FIELD OF INVENTION

[001] The present disclosure broadly relates to the field of synthetic biology. Particularly, the present disclosure provides materials and methods for the synthesis of aleuritic acid.

BACKGROUND OF INVENTION

[002] The primary acid ingredient of the lac resin generated by the Indian lac bug Kerria lacca is aleuritic acid (9,10,16-trihydroxyhexadecanoic acid). Aleuritic acid is a unique acid containing three hydroxyl groups of which two are adjacent carbon atoms. Aleuritic acid is a high-value molecule since it is used as a starting material for synthesis of macrocyclic perfumery compounds such as ambrettolide, iso- ambrettolide, civetone, dehydiocivetone, exaltone, glucose manoaleuritate and related lactones, insect sex pheromones, pharmaceutical chemicals, cosmetics, esters, metallic salts and stabilizers, plant growth regulators and biodegradable polymers like poly (aleuritic acid), polyhydroxyalkanoic acid (PHA) etc. (Kun Li et al 2019 Mater. Res. Express 6075328). The existing approach of purifying aleuritic acid to the necessary amount by alkaline hydrolysis of lac resin takes a complex and long route ( Characterization of Different Shellac Types and Development of Shellac- Coated Dosage Forms, Dissertation, Hamburg 2010). Therefore, there remains a need for an alternative route for bio conversion or de novo biosynthesis of aleuritic acid.

[003] The production of one of the aleuritic acid pathway intermediate, the 9, 10- dihydroxyhexadecanoic acid, was demonstrated by in vitro reconstitution of three different enzymes using 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/s 12934-017-0696-7).

[004] Patent document WO2019217226 Al discloses recombinant microorganisms that expresses 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 co-hydroxylase from Marinobacter aquaeolei for producing 9,10,16 - trihydroxyhexadecanoic acid (aleuritic acid).

[005] However, the methods known in the art fail to provide an efficient and time saving method to produce aleuritic acid for its large-scale commercial applications. Therefore, there is an immediate need to precisely capture the aleuritic biosynthetic pathway in a recombinant host system in order to increase the efficiency of aleuritic acid production.

SUMMARY OF THE INVENTION

[006] In an aspect of 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), wherein the host system is transformed with: (i) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding for Epoxide hydrolase enzyme and Monooxygenase enzyme, or (ii) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and bifunctional Epoxygenase-hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme.

[007] In an aspect of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising: culturing the recombinant host system as disclosed herein in a culture medium comprising a simple carbon source, wherein said host system is capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, Epoxygenase enzyme, Epoxide hydrolase enzyme, Monooxygenase enzyme or combinations thereof.

[008] In an aspect of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising: culturing the recombinant host system as disclosed herein in a culture medium comprising a simple carbon source, wherein said host system is capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, bifunctional Epoxygenase-hydrolase enzyme, Monooxygenase enzyme or combinations thereof.

[009] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce 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 be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF DRAWINGS

[0010] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0011] Figure 1 illustrates the identified biosynthetic pathway route (1) for aleuritic acid showing involvement of at least four types of enzymes 1. Fatty acid desaturase, 2. Epoxygenase, 3. Epoxy hydrolase, and 4. Cytochrome P450 Monooxygenase (CYPs), in accordance with an embodiment of the disclosure.

[0012] Figure 2 illustrates the identified biosynthetic pathway route (2) for aleuritic acid showing involvement of three types of enzymes 1. Fatty acid desaturase, 2. Bifunctional epoxygenase/hydrolase, and 3. Cytochrome P450 Monooxygenase, in accordance with an embodiment of the disclosure.

[0013] Figures 3A-E illustrate the strategy for aleuritic acid production using the biosynthetic pathway routes 1 and 2, in accordance with an embodiment of the disclosure.

[0014] Figure 4 illustrates the fatty acid distribution profile for screening of delta 9 desaturases from various organisms for increased palmitoleic acid biosynthesis in E. coli, in accordance with an embodiment of the disclosure.

