A process for production of CIS-3-hexenol

Abstract

The present disclosure relates to production of cis-3-hexenol from unsaturated fatty acid, such as alpha linoleic acid and linoleic acid. The process of the present disclosure involves combinatorial and sequential action of recombinant enzymes, such as LOX, HPL and ADH for the production of cis-3-hexenol.

Description

[0001] “A PROCESS FOR PRODUCTION OF CIS- 3- HEXENOL”

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of recombinant technology and fermentation technology. The present disclosure particularly relates to production of cis-3 Hexenol from combination of recombinant enzymes.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Cis-3-hexenol is a natural compound found in green leaves, fruits and vegetables. Conventionally, cis-3-hexenol is produced through- i. extraction from natural sources; ii. chemical synthesis; and iii. enzymatic reactions from the enzymes extracted from natural sources, such as seed and fruit. These reported processes have various limitations for instance, extraction of cis-3-hexenol from natural sources is tedious because the natural extracts are a mix of various biomolecule and it necessitates processing and pre-treatment. Similarly, use of naturally occurring enzymes for production of cis-3-hexenol does not lead to desired result because the enzymes present in the natural source are in small quantities and the extracts would contain competing enzymes which gives rise to undesired products alongside cis-3-hexenol.

[0006] Thus, there is a need for a process to produce cis-3-hexenol which overcomes the limitations described above. The present disclosure describes an effective process of producing cis-3- hexenol from its corresponding substrate through combinatorial and sequential action of recombinant enzymes.

[0007] SUMMARY OF THE DISCLOSURE

[0008] Accordingly, the present disclosure relates to a process for production of cis-3 -hexenol from combination of recombinant enzymes, such as lipoxygenases (LOX), hydroperoxide lyases (HPL) and alcohol dehydrogenase (ADH1). The combination of said recombinant enzymes converts substrate, such as linolenic acid and alpha linoleic acid into cis-3-hexenol through intermediates such as 13-HPOT (13-hydroperoxyoctadeca-9, 11, 15-trienoic acid) and cis- 3-hexenal. In an embodiment, the process of producing cis-3-hexenol comprises- contacting a substrate such as alpha linoleic acid and linoleic acid with biomass comprising lipoxygenases (LOX) and providing optimum condition for producing 13-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT); adding biomass comprising hydroperoxide lyases (HPL), thereby leading to conversion of 13-HPOT to cis-3-hexenal; and adding biomass comprising alcohol dehydrogenase (ADH), thereby producing cis-3- hexenol.

[0009] The process of the present disclosure leads to reduced or negligible formation of by-products such as hexanol, trans-hexenol alongside the desired cis-3-hexenol. Accordingly, the process of the present disclosure leads to production of cis-3-hexenol at highest proportions.

[0010] BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0011] In order that the present disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:

[0012] FIGURE 1 provides an image of agarose gel describing PCR amplification of LOX and HLP genes.

[0013] FIGURE 2 provides an image of agarose gel describing PCR amplification of ADH1 gene from Saccharomyces genomic DNA with flanking sites compatible for cloning in yeast and E.coli vectors.

[0014] FIGURE 3 provides an image of agarose gel with bands of PCR amplification of pET vector preparation by digestion at flanking sites compatible for cloning LOX, HLP and ADH1 genes, respectively.

[0015] FIGURE 4a illustrates a design of expression vector for LOX in pET 28a without PelB signal sequence. FIGURE 4b illustrates a design of expression vector for HPL in pET20b without PelB signal sequence.

[0016] FIGURE 4c illustrates a design of expression vector for ADH in pET28a and pATUM.

[0017] FIGURE 5 provides an image of SDS-PAGE gel illustrating expression of LOX (Ps LOX) and HLP (Pg HPL) proteins with 500 pm IPTG induction in M9 clones.

[0018] FIGURE 6 provides an image of SDS-PAGE gel illustrating expression of LOX (CaLOX) and HLP (Pg HPL) proteins with 500 pm IPTG induction in M13 clones.

[0019] FIGURE 7 provides an image of SDS-PAGE gel illustrating expression of LOX (AtLOX, GmLOX and RiLOX) proteins with 100 pm IPTG induction in BL21 (DE3).

[0020] FIGURE 8 provides an image of SDS-PAGE gel illustrating expression of ADH1 in pET28a, ATUM-Ura3 and ATUM-Lue2 vectors.

[0021] FIGURE 9 provides a plot describing the activity of biomass comprising LOX at a concentration of 175g / L LOX (Ps LOX and Ca LOX) at varied pH of 4.8, 6.8, 7.4 and 8.6.

