadecanoic acid; B) shows the elution peaks of 1) aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, 4) 9,10-dihydroxyhexadecanoic acid, and 5) 9,10-epoxyhexadecanoic acid; C) represents the mass spectrum of aleuritic acid; D) represents the mass spectrum of palmitic acid; E) represents the mass spectrum of palmitoleic acid; F) represents the mass spectrum of 9,10-dihydroxyhexadecanoic acid; and G) represents the mass spectrum of 9,10-epoxyhexadecanoic acid. according to an embodiment of the disclosure. Fig. 6A-B show the plasmid map with the gene combinations according to route 1 in accordance with an embodiment of the disclosure. Fig.7A-B show the plasmid map with the gene combinations according to route 2 in accordance with an embodiment of the disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0011] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those expressly described. It is intended that the present disclosure encompass all such variations and modifications. The disclosure also encompasses all steps, features, compositions, and compounds referred to or recited in this specification, individually or collectively, as well as all combinations of any or all of these steps or features. Definitions

[0012] For convenience, before further describing the present disclosure, certain terms used in the description and examples are defined herein. These definitions should be read and understood in light of the remainder of the disclosure, as by a person skilled in the art. The terms used herein have the meaning known to those skilled in the art. However, for convenience and completeness, certain terms and their meanings are listed below.

[0013] The articles “a”, “an” and “der” are used to refer to one or more (i.e. at least one) of the grammatical objects of the article.

[0014] Throughout this specification, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "including" are used in a broad, open-ended sense and are to be understood as implying the inclusion of a specified element or step, or group of elements or steps, but not the exclusion of any other element or step, or group of elements or steps. It is not intended to mean "consists only of."

[0015] The term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0016] The term "nucleic acid," as used herein, refers to a combination of nucleotide monomers linked together by covalent bonds, as in DNA or RNA. The terms "nucleic acid" and "polynucleotide" are used interchangeably.

[0017] The term “gene” as used here refers to nucleic acid sequences, such as DNA sequences, that encode either an RNA product or a protein product.

[0018] As used herein, the term "recombinant host system" refers to a host cell that has been genetically modified or manipulated such that certain enzymatic activities within the host cell have been altered, added, and / or removed compared to the parent or native host cell. A genetically modified or genetically manipulated host cell is an example of a recombinant host system.

[0019] As used herein, the term "heterologous" means that a polynucleotide or polypeptide sequence originates from another species or organism, or from another source. As used herein, this term refers to a nucleotide sequence or polypeptide sequence that does not occur naturally in a particular organism.

[0020] The term "vector" as used here refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence to which it has been bound. One type of useful vector is an episome (i.e., a nucleic acid capable of extrachromosomal replication). Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." The terms "plasmid" and "vector" are used interchangeably here, as a plasmid is the most commonly used form of vector.

[0021] The term "expression," as used herein with reference to a gene, refers to the production of one or more transcription and / or translation products of a gene. The terms "expression" or "expressed" are used interchangeably.

[0022] As used herein, the term "simple carbon source" refers to a substrate or compound that serves as an energy source for the growth of prokaryotic or simple eukaryotic cells. A simple carbon source, as used herein, may be glucose, sucrose, galactose, lactose, fructose, or a combination thereof.

[0023] Ratios, concentrations, quantities, and other numerical data may be presented here in a range format. It is understood that such a range format is used solely for simplicity and brevity and should be flexibly interpreted to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly stated.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. While any methods and materials similar or equivalent to those described herein may be used in the practice or examination of the disclosure, the preferred methods and materials are described now. All publications mentioned herein are incorporated by reference.

[0025] The scope of the present disclosure is not limited to the specific embodiments described herein, which are merely illustrative. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure described herein.

[0026] As mentioned above, the conventional materials and methods for producing aleuritic acid suffer from various limitations. The traditional method of extracting aleuritic acid from insect resin is a laborious process. Alternative methods have been explored that utilize recombinant microorganisms to produce aleuritic acid in vitro, thus eliminating the dependence on insect resins. However, these genetically engineered alternatives need to be improved in terms of synthesis efficiency and cost-effectiveness.

[0027] To solve the above-mentioned problems, the present disclosure discloses recombinant host systems comprising heterologous enzymes from different organisms that can be cultured using a simple carbon source to produce aleuritic acid. The present disclosure also discloses a process for producing aleuritic acid.

[0028] Consequently, the present disclosure solves the existing problems by providing precise methods using recombinant host systems for efficient and large-scale production of aleuritic acid.

[0029] In one embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, monooxygenase enzyme, bifunctional epoxygenase hydrolase enzyme, or combinations thereof.

[0030] In one embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, and monooxygenase enzyme.

[0031] In another embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, bifunctional epoxygenase hydrolase enzyme, and monooxygenase enzyme.