[0015] Figures 5A-G illustrate the Gas Chromatography Mass Spectrometry (GC- MS) analysis of intermediates and end products of the different aleuritic acid production methods, wherein A) depicts the elution peaks of 1) aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, and 4) 9,10dihydroxy hexadecanoic acid; B) depicts the elution peaks of 1) aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, 4) 9,10-dihydroxy hexadecanoic acid and 5) 9,10-epoxy hexadecanoic acid; C) depicts the mass spectrum of aleuritic acid; D) depicts the mass spectrum of palmitic acid; E) depicts the mass spectrum of palmitoleic acid; F) depicts the mass spectrum of 9,10-dihydroxy hexadecanoic acid; and G) depicts the mass spectrum of 9,10-epoxy hexadecanoic acid; in accordance with an embodiment of the disclosure.

[0016] Figures 6A-B illustrate the plasmid map harbouring gene combinations as per pathway route 1, in accordance with an embodiment of the disclosure.

[0017] Figures 7A-B illustrate the plasmid map harbouring gene combinations as per pathway route 2, in accordance with an embodiment of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0019] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0020] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0021] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising” are used in the inclusive, open sense, and will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps. It is not intended to be construed as “consists of only”.

[0022] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0023] The term “nucleic acid”, as used herein refers to a combination of nucleotide monomers which are connected to each other through covalent bonds as in DNA or RNA. The terms “nucleic acid” and “polynucleotide” are used interchangeably.

[0024] The term “gene”, as used herein refers to nucleic acid sequences e.g., DNA sequences, which encode either an RNA product or a protein product.

[0025] The term “recombinant host system”, as used herein refers to a host cell that has been genetically modified or engineered such that certain enzymatic activities within the host cell have been altered, added and/or deleted relative to the parent cell or native host cell. A genetically modified or genetically engineered host cell is an example of a recombinant host system.

[0026] The term "heterologous”, as used herein is used to mean that a polynucleotide or a polypeptide sequence is derived from a different species or derived from a different organism or derived from a different source. As used herein it refers to a nucleotide sequence or a polypeptide sequence that is not naturally present in a particular organism.

[0027] The term “vector”, as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked. One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal 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 ¹ and “vector is used interchangeably herein, in as much as a plasmid is the most commonly used form of vector.

[0028] The term “expression”, as used herein refers to a gene, refers to the production of one or more transcriptional and / or translational product (s) of a gene. The term " expression " or " expressed” are used interchangeably.

[0029] The term “simple carbon source”, as used herein refers to a substrate or compound used as a source of fuel for prokaryotic or simple eukaryotic cell growth. A simple carbon source as per the present disclosure could be glucose, sucrose, galactose, lactose, fructose, or combinations thereof.

[0030] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0031] Unless defined otherwise, 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 any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0032] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0033] As discussed in the background, there are various limitations associated with the conventional materials, and methods to produce aleuritic acid. The conventional method of extracting aleuritic acid from insect lac resin is a laborious process. Alternative methodologies have been explored employing recombinant microorganisms to produce aleuritic acid in in vitro systems thereby eliminating the dependence on insect resins. However, such genetic engineering alternatives need improvement with respect to efficiency and economy of synthesis.

[0034] To address the aforementioned problems, the present disclosure discloses recombinant host systems comprising heterologous enzymes from various organisms that can be cultured using simple carbon source to produce aleuritic acid. The present disclosure also discloses a method for the production of aleuritic acid. [0035] As an outcome of this, the present disclosure solves the problems existing in the art by providing precise methods using recombinant host systems for efficient and high scale production of aleuritic acid.

[0036] In an embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), said host system is capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, Epoxygenase enzyme, Epoxide hydrolase enzyme, Monooxygenase enzyme, bifunctional Epoxygenase-hydrolase enzyme or combinations thereof.

[0037] In one embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), said host system is capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, Epoxygenase enzyme, Epoxide hydrolase enzyme, and Monooxygenase enzyme.

[0038] In one another embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), said host system capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, bifunctional Epoxygenase-hydrolase enzyme and monooxygenase enzyme.

