EP4028533A1 - Photo biocatalytic synthesis of secondary fatty alcohols from unsaturated fatty acids - Google Patents

Photo biocatalytic synthesis of secondary fatty alcohols from unsaturated fatty acids

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Publication number
EP4028533A1
EP4028533A1 EP20772137.4A EP20772137A EP4028533A1 EP 4028533 A1 EP4028533 A1 EP 4028533A1 EP 20772137 A EP20772137 A EP 20772137A EP 4028533 A1 EP4028533 A1 EP 4028533A1
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EP
European Patent Office
Prior art keywords
acid
compartment
decarboxylase
optionally containing
photoactivated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP20772137.4A
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German (de)
French (fr)
Inventor
Frank Hollmann
Wuyuan ZHANG
Robert KOURIST
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Technische Universiteit Delft
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Technische Universiteit Delft
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Publication of EP4028533A1 publication Critical patent/EP4028533A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)

Definitions

  • the invention relates to a method for the preparation secondary fatty alcohols.
  • Long-chain (fatty) secondary alcohols may be useful as active ingredients in cosmetic formulations, performance additives in oleochemicals, as building blocks in natural product synthesis, and in organic photosensitisers.
  • the present invention relates to a more practical and environmentally less demanding alternative also yielding secondary fatty alcohols.
  • the invention provides a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising the steps of (a) a hydroxyl introduction stage comprising hydration and/or hydroxylation of the unsaturated fatty acid with a first enzyme to provide a fatty hydroxy acid; and (b) a decarboxylation stage comprising decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
  • the invention provides a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising enzymatic hydration of the unsaturated fatty acid with a hydratase to form a fatty hydroxy acid and decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
  • the invention provides for a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising comprises enzymatic hydroxylation of the unsaturated fatty acid with a fatty acid hydroxylase and decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
  • these processes may even result into secondary fatty alcohols of high enantiomeric purity.
  • the invention provides a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising the steps of (a) a hydroxyl introduction stage comprising hydration and/or hydroxylation of the unsaturated fatty acid with a first enzyme to provide a fatty hydroxy acid; and (b) a decarboxylation stage comprising decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
  • the present invention relates to a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid, wherein the hydroxyl-introduction stage comprises enzymatic hydration of a double bond.
  • the present invention relates to a method for the preparation of a-secondary fatty alcohol from an unsaturated fatty acid, comprising the steps of enzymatic hydration of the unsaturated fatty acid to form a fatty hydroxy acid and decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
  • the unsaturated fatty acid may contain more than one double bond, such as two, three, four or five double bonds.
  • the unsaturated fatty acid may further contain one or more hydroxyl groups, such as one, two, three or four hydroxyl groups.
  • the invention provides a method for the preparation of an enantiomerically pure secondary fatty alcohol from an unsaturated fatty acid, wherein an unsaturated fatty acid of the general formula [1] is converted into a fatty hydroxy acid of formula [2] and the fatty hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [3] wherein R1 and R2 independently represent an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
  • the invention provides a method for the preparation of an enantiomerically pure secondary fatty alcohol from an unsaturated fatty acid, wherein an unsaturated fatty acid is first hydroxylated with a fatty acid hydroxylase and the resulting fatty hydroxy acid is decarboxyl ated with a photoactivated decarboxylase to form a secondary fatty alcohol.
  • Suitable unsaturated fatty acids which can be converted using the method according to the present invention may include for example myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, linoleic acid, docosadienoic acid, a-linolenic acid, pinolenic acid, a-eleostearic acid, mead acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid , adrenic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, docosahexaenoic acid, a-hydroxymyristic acid, b-hydroxymyristic acid, a-hydroxypalmitic acid, b-hydroxypalmitic acid, b- hydroxy
  • a hydroxyl group into the unsaturated fatty acid to form a fatty hydroxy acid can be carried out for example using a hydratase enzyme from theE.C. class 4.2.1.