[0032] In one embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, monooxygenase enzyme, or combinations thereof, wherein the host system is transformed with: (i) a recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, and monooxygenase enzyme, or (ii) a first recombinant expression vector comprising a nucleic acid sequence encoding the desaturase enzyme, a second recombinant expression vector comprising a nucleic acid sequence encoding the epoxygenase enzyme, a third recombinant expression vector comprising a nucleic acid sequence encoding the epoxide hydrolase enzyme,and a fourth recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme, or (iii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxide hydrolase enzyme and the monooxygenase enzyme, or (iv) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxide hydrolase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxygenase enzyme and the monooxygenase enzyme, or (v) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the monooxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences,which encode the epoxygenase enzyme and the epoxide hydrolase enzyme, or (vi) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme, monooxygenase enzyme and epoxygenase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the epoxide hydrolase enzyme, or (vii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme, monooxygenase enzyme and epoxide hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the epoxygenase enzyme, or (viii) a first recombinant expression vector comprising nucleic acid sequences encoding the epoxygenase enzyme, monooxygenase enzyme and epoxide hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the desaturase enzyme,or (ix) a first recombinant expression vector comprising nucleic acid sequences encoding the epoxygenase enzyme, desaturase enzyme, and epoxide hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme; wherein the nucleic acid sequence encoding the desaturase enzyme is selected from SEQ ID NO: 27, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31; wherein the nucleic acid sequence encoding the epoxygenase enzyme is selected from SEQ ID NO: 16, SEQ ID NO: 9, SEQ ID NO: 17, or SEQ ID NO: 18; wherein the nucleic acid sequence encoding the epoxide hydrolase enzyme is selected from SEQ ID NO: 19, SEQ ID NO: 15, SEQ ID NO: 21 or SEQ ID NO: 23; and wherein the nucleic acid sequence encoding the monooxygenase enzyme is selected from SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO:20 oder SEQ ID NO:24.,

[0033] In one embodiment of the present disclosure, a recombinant host system is provided which has been developed for the production of 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid), wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, bifunctional epoxygenase hydrolase enzyme, monooxygenase enzyme or combinations thereof, wherein the host system is transformed with: (i) a recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme, bifunctional epoxygenase hydrolase enzyme and monooxygenase enzyme, or (ii) a first recombinant expression vector comprising a nucleic acid sequence encoding the desaturase enzyme, a second recombinant expression vector comprising a nucleic acid sequence encoding the bifunctional epoxygenase hydrolase enzyme encoded, and a third recombinant expression vector comprising a nucleic acid sequence,which encodes the monooxygenase enzyme, or (iii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the bifunctional epoxygenase hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme, or (iv) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the monooxygenase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the bifunctional epoxygenase hydrolase enzyme, or (v) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and a second recombinant expression vector comprising nucleic acid sequences,which encode the monooxygenase enzyme and the bifunctional epoxygenase hydrolase enzyme; wherein the nucleic acid sequence encoding the desaturase enzyme is selected from SEQ ID NO: 27, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31; wherein the nucleic acid sequence encoding the bifunctional epoxygenase hydrolase enzyme is selected from SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and wherein the nucleic acid sequence encoding the monooxygenase enzyme is selected from SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 24.,

[0034] In one embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is transformed with: (i) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxide hydrolase enzyme and the monooxygenase enzyme, or (ii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the bifunctional epoxygenase hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme.

[0035] In a further embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is transformed with: a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxide hydrolase enzyme and the monooxygenase enzyme; wherein the nucleic acid sequence encoding the desaturase enzyme is selected from SEQ ID NO: 27, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31; wherein the nucleic acid sequence encoding the epoxygenase enzyme is selected from SEQ ID NO: 16, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 18;wherein the nucleic acid sequence encoding the epoxide hydrolase enzyme is selected from SEQ ID NO:19, SEQ ID NO:15, SEQ ID NO:21, or SEQ ID NO:23; and wherein the nucleic acid sequence encoding the monooxygenase enzyme is selected from SEQ ID NO:13, SEQ ID NO:22, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, or SEQ ID NO:24.

[0036] In a further embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is transformed with: a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the bifunctional epoxygenase hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme; wherein the nucleic acid sequence encoding the desaturase enzyme is selected from SEQ ID NO: 27, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31; wherein the nucleic acid sequence encoding the bifunctional epoxygenase hydrolase enzyme is selected from SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8;and wherein the nucleic acid sequence encoding the monooxygenase enzyme is selected from SEQ ID NO:13, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.;

[0037] In one embodiment of the present disclosure, a recombinant host system for producing 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid) is provided, wherein the host system is selected from E. coli, S. cerevisiae, Yarrowia lipolytica, Rhodotorula toruloides, Pichia pastoris, Candida boidinii or Kluyveromyces lactis.