[0039] In an embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), said host system is capable of heterologous expression of nucleic acids encoding for 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 for Desaturase enzyme, Epoxygenase enzyme, Epoxide hydrolase enzyme, and Monooxygenase enzyme, or (ii) a first recombinant expression vector comprising a nucleic acid sequence encoding for Desaturase enzyme, a second recombinant expression vector comprising a nucleic acid sequence encoding for Epoxygenase enzyme, a third recombinant expression vector comprising a nucleic acid sequence encoding for Epoxide hydrolase enzyme, and a fourth recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme, or (iii) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding for Epoxide hydrolase enzyme and Monooxygenase enzyme, or (iv) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Epoxide hydrolase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding for Epoxygenase enzyme and Monooxygenase enzyme, or (v) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Monooxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding for Epoxygenase enzyme and Epoxide hydrolase enzyme, or (vi) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme, Monooxygenase enzyme and Epoxygenase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Epoxide hydrolase enzyme, or (vii) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme, Monooxygenase enzyme and Epoxide hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Epoxygenase enzyme, or (viii) a first recombinant expression vector comprising nucleic acid sequences encoding for Epoxygenase enzyme, Monooxygenase enzyme and Epoxide hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Desaturase enzyme, or (ix) a first recombinant expression vector comprising nucleic acid sequences encoding for Epoxygenase enzyme, Desaturase enzyme and Epoxide hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme; wherein the nucleic acid sequence encoding for 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 for 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 for 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 for 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.

[0040] In an embodiment of the present disclosure, there is provided a recombinant host system engineered for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), said host system is capable of heterologous expression of nucleic acids encoding for 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 for Desaturase enzyme, bifunctional Epoxygenase-hydrolase enzyme and Monooxygenase enzyme, or (ii) a first recombinant expression vector comprising a nucleic acid sequence encoding for Desaturase enzyme, a second recombinant expression vector comprising a nucleic acid sequence encoding for bifunctional Epoxygenase-hydrolase enzyme and a third recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme, or (iii) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and bifunctional Epoxygenase-hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme, or (iv) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Monooxygenase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for bifunctional Epoxygenase-hydrolase enzyme, or (v) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and a second recombinant expression vector comprising nucleic acid sequences encoding for Monooxygenase enzyme and bifunctional Epoxygenase-hydrolase enzyme; wherein the nucleic acid sequence encoding for 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 for 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 for 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.

[0041] In one embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), wherein the host system is transformed with: (i) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding for Epoxide hydrolase enzyme and Monooxygenase enzyme, or (ii) a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and bifunctional Epoxygenase-hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme.

[0042] In one another embodiment of the present disclosure, there is provided a recombinant host system for producing 9, 10, 16-trihydroxy hexadecanoic acid (Aleuritic acid), wherein the host system is transformed with: a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and Epoxygenase enzyme, and a second recombinant expression vector comprising nucleic acid sequences encoding for Epoxide hydrolase enzyme and Monooxygenase enzyme; wherein the nucleic acid sequence encoding for 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 for 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 for 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 for 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. [0043] In yet another embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), wherein the host system is transformed with: a first recombinant expression vector comprising nucleic acid sequences encoding for Desaturase enzyme and bifunctional Epoxygenase-hydrolase enzyme and a second recombinant expression vector comprising a nucleic acid sequence encoding for Monooxygenase enzyme; wherein the nucleic acid sequence encoding for 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 for 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 for 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.

[0044] In an embodiment of the present disclosure, there is provided a recombinant host system for producing 9,10,16-trihydroxy hexadecanoic acid (Aleuritic acid), wherein the host system is selected from E. coli, S. cerevisiae, Yarrowia lipolytica, Rhodotorula toruloides, Pichia pastoris, Candida boidinii, or Kluyveromyces lactis. [0045] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid using the recombinant host system as disclosed herein.

[0046] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising: culturing the recombinant host system as disclosed herein in a culture medium comprising a simple carbon source.

[0047] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising: culturing a recombinant host system in a culture medium comprising a simple carbon source, wherein said host system is capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, Epoxygenase enzyme, Epoxide hydrolase enzyme, Monooxygenase enzyme or combinations thereof.

[0048] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid, wherein the culture medium is optionally supplemented with Hexadecanoic acid.

[0049] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid, wherein the culture medium is optionally supplemented with 9 Hexadecenoic acid.

[0050] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising: culturing a recombinant host system in a culture medium comprising a simple carbon source, wherein said host system is capable of heterologous expression of nucleic acids encoding for 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.