  • suitable enzymes may include fumarate hydratase (E.C. 4.2.1.2), aconitate hydratase (E.C. 4.2.1.3), enoyl-CoA hydratase (E.C. 4.2.1.17), maleate hydratase (E.C. 4.2.1.31), oleate hydratase (E.C. 4.2.1.53), 3- hydroxybutyryl-CoA dehydratase (E.C.
  • lipoxygenases may be useful enzymes for the transformation of unsaturated fatty acids according to the present invention.
  • Exemplary lipoxygenase enzymes may include: linoleate 13S-lipoxygenase; arachidonate 12-lipoxygenase, arachidonate 15 -lipoxygenase, arachidonate 5 -lipoxygenase, arachidonate 8-lipoxygenase, linoleate 11 -lipoxygenase, linoleate 98- lipoxygenase, linoleate 8R-lipoxygenase, linolenate 9R-lipoxygenase, linoleate 10R- lipoxygenase, oleate 1 OS-lipoxygenase and linoleate 9/13 -lipoxygenase.
  • enzymes suitable for enzymes for the transformation of unsaturated fatty acids into fatty alcohols according to the present invention are 7,10-diol synthases and 5,8-diol synthases.
  • These enzymes can be obtained from various sources including (but not limited to): Arabidopsis thaliana , Aspergillus species, such as Aspergillus flavus , Aspergillus nidulans, Brassica oleracea var.
  • gemmifera Casuarina glauca, Cucumis sativus , Datisca glomerata , Elizabethkingia meningoseptica , Fusarium oxysporum , Glycine max , Flomo sapiens , Hordeum vulgare , Lactobaccilus species, Lysinibacillus fusiformis , Macrococcus caseolyticus, Momordica char anti a, Nicotiana attenuata , Nostoc punctiforme , Olea europaea , Oryza sativa, Pseudomonas species, such as Pseudomonas aeruginosa , Solanum lycopersicum , Solanum tuberosumand Stenotrophomonas maltophilia.
  • Suitable enzymes for the hydratase reaction are for example an oleate hydratase derived from Elizabethkingia meningoseptica (EmO ) (Demming, 2017) (SEQ ID NO: 6) or an oleate hydratase derived from a Lactobacillus species (ZrOH) (SEQ ID NO:2).
  • the present invention relates to a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid, wherein the hydroxyl-introduction stage comprises hydroxylation, and wherein the first enzyme comprises a fatty acid hydroxylase.
  • Suitable enzymes for the hydroxylation reaction of the fatty acid are 7,10-diol synthase from Pseudomonas aeruginosa (Estupinan, 2014), and 5,8-diol synthase from Aspergillus nidulans (Seo, 2014).
  • 5,8-diol synthase can be applied as this bifunctional enzyme adds two instead of only one new OH functionalities into an unsaturated fatty acid by a two- step reaction, according to the following scheme:
  • the fatty acid according to formula (4) is first converted by the 5,8-diol synthase into an intermediary compound (5) wherein it is further converted by the 5,8-diol synthase into the diol (6) and this fatty di-hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [7] wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
  • a fatty acid according to formula (4) is converted by the 5,8-diol synthase into the diol (6) and this fatty di-hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [7] wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
  • a suitable photoactivated decarboxylase for use according to the present invention is for example photoactivated decarboxylase from Chlorella variabilis NC64A (CvFAP) and photoactivated decarboxylase enzymes having a sequence identity of at least 50 %, prefered 65%, more preferred 80%, most preferred 85 % with the reported sequence of decarboxylase from Chlorella variabilis NC64A (CvFAP) (Sorigue, 2017).
  • An enzyme used in the process according to the present invention may be obtained from the organism which naturally produces such enzyme.
  • the enzyme may be produced by a host cell which is transformed to produce the enzyme or several of the enzymes needed in the process of the invention by recombinant techniques known in the art.
  • an enzyme may be applied during the process of the invention in the form of whole cells producing the enzyme.
  • an enzyme may be applied during the process of the invention in the form of a lysate of the cells producing the enzyme.