[0022] FIGURE 10 provides a plot describing the activity of biomass comprising LOX at a concentration of 175g / L LOX and biomass comprising LOX at a concentration of 175g / L HPL at pH of 6.8.

[0023] FIGURE 11 provides a plot describing the conversion of hexanal to cis-3-hexanol by the action of biomass comprising ADH1 (Test) and a plot showing no formation of cis-3-hexanol (Control).

[0024] FIGURE 12 provides a gas chromatography (GC) analysis plot of the conversion of the alpha linolenic acid (ALA) and Linoleic acid (LA) to cis-3-hexenol, respectively.

[0025] FIGURE 13 provides a gas chromatograph (GS) analysis plot of cis-3 -hexenol along with other side products. FIGURE 14 provides a gas chromatography mass spectroscopy (GC-MS) plot confirming the formation of cis-3 -hexenol.

[0026] BRIEF DESCRIPTION OF THE NUCLEOTIDE SEQUENCES AND PEPTIDE

[0027] SEQUENCE

[0028] SEQ ID NO. l: Ps LOX peptide sequence.

[0029] SEQ ID NO.2: Ps LOX nucleotide sequence.

[0030] SEQ ID NO.3: Ca LOX peptide sequence.

[0031] SEQ ID NO.4: Ca LOX nucleotide sequence.

[0032] SEQ ID NO.5: Ri LOX peptide sequence.

[0033] SEQ ID NO.6: Ri LOX nucleotide sequence.

[0034] SEQ ID NO.7: Gm LOX peptide sequence.

[0035] SEQ ID NO.8: Gm LOX nucleotide sequence.

[0036] SEQ ID NO.9: At LOX peptide sequence.

[0037] SEQ ID NO. 10: At LOX nucleotide sequence.

[0038] SEQ ID NO. 11 : Pg HPL peptide sequence.

[0039] SEQ ID NO. 12: Pg HPL nucleotide sequence.

[0040] SEQ ID NO. 13: Ma HPL peptide sequence.

[0041] SEQ ID NO. 14: Ma HPL nucleotide sequence.

[0042] SEQ ID NO. 15: Si HPL peptide sequence.

[0043] SEQ ID NO. 16: Si HPL nucleotide sequence.

[0044] SEQ ID NO. 17: Sc ADH1 peptide sequence.

[0045] SEQ ID NO. 18: Sc ADHI nucleotide sequence.

[0046] Table 1 describes sources of the nucleotide sequences of lipoxygenases (LOX), hydroperoxide lyases (HPL) and alcohol dehydrogenase (ADH).

[0047] Table 1:

[0048] DETAILED DESCRIPTION OF THE DISCLOSURE

[0049] Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. By means of further guidance, term definitions are included for better understanding of the present disclosure.

[0050] As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

[0051] The term ‘comprising’, ‘comprises’ or ‘comprised of as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps.

[0052] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0053] The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically, and preferably disclosed.

[0054] The term ‘exemplary’ or ‘exemplary embodiment’ as used herein refers to ‘serving as an example, instance, or illustration.’ Any embodiment of implementation of the present subject matter described herein as ‘exemplary’ is not necessarily to be construed as preferred or advantageous over other embodiments.

[0055] As used herein, the term ‘reaction mixture’ refers to a starting material of the reaction comprising substrate, such as alpha-linolenic acid (ALA) and linolenic acid (LA), which is used for producing cis-3 -hexenol. The reaction mixture may optionally comprise saline, buffer, additives etc, which aids in the reaction for producing cis-3 -hexenol.

[0056] Reference throughout this specification to ‘some embodiments’, ‘one embodiment’ or ‘an embodiment’ means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases ‘in some embodiments’, ‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0057] The present disclosure relates to an improved process of production of cis-3-hexenol (cis-3- hexen-l-ol) by combinatorial and sequential action of recombinant enzymes identified by the inventors of the present disclosure.

[0058] The present disclosure relates to an improved process for producing cis-3 -hexenol from an unsaturated fatty acid including but not limited to alpha-linolenic acid (ALA) and linolenic acid (LA). In some embodiments of the present disclosure, the process of producing cis-3 -hexenol involves oxidation of unsaturated fatty acid including but not limited to alpha-linolenic acid (ALA) and linolenic acid to 13-HPOT (13-hydroperoxyoctadeca-9, 11, 15- trienoic acid) by the action of lipoxygenases (LOX). Further, the 13-HPOT is converted to cis-3 -hexenal by the action of hydroperoxide lyases (HPL). Thereafter, the cis-3 -hexenal is converted to cis- 3 -hexenol by the action of alcohol dehydrogenase (ADH).