[0051] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding desaturase enzyme to a substrate comprising Hexadecanoic acid for conversion of Hexadecanoic Acid to 9 Hexadecenoic acid, adding Epoxygenase enzyme to 9 Hexadecenoic acid for conversion of 9 Hexadecenoic acid to 9,10-Epoxy hexadecanoic acid, adding Epoxide hydrolase enzyme to 9, 10 Epoxy hexadecanoic acid for conversion of 9,10-Epoxy hexadecanoic acid to 9,10-dihydroxy hexadecanoic acid, and adding Monooxygenase enzyme to 9,10-dihydroxy hexadecanoic acid for conversion of 9,10-dihydroxy hexadecanoic acid to 9,10,16 - trihydroxy hexadecanoic acid.

[0052] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding Epoxygenase enzyme to a substrate comprising 9-Hexadecenoic acid for conversion of 9 Hexadecenoic acid to 9, 10 Epoxy hexadecanoic acid, adding Epoxide hydrolase enzyme to 9,10-Epoxy hexadecanoic acid for conversion of 9,10-Epoxy hexadecanoic acid to 9,10-dihydroxy hexadecanoic acid, and adding Monooxygenase enzyme to 9,10-dihydroxy hexadecanoic acid for conversion of 9, 10-dihydroxy hexadecanoic acid to 9,10,16-trihydroxy hexadecanoic acid.

[0053] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding Epoxide hydrolase enzyme to a substrate comprising 9,10-Epoxy hexadecanoic acid for conversion of 9,10-Epoxy hexadecanoic acid to 9,10-dihydroxy hexadecanoic acid, and adding Monooxygenase enzyme to 9,10-dihydroxy hexadecanoic acid for conversion of 9,10-dihydroxy hexadecenoic acid to 9,10,16-trihydroxy hexadecanoic acid.

[0054] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding Monooxygenase enzyme to a substrate comprising 9,10-dihydroxy hexadecanoic acid for conversion of 9,10-dihydroxy hexadecanoic acid to 9,10,16-trihydroxy hexadecanoic acid.

[0055] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding Desaturase enzyme, Epoxygenase enzyme, Epoxide hydrolase and Monooxygenase enzyme to a substrate comprising Hexadecanoic acid.

[0056] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding Epoxygenase enzyme, Epoxide hydrolase enzyme and Monooxygenase enzyme to a substrate comprising 9-Hexadecenoic acid.

[0057] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: culturing a recombinant host system in a culture medium comprising a simple carbon source, wherein said host system is capable of heterologous expression of nucleic acids encoding for Desaturase enzyme, bifunctional Epoxygenase-hydrolase enzyme, Monooxygenase enzyme or combinations thereof. [0058] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid as disclosed herein, wherein the culture medium is optionally supplemented with Hexadecanoic acid.

[0059] In another embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid as disclosed herein, wherein the culture medium is supplemented with Hexadecanoic acid.

[0060] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid as disclosed herein, wherein the culture medium is optionally supplemented with 9-Hexadecenoic acid.

[0061] In another embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid as disclosed herein, wherein the culture medium is supplemented with 9-Hexadecenoic acid.

[0062] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding desaturase enzyme to a substrate comprising Hexadecanoic acid for conversion of Hexadecanoic acid to 9-Hexadecenoic acid, adding bifunctional Epoxygenase- hydrolase enzyme to 9-Hexadecenoic acid for conversion of 9-Hexadecenoic acid to 9,10,16-dihydroxy hexadecanoic acid, and adding Monooxygenase enzyme to 9,10- dihydroxy hexadecanoic acid for conversion of 9, 10 dihydroxy hexadecanoic acid to 9,10,16-trihydroxy hexadecanoic acid.

[0063] In one embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid comprising the steps of: adding bifunctional Epoxygenase-hydrolase enzyme to 9-Hexadecenoic acid for conversion of 9 Hexadecenoic acid to 9,10-dihydroxy hexadecanoic acid, and adding Monooxygenase enzyme to 9,10-dihydroxy hexadecanoic acid for conversion of 9, 10 dihydroxy hexadecanoic acid to 9,10,16-trihydroxy hexadecanoic acid.

[0064] In an embodiment of the present disclosure, there is provided a method of producing 9,10,16-trihydroxy hexadecanoic acid as disclosed herein, wherein the simple carbon source is selected from glucose, sucrose, galactose, lactose, fructose, or combinations thereof. [0065] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.