  • an enzyme may be applied during the process of the invention in a more or less purified form, such as in a form essentially free from particulate material from the producing cells. Purification of the enzyme may be performed by any method known in the art.
  • an enzyme used in the process according to the present invention may be a variant or mutant of a naturally occuring enzyme, still having the desired enzymatic activity.
  • Such variant enzyme may have a sequence identity as compared to the amino acid sequence of the naturally occuring enzyme of at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% and more preferably at least 99%.
  • the reactions can suitably be carried out in an emulsion of an aqueous phase and a water-immiscible organic phase.
  • the water-immiscible organic phase may also include a hydrophobic phase and an oil phase.
  • the water-immiscible organic phase may provide both a source of the (unsaturated fatty acid) substrate as well as a sink for the reaction product, whereas the enzymes will substantially reside in the aqueous phase.
  • a water-in-oil emulsion or an oil-in-water emulsion may be employed.
  • the reactions are carried out in an emulsion of an aqueous phase and a water-immiscible organic phase (such as an oil phase), more in particular the reactions may be carried out in a water-in-oil emulsion or an oil-in-water emulsion.
  • a water-immiscible organic phase such as an oil phase
  • the oil phase may for example comprise triolein. Accordingly, in one embodiment of the method of the invention the reactions are carried out in an emulsion of water and triolein.
  • the unsaturated fatty acids can be derived from a natural source, wherein the fatty acids may be found e.g. as esters, such as triglycerides, phospholipids or cholesterol esters.
  • the unsaturated fatty acids can be used directly as such or may be obtained from their natural source e.g. as amides, thioesters or esters of these unsaturated fatty acids.
  • the amides, thioesters or esters have to be converted to the unsaturated fatty acids e.g. by an enzymatic reaction such as: double bond can be (E) or (Z)-configured
  • the unsaturated fatty acid is obtained from a corresponding amide, thioester or ester.
  • Preferred sources of natural unsaturated fatty acids are mono-, di- or triesters of glycerol as present in natural fats and oils.
  • examples of such glycerides are (but are not limited to): soybean oil, tea seed oil, rapeseed oil, cottonseed oil, olive oil, castor oil, sunflower oil, peanut oil, canola oil, sunflower seed oil, sea buckthorn oil, linseed oil, palm oil or jojoba oil, including their (partially) hydrolysed forms.
  • hydrolases for preparing the free unsaturated fatty acid may be used any enzymes from the class of carboxylic acid hydrolases (E.C. 3.x.x.x).
  • carboxylic acid hydrolases comprises in particular so-called esterases, lipases, peptidases, amidases, thioesterases and glycosidases.
  • the unsaturated fatty acid is obtained from a corresponding mono-, di- or triester of glycerol
  • An enzyme used in the process according to the present invention may be obtained from the organism which naturally produces such enzyme.
  • the enzyme may be produced by a host cell which is transformed to produce the enzyme or several of the enzymes needed in the process of the invention by recombinant techniques known in the art.
  • an enzyme may be applied during the process of the invention in the form of whole cells producing the enzyme.
  • an enzyme may be applied during the process of the invention in the form of a lysate of the cells producing the enzyme.
  • an enzyme may be applied during the process of the invention in a more or less purified form, such as in a form essentially free from particulate material from the producing cells. Purification of the enzyme may be performed by any method known in the art.
  • an enzyme may be applied during the process of the invention in immobilized form.
  • Many useful methods for immobilization of enzymes are known in the art.
  • the concentrations of the enzymes may be chosen to be optimal for the enzymatic conversions, and may range between 0.000001 mM and 10 mM, preferably between 0.001 mM and 1 mM, more preferably between 0.001 mM and 0.1 mM.
  • the process of to the present invention may be carried out as a one-pot two-step procedure.
  • Such one-pot two-step procedure may comprise first performing the hydroxyl-introducing step followed by the addition of the photoactivated decarboxylase and illumination to promote the decarboxylation reaction.