[0038] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid using the recombinant host system disclosed herein is provided.

[0039] In one embodiment of the present disclosure, there is provided a process for producing 9,10,16-trihydroxyhexadecanoic acid, comprising: growing the recombinant host system as disclosed herein in a culture medium comprising a simple carbon source.

[0040] In one embodiment of the present disclosure, a method for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising: growing a recombinant host system in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, monooxygenase enzyme, or combinations thereof.

[0041] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, wherein the culture medium is optionally supplemented with hexadecanoic acid.

[0042] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, wherein the culture medium is optionally supplemented with 9-hexadecenoic acid.

[0043] In one embodiment of the present disclosure, a method for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising: culturing a recombinant host system in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, monooxygenase enzyme, or combinations thereof; wherein the culture medium is optionally supplemented with hexadecanoic acid; and wherein the culture medium is optionally supplemented with 9-hexadecenoic acid.

[0044] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding a desaturase enzyme to a substrate comprising hexadecanoic acid to convert hexadecanoic acid to 9-hexadecanoic acid, adding an epoxygenase enzyme to 9-hexadecenoic acid to convert 9-hexadecenoic acid to 9,10-epoxyhexadecanoic acid, adding an epoxide hydrolase enzyme to 9,10-epoxyhexadecanoic acid to convert 9,10-epoxyhexadecanoic acid to 9,10-dihydroxyhexadecanoic acid, and adding a monooxygenase enzyme to 9,10-dihydroxyhexadecanoic acid to convert 9,10-dihydroxyhexadecanoic acid to 9,10,16-trihydroxyhexadecanoic acid.

[0045] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding an epoxygenase enzyme to a substrate comprising 9-hexadecenoic acid to convert 9-hexadecenoic acid to 9,10-epoxyhexadecanoic acid, adding an epoxide hydrolase enzyme to 9,10-epoxyhexadecanoic acid to convert 9,10-epoxyhexadecanoic acid to 9,10-dihydroxyhexadecanoic acid, and adding a monooxygenase enzyme to 9,10-dihydroxyhexadecanoic acid to convert 9,10-dihydroxyhexadecanoic acid to 9,10,16-trihydroxyhexadecanoic acid.

[0046] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding an epoxide hydrolase enzyme to a substrate comprising 9,10-epoxyhexadecanoic acid to convert 9,10-epoxyhexadecanoic acid to 9,10-dihydroxyhexadecanoic acid, and adding a monooxygenase enzyme to 9,10-dihydroxyhexadecanoic acid to convert 9,10-dihydroxyhexadecanoic acid to 9,10,16-trihydroxyhexadecanoic acid.

[0047] In one embodiment of the present disclosure, a process for preparing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of: adding a monooxygenase enzyme to a substrate comprising 9,10-dihydroxyhexadecanoic acid to convert 9,10-dihydroxyhexadecanoic acid to 9,10,16-trihydroxyhexadecanoic acid.

[0048] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding a desaturase enzyme, epoxygenase enzyme, epoxide hydrolase, and monooxygenase enzyme to a substrate comprising hexadecanoic acid.

[0049] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding an epoxygenase enzyme, epoxide hydrolase enzyme, and monooxygenase enzyme to a substrate comprising 9-hexadecenoic acid.

[0050] In one embodiment of the present disclosure, a method for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of: culturing a recombinant host system in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, bifunctional epoxygenase hydrolase enzyme, monooxygenase enzyme, or combinations thereof.

[0051] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid as disclosed herein is provided, wherein the culture medium is optionally supplemented with hexadecanoic acid.

[0052] In another embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid as disclosed herein is provided, wherein the culture medium is supplemented with hexadecanoic acid.

[0053] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid as disclosed herein is provided, wherein the culture medium is optionally supplemented with 9-hexadecenoic acid.

[0054] In another embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid as disclosed herein is provided, wherein the culture medium is supplemented with 9-hexadecenoic acid.

[0055] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding a desaturase enzyme to a substrate comprising hexadecanoic acid to convert hexadecanoic acid to 9-hexadecenoic acid, adding a bifunctional epoxygenase hydrolase enzyme to 9-hexadecenoic acid to convert 9-hexadecenoic acid to 9,10,16-dihydroxyhexadecanoic acid, and adding a monooxygenase enzyme to 9,10-dihydroxyhexadecanoic acid to convert 9,10-dihydroxyhexadecanoic acid to 9,10,16-trihydroxyhexadecanoic acid.