[0059] According to the present disclosure, the enzymes, such as lipoxygenases (LOX), hydroperoxide lyases (HPL) and alcohol dehydrogenase (ADH) are recombinantly produced through fermentation process, respectively. Biomass comprising LOX, biomass comprising HPL, and biomass comprising ADH are individually and sequential employed for the production of cis-3 hexenol from the corresponding substrate.

[0060] The process of the present disclosure converts the unsaturated fatty acid including but not limited to alpha linolenic acid and linolenic acid to cis-3 -hexenol at highest proportion with reduced or negligible production of by-products, such as hexanol and trans-hexenol.

[0061] In some embodiments, the process of producing cis-3-hexenol comprises- contacting a substrate such as alpha linoleic acid and linoleic acid with biomass comprising LOX and providing optimum condition for producing 13-HPOT; adding biomass comprising HPL and providing optimum condition for conversion of 13-HPOT to cis-3-hexenal; and adding biomass comprising ADH and providing optimum condition for producing cis-3 -hexenol.

[0062] In some embodiments, the substrate, such as alpha linolenic acid and linoleic acid is contacted with the biomass comprising LOX, followed by allowing the reaction to occur for a duration ranging from about 90 minutes to 200 minutes, including all the values in the range, for instance, 91 minutes, 92 minutes, 93 minutes, 94 minutes and so on and so forth, up until 200 minutes, at a temperature ranging from about 20 °C to 40 °C. In an embodiment, reaction is carried out for said duration ranging from 90 minutes to 200 minutes under stirring condition at a speed ranging from about 150 rpm to 250 rpm, including all the values in the range, for instance, 151 rpm, 152 rpm, 153 rpm, 154 rpm and so on and so forth. In an embodiment, the reaction is allowed to occur at a temperature of about 20 °C, about 25 °C, about 30 °C, about 35 °C or about 40 °C.

[0063] In an embodiment, the substrate, such as alpha linolenic acid and linoleic acid is contacted with biomass comprising LOX and the reaction is allowed to occur for a duration of about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 180 minutes, about 190 minutes or about 200 minutes. In an embodiment, the reaction is carried out under stirring condition at a speed for about 150 rpm, about 160 rpm, about 170 rpm, about 180 rpm, 190 rpm, about 200 rpm, about 210 rpm, about 220 rpm, about 230 rpm, about 240 rpm or about 250 rpm.

[0064] In some embodiments, the reaction between the substrate, such as alpha linolenic acid and linoleic acid and the biomass comprising LOX is carried out at a pH ranging from about 4.8 to 8.6, including all the values in the range, for instance, 4.9, 5.0, 5.1, 5.2 and so on and so forth, up until 8.6.

[0065] In some embodiments, the biomass comprising LOX is employed at a concentration ranging from about 75 g / L to 200 g / L, including all the values in the range for instance, 76 g / L, 77 g / L, 78 g / L, 79g / L and so on and so forth. In some embodiment, the biomass comprising LOX is employed at a concentration of about 75 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L.

[0066] Accordingly, the substrate, such as alpha linolenic acid and linoleic acid is contacted with the biomass comprising LOX having concentration ranging from about 75 g / L to 200 g / L and the mixture is incubated for a duration ranging from about 100 minutes to 200 minutes, at a temperature ranging from about 20 °C to 40 °C and at pH ranging from about 4.8 to 8.6 for the formation of 13-HPOT.

[0067] In some embodiments of the present disclosure, the conversion of substrate, such as alpha linolenic acid and linoleic acid to 13-HPOT with the biomass comprising LOX is carried out in presence of oxygen. Accordingly, oxygen in the reaction is supplied in a range of about 0.5 LPM to 4 LPM, including all the values in the range, for instance, 0.6 LPM, 0.7 LPM, 0.8 LPM, 0.9 LPM and so on and so forth, up until 4 LPM. In an embodiment, addition of oxygen in an amount of about 1 LPM, about 2 LPM, about 3 LPM or about 4 LPM leads to 100% conversion of the substrate to 13-HPOT in about 30 minutes, about 60 minutes, about 90 minutes or about 150 minutes, respectively.

[0068] In some embodiments of the present disclosure, the conversion of substrate, such as alpha linolenic acid and linoleic acid to 13-HPOT with the biomass comprising LOX is carried out by adding hydrogen peroxide and catalase enzyme for providing oxygen during the reaction. In an embodiment, the catalase enzyme is added in an amount ranging from about 10 units to 50 units, including all the values in the range, for instance, 11 units, 12 units, 13 units, 14 units and so on and so forth, up until 50 units. In an embodiment, the hydrogen peroxide is added in an amount ranging from about 2mg to 20mg, including all the values in the range, for instance, 2. 1 mg, 2.2 mg, 2.3 mg, 2.4 mg and so on and so forth, up until 20 mg.