  • the one-pot two-step procedure may comprise first performing the hydroxyl-introducing step in the presence of both the hydroxyl-introducing enzyme in dark conditions and the photoactivated decarboxylase and later start illumination to promote the decarboxylation reaction.
  • the illumination will be started when substantially all of the unsaturated fatty acid is converted into the hydroxylated form.
  • reaction conditions such as temperature, pH, and concentrations
  • illumination relates to targeting a light source of the reaction medium with a wavelength of between 380 and 500 nm, more preferably near to 450 nm.
  • dark conditions may imply substantially the absence of illumination of the reaction medium with a light source at a wavelength of between 380 and 500 nm.
  • a flow-chemistry setup may be used.
  • the enzymes may be immobilised. Many methods for immobilisation of enzymes are known in the art.
  • the enzymes may be immobilized for example by covalent bonding to a suitable surface or a carrier, by adsorption to a suitable surface, by entrapment e.g. in microspheres, by cross-linking e.g. to each other and/or with a suitable matrix material, or by affinity binding.
  • the reaction medium in a continuous flow, may be conducted through a compartment containing immobilised hydratases and/or another immobilised hydroxyl-introducing enzyme. Subsequently, the reaction mixture may flow through a compartment containing an immobilised photodecarboxylase.
  • the residence time in each of the compartments may be controlled, for instance to provide for a substantially complete conversion of the starting compound into the end product of the relevant reaction e.g. by regulating the flow.
  • the dimension of the respective compartment may be geared to providing a substantially complete conversion of the starting compound into the end product of the relevant reaction.
  • the temperature in each of the compartments may be controlled. In specific embodiments, conditions like temperature and or residence time may be controlled for individual compartments, such that e.g. temperatures may differ between compartments.
  • the temperature of the reaction mixture may be chosen to be optimal for the enzymatic conversions, and may vary from 5 to 80 °C, more preferably from 15 to 50 °C, even more preferably from 20 to 40 °C.
  • the concentrations of the unsaturated fatty acids may be chosen to be optimal for the enzymatic conversions and may preferably range between 0.01 mM - 1000 mM; more preferred: 1 mM - 250 mM; even more preferred: 5- 100 mM.
  • the overall system operated optimally at slightly alkaline pH values, representing a compromise between the alkaline pH optimum for CvFAP and slightly acidic pH for the hydroxyl- introducing enzyme.
  • the pH of the reaction medium ranges between 3 and 12 more preferably between 4 and 11, even more preferably between 5 and 10, even more preferably between 5 and 9.
  • the present invention also relates to novel compounds, such as can be produced by the process according to the present invention.
  • the present invention relates to a compound obtainable by the method according to the invention, wherein the compound is selected from the group consisting of compound (3) wherein R1 and R2 independently represent an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and compound (7) wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
  • the present invention relates to a compound obtainable by the method according to the invention, wherein the compound is enantiomerically pure.
  • the present invention relates to the novel compounds (Z)- heptadec-6-en-9-ol; (7R,9R)-heptadecane-7,9-diol; (R,5Z, 1 lZ)-heptadeca-5, 1 l-dien-9-ol; (3Z,6Z)-heptadeca-3,6-dien-9-ol; (Z)-nonadec-6-en-9-ol; (6Z, 12Z)-nonadeca-6, 12-dien-9-ol.
  • the present invention relates to a flow-through device, especially configured for carrying out the process of the present invention.
  • This device comprises a multitude of compartments, arranged in series.
  • the compartments are fluidly (especially liquidly) coupled, such that a first compartment is in fluid connection with a second compartment; the second compartment may optionally be configured in fluid connection with an optional third compartment, etc...
  • Each compartment comprises an inlet and an outlet, and wherein the outlet of a first compartment is connected to the inlet of a second compartment.
  • At least two of the compartments of the device may house immobilized enzymes.
  • a first compartment may house immobilized enzymes useful for the hydroxyl introduction stage (such as immobilized hydratating and/or hydroxylating enzymes), and a second compartment may house immobilized enzymes useful for the decarboxylation stage (such as immobilized photoactivated decarboxylase).