EXAMPLES

[0066] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

Example 1

Identification of mechanism for aleuritic acid biosynthesis in the Indian lac bug Kerria lacca using bioinformatic tools

[0067] A mechanism for aleuritic acid biosynthesis 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 resin non-secreting (crawler) life stages of the lac insect, (b) use of broad spectrum inhibitors for epoxide hydrolase (EH) and epoxygenase (EPOX) in combination with trans-supply of probable intermediates, (c) enzymatic activity of fatty acid desaturase (FAD), epoxide hydrolase and monooxygenase (MONO) in adult lac insect extracts compared to crawler extracts (d) Western blotting for epoxygenase (EPOX), fatty acid desaturase, and epoxide hydrolase to check for the presence of these enzymes in adult lac insect, and (e) quantitative PCR for FAD, EH, EPOX, and MONO genes to detect transcripts in adult lac insect (Wang W, Liu P, Lu Q, Ling X, Zhang J, Chen MS, Chen H, Chen X. Potential Pathways and Genes Involved in Lac Synthesis and Secretion in Kerria chinensis (Hemiptera: Kerriidae) Based on Transcriptomic Analyses. Insects. 2019 Nov 28;10(12):430. doi: 10.3390/insectsl0120430). In aleuritic acid biosynthesis, the last two steps involve epoxygenation of palmitoleic acid at carbon 9, 10 position to 9,10-epoxy hexadecanoic acid followed by hydroxylation leading to 9,10-dihydroxy hexadecanoic acid production. Cytochrome P450 enzyme (CYP) class such as CYP94A1 are known to be bifunctional, catalyzing both, epoxidation and hydroxylation of fatty acids (Pinot F, Skrabs M, Compagnon V, Salaiin JP, Benveniste I, Schreiber L, Durst F. Omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence. Biochem Soc Trans. 2000 Dec;28(6):867-70). The third step is omega hydroxylation of 9,10-dihydroxy hexadecanoic acid which is catalyzed by Cytochrome P450 monooxygenase enzyme (CYPs). Fatty acids and their derivatives are subjected to many types of oxidation reaction including hydroxylation, epoxidation, dehydration, and reduction. Several forms of CYPs are suspected of being involved in these reactions (Arrieta-Baez D, Cruz-Carrillo M, Gomez-Patino 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/moleculesl606492). Cytochrome P450-dependent monooxygenases from plants and other bacterial species like Bacillus species including B. megaterium catalyse in-chain and omega hydroxylation as well as 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, Pagesl473 to 1485, https://doi.org/10.10167j.biotechadv.2013.07.004). There are multiple forms of cytochrome P450 involved in these reactions, each of which possesses distinguishable substrate specificity.

[0068] In order 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 protein motif fingerprint database. Protein Eng. 1994 Jul;7(7):841-8. doi: 10.1093/protein/7.7.841.) were used to identify relevant epoxygenase and epoxide hydrolase from lac insect transcriptome sequence. Biological sequence motif is short, usually fixed length, sequence patterns that represent important structural or functional features in nucleic acid and protein sequences are 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 Uniprot database, then the extracted proteins were given as a query in PRINTS. After identification of corresponding motif sequence from PRINTS database, the data was fed into a biopython program to extract the putative epoxide hydrolase, CYPs and epoxygenase from protein in lac insect transcriptome. In total, eight putative epoxygenase and hydrolase enzyme sequence (SEQ ID NOs: 1-8) were identified from Lac transcriptome. In general, epoxy fatty acid and hydrolyzed epoxy groupdiol is highly toxic to microorganisms, including E. coli and yeast. High levels of epoxy fatty acids can stop the growth and kill E. coli because of their physical effects on membranes and detergency and also possibly acidity (Royce LA, Liu P, Stebbins MJ, Hanson BC, Jarboe LR. The damaging 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 disclosure a shorter route has been identified by bypassing the epoxide formation using bifunctional epoxy hydrolases identified from the lac transcriptome.

[0069] Apart from enzymes identified from Lac transcriptome, functional homologs from other species reported for carrying similar fatty acid epoxidation and hydroxylation activities were also shortlisted (Table 1). All the shortlisted sequences were codon optimized for E. coli expression and gene synthesized.