[0069] In some embodiments, in the process of the present disclosure, the 13-HPOT is converted to cis-3- hexenal by adding biomass comprising HPL at a concentration ranging from about 50 g / 1 to 150 g / 1. In an embodiment, the reaction for obtaining the cis-3- hexenal is carried out for a duration ranging from about 30 mins to 150 mins, at a temperature ranging from about 10 °C to 40° C and pH ranging from about 4.5 to 8.5. In an embodiment, reaction is carried out for a duration of about 30 mins, about 40 mins, about 50 mins, about 60 mins, about 70 mins, about 80 mins, about 90 mins, about 100 mins, about 110 mins, about 120 mins, about 130 mins, about 140 mins or about 150 mins. In an embodiment, the reaction is carried out for a duration of about 10 °C, about 20 °C, about 30 °C or about 40 °C.. In an embodiment, the reaction is carried out at a pH of about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0 or about 8.0.

[0070] In some embodiments of the present disclosure, the conversion of the cis-3-hexenal to cis- 3-hexenol is carried out by adding biomass comprising ADH at a concentration ranging from about 50 g / 1 to 150 g / 1, including all the values in the range, for instance, 51 g / 1, 52 g / 1, 53 g / 1, 54 g / 1 and so on and so forth, up until 150 g / 1. In an embodiment, reaction for the conversion of the cis-3-hexenal to cis-3-hexenol is carried out for a duration ranging from about 5 mins to 120 mins. In an embodiment, the reaction for the conversion of the cis-3-hexenal to cis-3-hexenol is carried out at a temperature ranging from about 20 °C to 40 °C, including all the values in the range, for instance, 21 °C, 22 °C, 23 °C, 24 °C and so on and so forth, up until 40 °C. In an embodiment, the reaction is carried out at a temperature of about 20 °C, about 25 °C, about 30 °C, about 35 °C or about 40 °C.

[0071] In an embodiment, the reaction for the conversion of the cis-3-hexenal to cis-3-hexenol is carried out at a pH ranging from about 6.5 to 8.5, including all the values in the range, for instance, 6.6, 6.7, 6.8, 6.9 and so on and so forth.

[0072] Accordingly, the reaction for the conversion of the cis-3-hexenal to cis-3-hexenol is carried out by adding biomass comprising ADH at a concentration ranging from about 50 g / 1 to 150 g / 1. Upon adding the biomass comprising ADH, the mixture is incubated for a duration ranging from about 5 mins to 120 mins, at a temperature ranging from about 20 °C to 40 °C and at a pH ranging from about 6.5 to 8.5.

[0073] In some embodiments of the present disclosure, the production of cis-3 hexenol from an unsaturated fatty acid including but not limited to alpha-linolenic acid (ALA) and linolenic acid involves combinatorial addition of biomass comprising LOX, biomass comprising HPL and biomass comprising ADH. The biomass comprising LOX, biomass comprising HPL and biomass comprising ADH are added together to a reaction mixture comprising unsaturated fatty acid including but not limited alpha-linolenic acid (ALA) and linolenic acid. In an embodiment, the biomass comprising LOX is employed at a concentration ranging from about 75 g / L to 200 g / L. In an embodiment, the biomass comprising HPL is employed at a concentration ranging from about 50 g / L to 150 g / L. In an embodiment, the biomass comprising ADH is employed at a concentration ranging from about 50 g / L to 150 g / L. Accordingly, about 75 g / L to 200 g / L of the biomass comprising LOX, about 50 g / L to 150 g / L of the biomass comprising HPL and about 50 g / L to 150 g / L of the biomass comprising ADH are added simultaneously to a reaction mixture comprising substrate, such as alpha linolenic acid and linolenic acid for obtaining cis-3 hexenol. During reaction, the biomass comprising LOX converts said substrate to 13-HPOT. Subsequently, the biomass comprising HPL converts the 13-HPOT to cis-3-hexenal and thereafter the biomass comprising ADH converts the cis-3-hexenal to cis -3 -hexenol. The reaction for obtaining cis-3-hexenol from said substrate by employing combinatorial addition of biomass comprising LOX, biomass comprising HPL and biomass comprising ADH is carried out at a temperature ranging from about 10 °C to 37 °C, for a duration ranging from about 60 minutes to 120 minutes and at a pH ranging from about 5.0 to 9.0. Further, during the reaction, oxygen is supplied in an amount ranging from about 0.5 LPM to 4 LPM.

[0074] In some embodiments of the present disclosure, the genes of LOX, HPL and ADH from the sources as indicated in Table 1 described above is expressed as full-length peptides in prokaryotic and eukaryotic host system, individually. The prokaryotic host expression system includes but not limited to E.coli and the eukaryotic host expression system includes but not limited to Saccharomyces cerevisiae and Pichia pastoris.