[0056] In one embodiment of the present disclosure, a process for producing 9,10,16-trihydroxyhexadecanoic acid is provided, comprising the steps of adding a bifunctional epoxygenase hydrolase enzyme to 9-hexadecenoic acid to convert 9-hexadecenoic acid to 9,10-dihydroxyhexadecanoic acid, and adding monooxygenase enzyme to 9,10-dihydroxyhexadecanoic acid to convert 9,10-dihydroxyhexadecanoic acid to 9,10,16-trihydroxyhexadecanoic acid.

[0057] In one embodiment of the present disclosure, a process for preparing 9,10,16-trihydroxyhexadecanoic acid as disclosed herein is provided, wherein the simple carbon source is selected from glucose, sucrose, galactose, lactose, fructose, or combinations thereof.

[0058] Although the subject matter has been described in great detail with reference to specific examples and implementations, other implementations are also possible. EXAMPLES

[0059] The disclosure will now be illustrated with working examples which are intended to illustrate the operation of the disclosure and are not to be taken restrictively to imply any limitations on the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice of the disclosed methods and compositions, the exemplary methods, apparatus, and materials are described herein. It is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may apply. Example 1Identification of the mechanism for the biosynthesis of aleuritic acid in the Indian lac bug Kerria lacca using bioinformatics tools

[0060] A mechanism for the biosynthesis of aleuritic acid in the Indian lac bug Kerria lacca has been proposed based on evidence from five different techniques.(a) GC-MS analysis of fatty acid methyl ester (FAME) in the resin-secreting (adult) and non-resin-secreting (crawling) life stages of the Lac insect, (b) use of broad-spectrum inhibitors for epoxide hydrolase (EH) and epoxygenase (EPOX) in combination with transsupply of likely intermediates, (c) enzymatic activity of fatty acid desaturase (FAD), epoxide hydrolase, and monooxygenase (MONO) in extracts from adult Lac insects compared to crawler extracts, (d) Western blotting for epoxygenase (EPOX), fatty acid desaturase, and epoxide hydrolase to verify the presence of these enzymes in adult Lac insects, and (e) quantitative PCR for FAD, EH, EPOX, and MONO genes to detect transcripts in adult Lac insects (Wang W, Liu P, Lu Q, Ling X, Zhang J, Chen MS, Chen H, Chen Insects.2019 Nov 28;10(12):430. doi: . 10.3390 / insects10120430). In the biosynthesis of aleuritic acid, the last two steps involve the epoxygenation of palmitoleic acid at carbon position 9, 10 to 9,10-epoxyhexadecanoic acid, followed by hydroxylation leading to the formation of 9,10-dihydroxyhexadecanoic acid. Cytochrome P450 enzymes (CYP) such as CYP94A1 are known to be bifunctional, catalyzing both the epoxidation and hydroxylation of fatty acids (Pinot F, Skrabs M, Compagnon V, Salaün JP, Benveniste I, Schreiber L, Durst F. Omega-hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defense. Biochem Soc Trans. 2000 Dec;28(6):867-70). The third step is the omega-hydroxylation of 9,10-dihydroxyhexadecanoic acid, which is catalyzed by the enzyme cytochrome P450 monooxygenase (CYPs). Fatty acids and their derivatives undergo many types of oxidation reactions, including hydroxylation, epoxidation, dehydration, and reduction.It is suspected that several forms of CYPs are involved in these reactions (Arrieta-Baez D, Cruz-Carrillo M, G6mez-Patifio MB, Zepeda-Vallejo LG. Derivatives of 10,16-dihydroxyhexadecanoic acid isolated from tomato (Solanum lycopersicum) as potential material for aliphatic polyesters. Molecules. 2011 Jun 15;16(6):4923-36. doi: 10.3390 / molecules1606492). Cytochrome P450-dependent monooxygenases from plants and other bacterial species such as Bacillus species, including B. megaterium, catalyze the in-chain and omega hydroxylation, as well as the epoxidation, of medium- and long-chain fatty acids (Kyoung-Rok Kim, Deok-Kun Oh, Production of hydroxy fatty acids by microbial fatty acid hydroxylation enzymes, Biotechnology Advances, Volume 31, Issue 8, 2013, Pages 1473 to 1485, https: / / doi.org / 10.1016 / j.biotechadv.2013.07.004). Several forms of cytochrome P450 are involved in these reactions, each with different substrate specificity.