  • the compartment housing the immobilized photoactivated decarboxylase further comprises means for illuminating the content of the compartment with a suitable light source emitting light with a wavelength within the range as defined above.
  • the device may further comprise or be functionally coupled to a controlling system.
  • the controlling system may be configured to execute the process with the flow-through device. As indicated above, the controlling system may be configured to control one or more of residence time, temperature, etc.
  • the controlling system may also be configured to control the light source.
  • controlling and similar terms like “controlled” and “control” especially refer at least to determining the behavior or supervising the running of an element.
  • controlling and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc..
  • controlling and similar terms may additionally include monitoring.
  • controlling and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element.
  • the controlling of the element can be done with a controlling system, which may also be indicated as “controller”.
  • the controlling system and the element may thus at least temporarily, or permanently, functionally be coupled.
  • the element may comprise the controlling system.
  • the controlling system and element may not be physically coupled. Control can be done via wired and/or wireless control.
  • the term “controlling system” may also refer to a plurality of different controlling systems, which especially are functionally coupled, and of which e.g. one controlling system may be a master controlling system and one or more others may be slave controlling systems.
  • a controlling system may comprise or may be functionally coupled to a user interface.
  • the system, or apparatus, or device may execute an action in a “mode” or “operation mode” or “mode of operation”. Likewise, in a method an action or stage, or step may be executed in a “mode” or “operation mode” or “mode of operation”.
  • mode may also be indicated as “controlling mode”. This does not exclude that the system, or apparatus, or device may also be adapted for providing another controlling mode, or a plurality of other controlling modes. Likewise, this may not exclude that before executing the mode and/or after executing the mode one or more other modes may be executed.
  • a controlling system may be available, that is adapted to provide at least the controlling mode.
  • the choice of such modes may especially be executed via a user interface, though other options, like executing a mode in dependence of a sensor signal or a (time) scheme, may also be possible.
  • the operation mode may in embodiments also refer to a system, or apparatus, or device, that can only operate in a single operation mode (i.e. “on”, without further tunability).
  • the controlling system may control in dependence of one or more of an input signal of a user interface, a sensor signal (of a sensor), and a timer.
  • the term “timer” may refer to a clock and/or a predetermined time scheme.
  • the sensor signal may be the signal of a sensor configured to sense conversion rate and/or conversion efficiency.
  • coli ] 7 gL 1 , HEPS buffer pH 7.5 (50mM, with 10 % (v/v) DMSO): oleic acid ( ⁇ ), 8-hydroperoxy-9(Z)-octadecenoic acid (8-HPOME) (O), 5,8-dihydroxy-9(Z)-octadecenoic acid (5,8-diHOME) ( ⁇ ).
  • oleate hydratase from Lactobacillus reuteri (L/ ⁇ H) was chosen as the hydratase enzyme to catalyse the first step of the cascade in Examples 1-4.
  • Example 5 illustrates the diol synthesis as the first step, using a 5,8-diol synthase of Aspergillus nidulans (d//DS) as the hydroxyl-introducing enzyme.
  • Oleate hydratase from Lactobacillus reuteri was produced via recombinant expression of E. coli BL21 (DE3) cells harbouring pET28a(+) L/ ⁇ H.
  • the synthetic gene encoding L/ ⁇ H (Accession number: WP 109913811) (SEQ ID NO: 1) was cloned into a pET28 vector and the enzyme was recombinantly expressed in E. coli BL21 (DE3) cells.
  • the lyophilised cells overexpressing ZrOH were used for further reactions. Control experiments with empty E. coli cells (not containing the plasmid for ZrOH) exhibited no hydratase activity.
  • LB lysogeny broth
  • the pre-cultures were used to inoculate large cultures (1000 mL LB + 50 pg/mL kanamycin in 5 L shake flasks). Cells were grown at 37 °C, 180 rpm, until an OD 6OO between 0.6-0.8 was reached. Protein production was induced by the addition of 0.5 mM IPTG (final concentration) and the cells were left at 20 °C, 180 rpm, for overnight (18 hours).