[0075] In an embodiment, according to the present disclosure the LOX gene, HPL gene and ADH gene, respectively are cloned as full-length peptides and are expressed under signal peptides including but not limited to PelB signal sequence, MBP tag sequence and Thioredoxin A tag sequence.

[0076] In an embodiment, according to present disclosure the LOX gene, HPL gene and ADH gene, respectively are expressed alongside chaperones including but not limited to foldases, Protein Disulfide-Isomerase (PDI) and chaperonins, which ensures efficient folding of the peptides- LOX, HPL and ADH, respectively by forming proper secondary and tertiary structures in the protein.

[0077] In some embodiments of the present disclosure, the LOX gene is expressed in cytosolic region of the host cells wherein the peptide is folded under the influence of combination of chaperons and increased redox potential in the cytosolic region due to co-expression of the tag sequence.

[0078] In some embodiments of the present disclosure, the HPL gene is expressed in cytosolic region of the host cells wherein the peptide is folded under the influence of combination of chaperons and increased redox potential in the cytosolic region due to co-expression of the tag sequence. In some embodiments of the present disclosure, the ADH gene is expressed in cytosolic region of the host cells wherein the peptide is folded under the influence of combination of chaperons and increased redox potential in the cytosolic region due to coexpression of the tag sequence. In the present disclosure, the recombinant enzymes, such as LOX, HPL and ADH employed in the process for conversion of unsaturated fatty acid, such as alpha linoleic acid and linolenic acid into cis-3-hexenol, are without any competing enzymes. Thus, leading to an improved production of cis-3-hexenol, unlike the enzymes extracted from natural sources.

[0079] The process of the present disclosure provides for highly efficient conversion of unsaturated fatty acid, such as alpha linolenic acid and linolenic acid into cis-3 -hexenol, with reduced or negligible by products. Thus, making the process efficient and economical.

[0080] While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples (and drawings) described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims. The present disclosure is therefore further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

[0081] EXAMPLES

[0082] Example 1: Cloning of LOX gene

[0083] The CaLOX and PsLOX genes were cloned in the pET28a vector system as per the cloning strategies and design of constructs mentioned in Figure 4a between restriction endonuclease Ncol & EcoRI sites. The clones were obtained upon bacterial transformation by selection of kanamycin antibiotic at lOOug / mL. The host chosen was E.coli BL21 (DE). The strain expresses the ORF of the gene by addition of IPTG which is unnatural inducer and mimic of lactose in the natural system which trigger Lac operon. This lac operon finally triggers the production of T7 RNA polymerase which translates the genes which were cloned downstream of T7 promoter.

[0084] Similarly, the CaLOX and PsLOX constructs were generated under PelB signal sequence in pET28a and pET20b vector to channelize the nascent peptide toward periplasmic region of the host. Example 2: Cloning of HPL gene

[0085] The PgHPL and MaHPL genes were cloned in the pET20b vector system as per the cloning strategies and design of constructs mentioned in Figure 4b between restriction endonuclease Ndel & EcoRI sites. The clones were obtained upon bacterial transformation by selection of ampicillin antibiotic at lOOug / mL. The host chosen was E.coli BL21 (DE).

[0086] Similarly, the pgHPL and MaHPL constructs were generated under PelB signal sequence in pET28a and pET20b vector to channelize the nascent peptide toward periplasmic region of the host.

[0087] Example 3: Cloning of ADH gene

[0088] The ADH1 genes were cloned in pET28a and pATUM vector system as per the cloning strategies and design constructs mentioned in Figure 4c between restriction endonuclease Ndel-BamHI sites and Sapl site, respectively.

[0089] Example 4: Expression of LOX gene

[0090] E.coli clones obtained according to Example 1 were patched onto LB agar plates containing appropriate concentration of antibiotic. The patches were further inoculated into LB broth with antibiotic and grown overnight in a shaker incubator at 37 °C temperature and 200rpm.

[0091] The overnight grown inoculum was used to further inoculate into lOOmL of LB broth with antibiotic with the starting OD of 0.2 in shaker incubator at 37 °C temperature and 200rpm. Upon reaching the OD of 1.2 - 1.4, 500uM IPTG was added aseptically and further incubated for another 24-48 hours. Once the final OD reaches up to in the range of 7.0-10, the sample was centrifuged at 10,000 rpm using table-top centrifuge for lOmins at 4 °C. The supernatant was discarded, and the cell pellet biomass was collected and stored at -20 °C freezer until further use. Example 5: Expression of HPL gene

[0092] E.coli clones obtained according to Example 2 were patched onto LB agar plates containing appropriate concentration of antibiotic. The patches were further inoculated into LB broth with antibiotic and grown overnight in a shaker incubator at 37 °C temperature and 200 rpm.