[0061] To search for similar enzymes, bioinformatics tools such as CLC genomic workbench and other additional alignment software tools such as PRINTS (Attwood TK, Beck ME. PRINTS - a database for protein motif fingerprints. Protein Eng. 1994 Jul;7(7):841-8. doi: 10.1093 / protein / 7.7.841.) were used to identify relevant epoxygenases and epoxide hydrolases from the transcriptome sequence of Lac insects. Biological sequence motifs are short sequence patterns, usually of fixed length, that represent important structural or functional features in nucleic acid and protein sequences, e.g., transcription binding sites, splice junctions, active sites, or interaction interfaces. Motif sequence analysis was performed by extracting known epoxygenase-, hydrolase-, and omega-hydroxylation-specific CYP proteins from the Uniprot database and entering the extracted proteins as a query in PRINTS.After identifying the corresponding motif sequence from the PRINTS database, the data were entered into a Biopython program to extract the putative epoxide hydrolase, CYP, and epoxygenase proteins from the transcriptome of Lac insects. A total of eight putative epoxygenase and hydrolase enzyme sequences (SEQ ID NOs: 1-8) were identified in the Lac transcriptome. In general, epoxy fatty acids and hydrolyzed epoxy group diols are highly toxic to microorganisms, including E. coli and yeast. High concentrations of epoxy fatty acids can inhibit the growth of E. coli and kill it because of their physical effects on membranes and detergent properties and because they may also be acidic (Royce LA, Liu P, Stebbins MJ, Hanson BC, Jarboe LR. The deleterious effects of short-chain fatty acids on Escherichia coli membranes. Appl Microbiol Biotechnol. 2013 Sep;97(18):8317-27. doi: 10.1007 / s00253-013-5113-5).In the present publication, a shorter pathway bypassing epoxide formation was identified using bifunctional epoxy hydrolases from the lac transcriptome.

[0062] In addition to the enzymes identified from the Lac transcriptome, functional homologs from other species were also shortlisted, which were reported to exhibit similar fatty acid epoxidation and hydroxylation activities (Table 1). All shortlisted sequences were codon-optimized for expression in E. coli, and the gene was synthesized. Table 1 SEQ-ID ENZYME NAME ORGANISM SEQ ID NO: 1 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:2 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:3 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:4 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:5 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:6 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:7 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:8 Bifunctional Epoxyhydrolase Kerria lac SEQ ID NO:9 Epoxygenase White vetch SEQ ID NO:10 Monooxygenase Arabidopsis thaliana SEQ ID NO:11 Monooxygenase Glycine soja SEQ ID NO:12 Monooxygenase Arabidopsis thaliana SEQ ID NO:13 Monooxygenase Citrus clementine SEQ ID NO:14 Monooxygenase Citrus clementine SEQ ID NO:15 Epoxide Hydrolase Arabidopsis thaliana SEQ ID NO:16 Epoxygenase Euphorbia lagasca SEQ ID NO:17 Epoxygenase Homo sapiens SEQ ID NO:18 Epoxygenase Homo sapiens SEQ ID NO:19 Epoxide Hydrolase Myxococcus xanthus SEQ ID NO:20 Monooxygenase White rice SEQ ID NO:21 Epoxide Hydrolase Streptomyces peucetius SEQ ID NO:22 Monooxygenase Marinobacterhydrocarbonoclasticus SEQ ID NO:23 Epoxide-Hydrolase Mangifera indica SEQ ID NO:24 Monooxygenase Gordonia alcanivorans SEQ ID NO:25 Delta-9-Desaturation Psychrobacterurativorans SEQ ID NO:26 Delta-9-Desaturation Choristoneuraoccidentalis SEQ ID NO:27 Delta-9-Desaturation Pseudomonas aeruginosa SEQ ID NO:28 Delta-9-Desaturation Arabidopsis thaliana SEQ ID NO:29 Delta-9-Desaturation Caenorhabditis elegans SEQ ID NO:30 Delta-9-Desaturation Trypanosoma cruzi SEQ ID NO:31 Delta-9-Desaturation Linnemannia wins Example 2Identification of the best gene / pathway combination for the production of aleuritic acid in E. coli through in vitro studies

[0063] The aleuritic acid producing E. coli strain (recombinant host system) was constructed for experiments aimed at finding optimal combinations of biosynthesis genes derived from epoxygenase ( Fig. 3C), epoxide hydrolase ( Fig. 3D), bifunctional epoxy hydrolase ( Fig. 3A) and CYPs ( Fig.3B and E). The shortlisted genes were codon-optimized and gene-synthesized for E. coli. The individual genes were expressed under the T7 promoter in an episomal E. coli expression vector pET28+ and transformed into E. coli BL21 cells. The E. coli transformants were grown overnight at 37 °C in 1 ml of M9 minimal medium containing appropriate antibiotics (ampicillin (100 mg / L) and glucose (single carbon source)) in a 96-well format. The next day, 150 µl of each culture was inoculated into 3 ml of M9 minimal medium containing ampicillin (100 mg / L) and isopropyl-β-D-thiogalactopyranoside (IPTG) 0.5 mM in a 24-well format and incubated for 20 h at 30 °C and 250 rpm. Using the whole-cell lysate of the induced recombinant cultures, the feasibility of obtaining 9,10,16-dihydroxyhexadecanoic acid together with the provision of 9-hexadecenoic acid (palmitoleic acid) as a substrate was investigated.The induced cultures were lysed with the protein extraction reagent Bugbuster (Merck), and the lysed cultures were mixed in equal amounts with substrates such as 9-hexadecenoic acid / palmitoleic acid, 9,10-dihydroxyhexadecanoic acid, 9,10-epoxyhexadecanoic acid (200 µM each), and NADPH (0.1 µM) and incubated at 30 °C for 6 hours (Fig. Fig. 3A-3E). The cultured culture lysates were then extracted with hexane and ethyl acetate, and the extract was subjected to GC-MS analysis. GC-MS analysis