[0070] Table 1

Example 2

Identification of best gene/pathway combination for production of aleuritic acid in E. coli through in vitro studies

[0071] Aleuritic acid producing E. coli strain (recombinant host system) was constructed for experiments designed to find optimal combinations of biosynthetic genes composed of epoxygenase (Figure 3C), Epoxide hydrolase (Figure 3D), bifunctional-epoxy hydrolase (Figure 3A), and CYPs (Figure 3B and E). Shortlisted pathway genes were codon optimized for E. coli and gene synthesized. The individual genes were expressed under T7 promoter in an episomal E. coli expression vector pET28+ under T7 promoter and transformed in E. coli BE21 cells. The E. coli transformants were grown overnight at 37 °C in 1 ml of M9 minimal media containing appropriate antibiotics (ampicillin (100 mg/1), and glucose (simple carbon source) in 96-well format. The next day, 150 pl of each culture was inoculated into 3 ml M9 minimal media containing ampicillin (100 mg/1), isopropyl-P-D- thiogalactopyranoside (IPTG) 0.5 mM in 24-well format and incubated at 30° C and 250 rpm for 20 h. Using the whole cell lysate of the induced recombinant cultures, the possibility of obtaining 9,10,16-dihydroxyhexadecanoic acid was explored along with the supply of 9-hexadecenoic acid (palmitoleic acid) as substrate. The induced cultures were lysed using Bugbuster Protein extraction reagent (Merck) and the lysed cultures were mixed in equal quantity with addition of substrates, such as 9- hexadecenoic acid/palmitoleic acid, 9,10-dihydroxy hexadecanoic acid, 9,10-epoxy hexadecanoic acid (200 pM each) and NADPH (0.1 pM) and incubated at 30 °C for 6 hrs (Figures 3A-3E). The incubated culture lysates were then extracted with hexane and ethyl acetate, and the extract was subjected to GC-MS analysis.

GC-MS Analysis

[0072] Metabolites were estimated using Agilent GCMS-6890N-5973 Plus as per the following conditions. Column: RTX-5MS, 30 m; Column Oven Temperature: 140 °C; Injection Temperature: 260 °C; Injection Mode: Splitless; Carrier gas: Helium; Oven Programme: 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 depicted in Figures 5A-G. [0073] Table 2 below provides the details of enzymes identified for substrates tested and product formed.

[0074] Table 2

[0075] The list of enzymes shortlisted in Table 2 were further optimized with potential desaturase enzymes to obtain gene combinations to catalyze the conversion of palmitic acid to palmitoleic acid and finally the desired product of aleuritic acid in ensuing steps of the biosynthetic pathway as described in Figure 1 and Figure 2.

Example 3:

Screening of delta 9 desaturases for increased palmitoleic acid biosynthesis in E, coli

[0076] Palmitoleic acid (16: 1A9), a kind of monounsaturated co-7 fatty acid, possesses a double bond at the seventh carbon atom starting from the methyl end of the acyl chain. It is initially biosynthesized by a desaturase known as A9-16:0- desaturase, using 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 A9 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 A9-desaturases have been reported across living kingdoms including plants, mammals, fungi, and some bacteria; however very limited data is known for their expression and activity towards palmitic acid in E. coli. In order to increase the free fatty acid precursor 9- hexadecenoic acid (palmitoleic acid) pool and in turn aleuritic acid production (Figure 1 and Figure 2), the functional homologs of palmitic acid specific desaturase enzymes from various prokaryotic species (Table 3) were gene synthesized. The genes were cloned under T7 promoter in an episomal E. coli expression vector pET28+ and transformed in E. coli BL21 cells. Under shake-flask conditions, the bacterial cultures were first grown at 37 °C and 200 rpm. 0.5 mM of IPTG was added at an ODeoo of about 0.6 to induce the expression of recombinant proteins and production of palmitoleic acid. Then the culture temperature was shifted to 30 °C after adding the inducer. The induced cells were extracted with 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. In Figure 4, fatty acid distribution percentage values are the average of at least three biological replicates with the associated standard deviation indicated. One of the desaturase genes from Pseudomonas aeruginosa (SEQ ID NO : 27 in Table 3 and Figure 4) showed 29.2% increase in palmitoleic acid titer as compared to wild type, and wherein 29.2% indicates total percentage amount of palmitoleic acid (C 16: 1) in comparison with palmitic acid (C16:0). [0077] Table 3

[0078] Therefore, desaturase gene from Pseudomonas aeruginosa was preferable to be included as part of the gene combinations as described in earlier examples to produce aleuritic acid.