[0093] The overnight grown inoculum was used to further inoculate into lOOmL of LB broth with antibiotic with the starting OD of 0.2 in shaker incubator at 37 °C temperature and 200rpm. Upon reaching the OD of 1.2 - 1.4, 500uM IPTG was added aseptically and further incubated for another 24-48 hours. Once the final OD reaches up to in the range of 7.0-10, the sample was centrifuged at 10,000 rpm using table-top centrifuge for lOmins at 4 °C. The supernatant was discarded, and the cell pellet biomass was collected and stored at -20 °C freezer until further use.

[0094] Example 6: Expression of ADH gene

[0095] Saccharomyces cerevisiae and Pichia pastoris clones obtained according to Example 3 were patched onto SC dropout agar plates containing appropriate amino-acid dropout for selection of clones with complemented autotrophic marker present in the vector. The patches were further inoculated into YPD broth and grown overnight in a shaker incubator at 30 °C temperature and 200rpm.

[0096] The overnight grown inoculum was used to further inoculate into lOOmL of YPD broth with the starting OD of 0.2 in shaker incubator at 30 °C temperature & 200rpm for 48 to 72 hours. Upon reaching the OD of 20 and above, Cell mass was harvested by centrifuging the culture sample at 10,000 rpm using table-top centrifuge for lOmins at 4 °C. The supernatant was discarded, and the cell pellet biomass was collected and stored at -20 °C freezer until further use

[0097] Example 7: Evaluating expression of LOX, HPL and ADH enzymes lOmg of the wet cell pellet biomass from Examples 4, 5 and 6, respectively were taken and samples were prepared for SDS-PAGE analysis. The cell pellet biomass was dissolved into 160ul of TBS buffer and 40pl of Laemmli buffer which contains an electrophoretic dye for denaturation of proteins and monitoring the front of running gel. The dissolved pellets containing the Laemmli buffer were mixed well and incubated at 95 °C for 10 mins with intermitent tapping of the samples. 10% SDS-PAGE gels were prepared, and the samples were loaded and run in the gel electrophoresis chamber containing buffer for 60 minutes at 100 volts seting.

[0098] The gels after electrophoretic separation were taken out and kept in CBB (Coomassie brilliant blue) staining dye which imparts blue color to the protein bands. Figures 5, 6, 7 and 8 show the well separated recombinant proteins (LOX, HPL and ADH) and identified by the protein maker with different reference sizes.

[0099] Example 8: Conversion of unsaturated fatty acid to 13-HPOT

[0100] 10 ml induced biomass comprising LOX (from Example 4) having a concentration of 175 g / 1 was mixed with about 5% to 20% of the alpha linolenic acid (ALA) and incubated at 30 °C at pH 4.8 to 8.6, for a duration of about 150 minutes under shaking at 220 rpm.

[0101] 100% conversion of ALA to 13-HPOT was noted at 150 minutes at a pH of 6.8 from the PsLOX biomass

[0102] 80% conversion of ALA to 13-HPOT was noted at 150 minutes at a pH of 7.4 from the CaLOX biomass.

[0103] 60% conversion of ALA to 13-HPOT was noted at 150 minutes at a pH of 6.8 and 7.4, respectively from the CaLOX biomass

[0104] Example 9: Conversion of unsaturated fatty acid to 13-HPOT

[0105] 10 ml induced biomass comprising LOX (from Example 4) having concentration of 175 g / 1 was mixed with about 5% to 20% of the alpha linolenic acid (ALA) and incubated at 30 °C at pH 4.8 to 8.6, for a duration of about 150 minutes under shaking at 220 rpm. During the reaction, about 10 to 50 units of catalyse enzyme and 10 mg of hydrogen peroxide were added every 10 minutes.

[0106] 100% conversion of ALA to 13 -HPOT was noted in about 40 minutes to 60 minutes . Further, it was noted that increasing the addition of catalyse enzyme considerably lowered the activity due to toxicity effect of the catalase enzyme.

[0107] Figure 9 illustrates the conversion of ALA to 13-HPOT at varied pH. Example 10: Conversion of 13-HPOT to cis-3-hexenal

[0108] For the 13-HPOT obtained in the Examples 8 and 9, 5ml of biomass comprising HPL at a concentration of about 100 g / L was added, followed by mixing and incubating at a temperature of about 10° C, duration of about 60 mins and pH of about 7.5 to obtain cis-3- hexenal.

[0109] Figure 10 illustrates conversion of 13-HPOT to cis-3-hexenal.