[0064] The metabolites were determined using the Agilent GCMS-6890N-5973 Plus under the following conditions: Column: RTX-5MS, 30 m; column oven temperature: 140 °C; injection temperature: 260 °C; injection mode: splitless; carrier gas: helium; oven program: 140 °C hold for 5 min; 4 °C / min 240 °C hold for 5 min; diluent: n-hexane; scan range: 40-650 m / z. The GC-MS results are shown in the Fig. 5A-G.

[0065] Table 2 provides details of the enzymes identified for the tested substrates and the products formed. Table 2 Enzyme Organismus SEQ ID GetSubstrate ReformedProduct Epoxygenase Euphorbialagascae SEQ IDNO: 16 Palmitolein saucers 9.10Epoxyhexadecane Epoxide-Hydrolase Arabidopsisthaliana SEQ IDNO: 15 9.10Epoxyhexadecane 9,10Dihydroxyhexadecancer Epoxide-Hydrolase Myxococcusxanthus SEQ IDNO: 19 9.10Epoxyhexadecane 9,10Dihydroxyhexadecancer BifunctionalEpoxyhydrolase Kerria Lacca SEQ IDNO: 03 Palmitolein saucers 9,10Dihydroxyhexadecancer Monooxygenase Marinobacterhydrocarbonoclasticus SEQ IDNO: 22 9,10Dihydroxyhexadecancer AleuriticHealth Monooxygenase Citrusclementia SEQ IDNO: 13 9,10Dihydroxyhexadecancer AleuriticHealth

[0066] The enzymes listed in Table 2 were further optimized with potential desaturase enzymes to obtain gene combinations that catalyze the conversion of palmitic acid to palmitoleic acid and finally the desired product aleuritic acid in the subsequent steps of the biosynthetic pathway, as described in Fig. 1 and Fig. 2 described. Example 3: Screening of delta-9 desaturases to enhance palmitoleic acid biosynthesis in E. coli

[0067] Palmitoleic acid (16:1Δ9), a monounsaturated ω-7 fatty acid, possesses a double bond at the seventh carbon atom, starting at the methyl end of the acyl chain. It is initially biosynthesized by a desaturase known as Δ9-16:0 desaturase, which uses saturated palmitic acid (16:0) as a substrate (Liu B, Sun Y, Hang W, Wang X, Xue J, Ma R, Jia X, Li R. Characterization of a Novel Acyl-ACP Δ9 Desaturase Gene Responsible for Palmitoleic Acid Accumulation in a Diatom Phaeodactylum tricornutum. Front Microbiol. 2020 Dec 16; 11:584589. doi: 10.3389 / fmicb.2020.584589.). Although many genes for Δ9-desaturases are known in all living kingdoms, including plants, mammals, fungi, and some bacteria, very little data are available on their expression and activity towards palmitic acid in E. coli. To increase the pool of the free fatty acid precursor 9-hexadecenoic acid (palmitoleic acid) and thus aleuritic acid production ( Fig. 1 and Fig.2), the functional homologs of the palmitic acid-specific desaturase enzymes from various prokaryotic species (Table 3) were gene-synthesized. The genes were cloned under the T7 promoter into an episomal E. coli expression vector pET28+ and transformed into E. coli BL21 cells. The bacterial cultures were initially grown under shake flask conditions at 37 °C and 200 rpm. At an OD 600 of approximately 0.6, 0.5 mM IPTG was added to induce the expression of the recombinant proteins and the production of palmitoleic acid. After addition of the inducer, the culture temperature was increased to 30 °C. The induced cells were extracted with an equal volume of ethyl acetate. The collected organic layer was then subjected to GCMS analysis as described in Example 2 to determine the production levels of palmitoleic acid. Fig.4, the percentage values ​​of the fatty acid distribution are the average of at least three biological replicates with the corresponding standard deviation. One of the desaturase genes of Pseudomonas aeruginosa (SEQ ID NO: 27 in Table 3 and Fig. 4) showed an increase in palmitoleic acid titer by 29.2% compared to wild type, where 29.2% represents the percentage of total palmitoleic acid (C16:1) compared to palmitic acid (C16:0). Table 3 SEQ ID ORGANISM SEQ ID NO:25 Psychrobacter urativorans SEQ ID NO:26 Choristoneura western SEQ ID NO:27 Pseudomonas aeruginosa SEQ ID NO:28 Arabidopsis thaliana SEQ ID NO:29 Caenorhabditis elegans SEQ ID NO:30 Trypanosoma cruzi SEQ ID NO:31 Linnemannia wins