Example 4 Reconstitution of Aleuritic acid biosynthesis pathway in E. coli

[0079] The optimized gene combination identified through Examples 1, 2 and 3 was used to construct the aleuritic acid biosynthesis in E. coli (host system). The genes identified were assembled in episomal expression vectors pETDuet and pACYCDuet vectors (Merck-No vagen). Both the pETDuet and pACYCDuet plasmids harbouring relevant gene combinations (Figure 6A-B; Figure 7A-B) was cotransformed and recombinant cells were selected on appropriate antibiotic markers containing Furia Bertani (FB) agar plates. The E. coli induction experiments and GC-MS analysis were carried out as described in Example 2. The aleuritic acid and its precursor (9,10- dihydroxy hexadecanoic acid) production in E. coli were confirmed as compared to standard.

[0080] E. coli (recombinant host system) biosynthesis of aleuritic acid and its intermediates 9,10-dihydroxy hexadecanoic acid and 9,10-Epoxyhexadecanoic acid expressing the following gene combinations (Figure 1 -pathway route 1): (i) Desaturase (SEQ ID:27) (ii) Epoxygenase (SEQ ID: 16) (iii) Epoxide hydrolase (SEQ ID: 19) (iv) Monooxygenase (SEQ OID: 13) (Figure 6A-B) is confirmed in the GC-MS analysis elution peaks as depicted in Figure 5B and corresponding mass spectrum images illustrated in Figure 5C-5G.

[0081] Figures 5B-G illustrate the Gas Chromatography Mass Spectrometry (GC- MS) analysis of intermediates and end products of the aleuritic acid production based on pathway route 1 using the recombinant host system of the present disclosure, wherein A) depicts the elution peaks of 1) aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, and 4) 9,10-dihydroxy hexadecanoic acid; and C) depicts the mass spectrum of aleuritic acid; D) depicts the mass spectrum of palmitic acid; E) depicts the mass spectrum of palmitoleic acid; and G) depicts the mass spectrum of 9,10- epoxy hexadecanoic acid. It is essential to note the production of 9,10-epoxy hexadecanoic acid intermediate due to the action of epoxygenase and epoxide hydrolase enzymes.

[0082] E. coli (recombinant host system) biosynthesis of aleuritic acid and its intermediates 9,10-dihydroxy hexadecanoic acid and palmitoleic acid expressing the following gene combinations (Figure 2-pathway route 2): (i) Desaturase (SEQ ID:27) (ii) Bifunctional Epoxy hydrolase (SEQ ID: 03) (iii) Monooxygenase (SEQ ID: 13) (Figure 7A-B) was confirmed in the GC-MS analysis elution peaks as depicted in Figure 5A and corresponding spectral images illustrated in Figure 5C- 5F. [0083] Figures 5B-F illustrate the Gas Chromatography Mass Spectrometry (GC- MS) analysis of intermediates and end products of the aleuritic acid production method based on pathway route 2 using the recombinant host system of the present disclosure , wherein A) depicts the elution peaks of 1 -aleuritic acid, 2) palmitic acid, 3) palmitoleic acid, and 4) 9,10- dihydroxy hexadecanoic acid; C) depicts the mass spectrum of aleuritic acid; D) depicts the mass spectrum of palmitic acid; E) depicts the mass spectrum of palmitoleic acid; and F) depicts the mass spectrum of 9,10- dihydroxy hexadecanoic acid. It is essential to note the absence of 9,10-epoxy hexadecanoic acid intermediate in the elution peaks due to the action of bifunctional epoxide hydrolase enzymes in place of epoxygenase and epoxide hydrolase enzymes.

[0084] Overall, it can be inferred from the above-described examples that the recombinant host system of the present disclosure transformed with the specific gene combinations have the potential to provide alternate means for the production of aleuritic acid to meet the industrial needs.

Advantages of the present disclosure

[0085] The present disclosure provides a recombinant system for producing aleuritic acid with the following advantages. a) The disclosed system yields aleuritic acid with good purity and yield in a time efficient manner compared to conventional approaches. b) The disclosed method for producing aleuritic acid can be scaled up to meet the industrial needs of aleuritic acid.