[0110] Example 11: Conversion of cis-3-hexenal to cis-3-hexenol

[0111] For the cis-3-hexenal obtained in Example 10, about 9.5ml of 100 g / L of biomass comprising ADH was added, followed by mixing and incubated for a duration of about 60 mins, at a temperature of about 30° C and pH of 7.5 to obtain cis-3-hexenol. Figure 11 illustrates the conversion of cis-3-hexenal to cis-3-hexenol (in Test) and no conversion in the Control.

[0112] Example 12: Conversion of unsaturated fatty acid to cis-3-hexenol

[0113] Reaction was carried at volumes ranging from 10ml to 500ml at pH 6.8 and at a temperature of 30 °C.

[0114] Induced LOX biomass was dissolved in 10ml at concentration of 75 gm / L with 52.5 mM substrate dosed over (added in parts, 2.5 mM ALA (25 pmol ALA) each at the interval of 10 minutes over 3 hrs period with;

[0115] (1) Oxygen supply at 2 LPM; or

[0116] (2) 20 Units of catalase enzyme and 10 mg Hydrogen peroxide was added every 10 min. 5ml each of cell biomass HPL & ADH1 cell biomass with the concentration of 75 g / 1 were added and incubated for 60 minutes at 10°C temperature for HPL reaction and then changed the temperature to 30°C temperature and further incubated for 60 minutes.

[0117] The data showed that the complete reaction (conversion of ALA to cis-3 -hexenol) was completed in about 300 minutes from the start of the experiment in the given conditions.

[0118] The reaction mixture was extracted using ethyl acetate solvent at 1: 1, 1:2 and 1:3 ratio volumes and used for GC analysis. The reaction with alpha linolenic acid (ALA), the product formation cis-3- hexenol was evident by RT (retention time) in the gas chromatogram which matches reference RT of the product.

[0119] The data showed that the recovery of the final product was 100% extracted into organic phase with 1:2 and 1:3 ratios and 85% extracted in 1: 1 ratio. Though the extraction process is 100% in higher volumes, it is eventually less efficient in the downstream as more solvent will have more losses in the purification process.

[0120] Figure 12 describes GC analysis plots illustrating formation of cis-3-hexanol.

[0121] Further, the reaction mixture was analysed in GC-MS, which again confirmed the formation of product (cis-3-hexenol). Figure 14 describes GC-MS analysis plot illustrating formation of cis-3-hexenol.

[0122] Example 13: Process of producing cis-3- hexenol from unsaturated fatty acid

[0123] About 100-300 g / L of the biomass comprising LOX, about 200-300 g / L of the biomass comprising HPL and about 100-300 g / L of the biomass comprising ADH is added to a reaction comprising unsaturated fatty acid, such as alpha linolenic acid (5% to 20%), followed by mixing. About 1-5 LPM of oxygen was supplied. The reaction was initiated at a temperature of about 10-25 °C and maintained for a duration of about 60-120 minutes. The pH of the reaction mixture was maintained at 5-8. After the completion of 120 minutes, cis- 3 -hexenol was obtained.

[0124] The present disclosure is further explained by the following numbered embodiments. These embodiments represent important aspects of the present disclosure, but should not be construed to limit its scope:

[0125] 1. A process for producing cis-3-hexenol, said process comprises- contacting a substrate such as alpha linoleic acid and linoleic acid with biomass comprising LOX and providing optimum condition for producing 13-HPOT; adding biomass comprising HPL and providing optimum condition for conversion of 13-HPOT to cis-3-hexenal; and adding biomass comprising ADH and providing optimum condition for the production of cis-3 -hexenol.

[0126] 2. The process as defined in embodiment 1, the biomass comprising LOX is contacted with the substrate, and reaction is carried out for a duration ranging from about 90 minutes to 200 minutes, at a temperature ranging from about 20 °C to 40 °C and pH ranging from about 4.8 to 8.6, under stirring at a speed ranging from about 150 rpm to 250 rpm.

[0127] 3. The process as defined in embodiment 2, wherein the process involves supplying oxygen in range of about 0.5 LPM to 4 LPM.

[0128] 4. The process as defined in embodiment 2, wherein the process involves adding 10 to 50 units of catalase enzyme and about 5mg to 15 mg of hydrogen peroxide.

[0129] 5. The process as defined in embodiment 1, wherein the conversion of 13-HPOT to cis- 3-hexenal is carried out by adding biomass comprising HPL at a concentration ranging from about 50 g / 1 to 150 g / 1.

[0130] 6. The process as defined in embodiment 1, wherein the conversion of the cis-3-hexenal to cis-3 -hexenol is carried out by adding biomass comprising ADH at a concentration ranging from about 50 g / 1 to 150 g / 1.