[0068] Therefore, the desaturase gene from Pseudomonas aeruginosa was preferably used as part of the gene combinations described in previous examples for the production of aleuritic acid. Example 4Reconstruction of the aleuritic acid biosynthesis pathway in E. coli

[0069] The optimized gene combination determined in Examples 1, 2, and 3 was used to construct aleuritic acid biosynthesis in E. coli (host system). The identified genes were incorporated into the episomal expression vectors pETDuet and pACYCDuet (Merck-Novagen). Both the pETDuet and pACYCDuet plasmids, which harbor relevant gene combinations ( Fig. 6A-B; Fig. 7A-B), were cotransformed, and the recombinant cells were selected on Luria-Bertani (LB) agar plates with appropriate antibiotic markers. E. coli induction experiments and GC-MS analysis were performed as described in Example 2. The production of aleuritic acid and its precursor (9,10-dihydroxyhexadecanoic acid) in E. coli was confirmed by comparison to the standard.

[0070] E. coli (recombinant host system) Biosynthesis of aleuritic acid and its intermediates 9,10-dihydroxyhexadecanoic acid and 9,10-epoxyhexadecanoic acid by expression of the following gene combinations ( Fig. 1-Way 1): (i) desaturase (SEQ ID: 27) (ii) epoxygenase (SEQ ID: 16) (iii) epoxide hydrolase (SEQ ID: 19) (iv) monooxygenase (SEQ ID: 13) ( Fig. 6A-B) is replaced by the Fig. 5B shown elution peaks of the GC-MS analysis and the Fig. 5C-5G shown corresponding mass spectra.

[0071] The Fig.5B-G show the gas chromatography-mass spectrometry (GC-MS) analysis of intermediates and final products of aleuritic acid production based on Route 1 using the recombinant host system of the present disclosure, where A) shows the elution peaks of 1) aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, and 4) 9,10-dihydroxyhexadecanoic acid; and C) shows the mass spectrum of aleuritic acid; D) shows the mass spectrum of palmitic acid; E) shows the mass spectrum of palmitoleic acid; and G) shows the mass spectrum of 9,10-epoxyhexadecanoic acid. It is important to note that the intermediate 9,10-epoxyhexadecanoic acid is produced by the action of the enzymes epoxygenase and epoxide hydrolase.

[0072] E. coli (recombinant host system) Biosynthesis of aleuritic acid and its intermediates 9,10-dihydroxyhexadecanoic acid and palmitoleic acid by expression of the following gene combinations ( Fig.2-Way 2): (i) Desaturase (SEQ ID: 27) (ii) Bifunctional Epoxyhydrolase (SEQ ID: 03) (iii) Monooxygenase (SEQ ID: 13) ( Fig. 7A-B) was carried out by the Fig. 5A shown elution peaks of the GC-MS analysis and the Fig. 5C-5F shown corresponding spectral images.

[0073] The Fig.5B-F show the gas chromatography-mass spectrometry (GC-MS) analysis of intermediates and final products of the process for producing aleuritic acid based on Route 2 using the recombinant host system of the present disclosure, where A) shows the elution peaks of 1-aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, and 4) 9,10-dihydroxyhexadecanoic acid; C) shows the mass spectrum of aleuritic acid; D) shows the mass spectrum of palmitic acid; E) shows the mass spectrum of palmitoleic acid; and F) shows the mass spectrum of 9,10-dihydroxyhexadecanoic acid. It is important to note that no 9,10-epoxyhexadecanoic acid intermediate is present in the elution peaks, which is due to the action of bifunctional epoxide hydrolase enzymes instead of epoxygenase and epoxide hydrolase enzymes.

[0074] Overall, it can be concluded from the examples described above that the recombinant host system of the present disclosure transformed with the specific gene combinations has the potential to provide alternative means for the production of aleuritic acid to meet industrial needs. Benefits of this disclosure

[0075] The present disclosure provides a recombinant system for producing aleuritic acid with the following advantages. a) The disclosed system provides aleuritic acid with good purity and yield and is time-saving compared to conventional processes. b) The described process for the production of aleuritic acid can be extended to the industrial demand for aleuritic acid. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] WO 2019217226A1

[0004] Cited non-patent literature

[0000] Kaprakkaden A, Srivastava P, Bisaria VS. In vitro synthesis of 9, 10-dihydroxyhexadecanoic acid using recombinant Escherichia coli. Microb Cell Fact. 2017 May 18; 16(1):85. doi: 10.1186 / s12934-017-0696-7

[0003] Pinot F, Skrabs M, Compagnon V, Salaün JP, Benveniste I, Schreiber L, Durst F. Omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defense. Biochem Soc Trans. 2000 Dec;28(6):867-70

[0060] Arrieta-Baez D, Cruz-Carrillo M, Gomez-Patifio MB, Zepeda-Vallejo LG. Derivatives of 10,16-dihydroxyhexadecanoic acid isolated from tomato (Solanum lycopersicum) as potential materials for aliphatic polyesters. Molecules. 2011 Jun 15;16(6):4923-36. doi: 10.3390 / molecules1606492

[0060] Kyoung-Rok Kim, Deok-Kun Oh, Production of hydroxy fatty acids by microbial fatty acid hydroxylation enzymes, Biotechnology Advances, Volume 31, Issue 8, 2013, Pages 1473 to 1485, https: / / doi.org / 10.1016 / j.biotechadv.2013.07.004

[0060] Attwood TK, Beck ME. PRINTS - a database for protein motif fingerprints. Protein Eng. 1994 Jul;7(7):841-8. doi: 10.1093 / protein / 7.7.841

[0061] Royce LA, Liu P, Stebbins MJ, Hanson BC, Jarboe LR. The deleterious effects of short-chain fatty acids on Escherichia coli membranes. Appl Microbiol Biotechnol. 2013 Sep;97(18):8317-27. doi: 10.1007 / s00253-013-5113-5

[0061] Liu B, Sun Y, Hang W, Wang X, Xue J, Ma R, Jia X, Li R. Characterization of a Novel Acyl-ACP Δ9 Desaturase Gene Responsible for Palmitoleic Acid Accumulation in a Diatom Phaeodactylum tricornutum. Front Microbiol. 2020 Dec 16; 11:584589. doi: 10.3389 / fmicb.2020.584589.

[0067]

Claims

[1] Recombinant host system for the production of 9,10,16-trihydroxyhexadecanoic acid (aleuritic acid), wherein the host system is transformed with: (i) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding the epoxide hydrolase enzyme and the monooxygenase enzyme, or (ii) a first recombinant expression vector comprising nucleic acid sequences encoding the desaturase enzyme and the bifunctional epoxygenase hydrolase enzyme, and a second recombinant expression vector comprising a nucleic acid sequence encoding the monooxygenase enzyme. [2] The recombinant host system according to claim 1, wherein the nucleic acid sequence encoding the desaturase enzyme is selected from SEQ ID NO: 27, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO:

31. [3] The recombinant host system of claim 1, wherein the nucleic acid sequence encoding the epoxygenase enzyme is selected from SEQ ID NO: 16, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO:

18. [4] The recombinant host system of claim 1, wherein the nucleic acid sequence encoding the epoxide hydrolase enzyme is selected from SEQ ID NO:19, SEQ ID NO:15, SEQ ID NO:21 or SEQ ID NO:

23. [5] The recombinant host system of claim 1, wherein the nucleic acid sequence encoding the monooxygenase enzyme is selected from SEQ ID NO:13, SEQ ID NO:22, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20 or SEQ ID NO:

24. [6] The recombinant host system of claim 1, wherein the nucleic acid sequence encoding bifunctional epoxygenase hydrolase enzyme is selected from SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8. [7] The recombinant host system according to claim 1, wherein the host system is selected from E. coli, S. cerevisiae, Yarrowia lipolytica, Rhodotorula toruloides, Pichia pastoris, Candida boidinii or Kluyveromyces lactis. [8] A process for the preparation of 9,10,16-trihydroxyhexadecanoic acid, comprising: Cultivating the recombinant host system of claim 1 in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, epoxygenase enzyme, epoxide hydrolase enzyme, monooxygenase enzyme, or combinations thereof. [9] A process according to claim 8, wherein the culture medium is optionally supplemented with hexadecanoic acid. [10] The method of claim 8, wherein the culture medium is optionally supplemented with 9-hexadecenoic acid. [11] A process for the preparation of 9,10,16-trihydroxyhexadecanoic acid, comprising: Cultivating the recombinant host system of claim 1 in a culture medium comprising a simple carbon source, wherein the host system is capable of heterologous expression of nucleic acids encoding the desaturase enzyme, bifunctional epoxygenase hydrolase enzyme, monooxygenase enzyme, or combinations thereof. [12] The process according to claim 11, wherein the culture medium is optionally supplemented with hexadecanoic acid. [13] The method of claim 8 or 11, wherein the carbon source is selected from glucose, sucrose, galactose, lactose, fructose or combinations thereof.

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