[0131] 7. The process as defined in embodiment 6, wherein the conversion of the cis-3-hexenal to cis-3-hexenol is carried out at a temperature ranging from about 20 °C to 40 °C and at a pH ranging from about 6.5 to 8.5.

[0132] 8. The process as defined in embodiment 1, wherein the biomass comprising LOX is selected from a group comprising biomass comprising PsLOX, biomass comprising CaLOX biomass, biomass comprising RiLOX, biomass comprising GmLOX and combinations thereof; and wherein the biomass comprising HPL is selected from a group comprising biomass comprising PgHPL, biomass comprising MaHPL, biomass comprising SiPHL biomass and combinations thereof. 9. The process as defined in embodiment 1, wherein the process leads to reduced or negligible formation of by-product selected from a group comprising hexanol, transhexenol and a combination thereof.

Claims

We Claim:

1. A process for producing cis-3-hexenol, said process comprises adding biomass comprising lipoxygenase (LOX), biomass comprising hydroperoxide lyase (HPL), biomass comprising alcohol dehydrogenase (ADH) or a combination thereof to a reaction mixture comprising substrate selected from a group comprising linolenic acid and alpha-linolenic acid.

2. The process as claimed in claim 1, wherein said process comprises- a) contacting a substrate selected from a group comprising alpha-linolenic acid and linolenic acid with biomass comprising lipoxygenase (LOX) under condition suitable for a reaction for formation of 13 -hydroperoxyoctadeca-9, 11,15 -trienoic acid (13-HPOT); b) adding biomass comprising hydroperoxide lyase (HPL) to reaction mixture of step a) under conditions suitable for a reaction for conversion of 13-HPOT to cis-3 - hexenal; and c) adding biomass comprising alcohol dehydrogenase (ADH) to reaction mixture of step b) under conditions suitable for a reaction for conversion of cis-3-hexenal to cis-3-hexenol.

3. The process as claimed in claim 1, wherein the process comprises adding biomass comprising lipoxygenase (LOX), biomass comprising hydroperoxide lyase (HPL) and biomass comprising alcohol dehydrogenase (ADH) to the reaction mixture, wherein the biomass comprising (LOX) converts the substrate to 13-HPOT, the biomass comprising HPL converts 13-HPOT to cis-3 hexenal and the biomass comprising ADH converts the cis-3 hexenal to cis-3 hexenol.

4. The process as claimed in claim 1, wherein the lipoxygenase (LOX), the hydroperoxide lyase (HPL) and the alcohol dehydrogenase (ADH) is independently produced recombinantly.

5. The process as claimed in claim 1, wherein the substrate is at a concentration ranging from about 5% to 20%.

6. The process as claimed in claim 1, wherein the biomass comprising LOX is at a concentration ranging from about 75 g / L to 200 g / L.

7. The process as claimed in claim 1, wherein the biomass comprising HPL is at a concentration ranging from about 50 g / L to 150 g / L.

8. The process as claimed in claim 1, wherein the biomass comprising ADH is at a concentration ranging from about 50 g / L to 150 g / L.

9. The process as claimed in claim 2, wherein the condition suitable for formation of 13- HPOT is temperature ranging from 20°C to 40°C, duration of 90 to 200 minutes and pH 4.8 to 8.6.

10. The process as claimed in claim 2, wherein the reaction in step a) comprises further addition of hydrogen peroxide and catalase, wherein the hydrogen peroxide is added in an amount of 10 to 50 units and hydrogen peroxide is added in an amount of about 2 mg to 20 mg for producing oxygen during the reaction.

11. The process as claimed in claim 2, wherein the reaction in step a) comprises supplying oxygen at a flow rate of about 0.5 to 4 LPM.

12. The process as claimed in claim 2, wherein the condition suitable for the reaction in step b) is temperature ranging from about 10 °C to 40 °C and pH ranging from about4.5 to 8.5 and duration is ranging from about 30 minutes to 150 minutes.

13. The process as claimed in claim 2, wherein the condition suitable for the reaction in step c) is temperature ranging from about 20 °C to 40 °C, duration ranging from about6.5 to 8.5 and duration ranging from about 5 minutes to 120 minutes.

14. The process as claimed in claim 3, wherein the process comprises supplying oxygen at a flow rate of about 0.5 to 4 LPM.

15. The process as claimed in claim 3, wherein the process is carried out a temperature ranging from about 10 °C to 37 °C, for a duration ranging from about 60 minutes to 120 minutes and at a pH ranging from about 5 to 8.

16. The process as claimed in claim 1, wherein the process further comprises extracting the cis-3-hexenol using ethyl acetate at a ratio of 1: 1 to 1:3 (v / v).

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