  • Cells were harvested by centrifugation (11000 g at 4 °C for 10 min), washed with Tris- HC1 buffer (50 mM, pH 7.5, 100 mM NaCl) and centrifuged again. The cell pellets were collected and stored at -80 °C for further use.
  • the fatty acid photo decarboxylase from Chlorella variabilis NC64A (CvFAP) using a nucleotide sequence coding for the decarboxylase according to SEQ ID NO: 3 was produced following a previously established protocol (Huijbers, 2018) In short, 10 mL precultures of E. coli BL21 (DE3) cells harboring the designed pET28a-His-TrxA-CvFAP plasmid were grown overnight in terrific broth (TB) medium, containing 50 pg/mL kanamycin.
  • TB terrific broth
  • 500 mL cultures (TB + 50 pg/mL kanamycin in 2 L shake flasks) were prepared (cell growth at 37 °C, 180 rpm, until an ODeoo between 0.7-0.8 followed by induction by the addition of 0.5 mM IPTG). The cultures were incubated at 17 °C, 180 rpm, for another 20 hours. Cells were harvested (centrifugation at 11,000 g, 4 °C for 10 min), washed with Tris- HC1 buffer (50 mM, pH 8, 100 mM NaCl) and centrifuged again. The cell pellet was suspended in the same buffer, and 1 mM PMSF was added.
  • Tris- HC1 buffer 50 mM, pH 8, 100 mM NaCl
  • the total protein content of the cell extract was determined by a BCA Assay (Interchim), using BSA as a standard.
  • CvFAP production was analysed by SDS-PAGE using a CriterionTM Cell electrophoresis system (Bio-Rad).
  • E. coli BL21(DE3) pACYC- PelBSS-A/rDS displayed approximately 10-fold greater transformation rates and 4-fold higher final product concentration, as compared to the control strain E. coli BL21(DE3) pET21a- H «DS ( Figure 4).
  • the ri//DS-catalysed diol synthetic reaction was combined with the photodecarboxylation by CvFAP.
  • the first and second reactions by H «DS were initiated by adding 15 mM oleic acid into the reaction medium (HEPS buffer pH 7.5 (50mM, with 10 % (v/v) DMSO), containing the recombinant E. coli BL21(DE3) pACYC-PelBSS-/l//DS.
  • the mixture was extracted with ethyl acetate (75 mL, 2x).
  • the extraction solvent of the combined phases was removed under reduced pressure.
  • the crude product was purified via flash chromatography (liquid loading) on silica gel using heptane/ethyl acetate 40:1 as eluent for 15 min, followed by a programmed gradient for 10 min (ethyl acetate / heptane (2.5 to 80% ethyl acetate / heptane gradient). 82.5 mg (32.5% isolated yield) of the corresponding alcohol was obtained starting from linoleic acid.
  • one-pot one-step procedures i.e. performing the hydration and the decarboxylation reaction at the same time
  • the next step was to precede the cascade by a hydrolase step to enable triglycerides as starting materials (Figure 2).
  • a hydrolase step to enable triglycerides as starting materials (Figure 2).
  • the lipase was commercially obtained from Sigma-Aldrich (Triacylglycerol acylhydrolase, EC Number 3.1.1.3) and used according to recommendations of the supplier.
  • A9-un saturated fatty acids showed relative high conversion ranging from 24- 74%.
  • Four of the alcohol products were submitted to O-acylation using ((S)-(+)-0- acetylmandelic acid for NMR analysis to determine their optical purity. All alcohol products were essentially optically pure, which is in line with the reported high stereospecificity of FAHs.
  • the mixture was extracted with ethyl acetate (75 mL, 2x).
  • the extraction solvent of the combined phases was removed under reduced pressure.
  • the crude product was purified via flash chromatography (liquid loading) on silica gel using heptane/ethyl acetate 40:1 as eluent for 15 min, followed by a programmed gradient for 10 min (ethyl acetate / heptane (2.5 to 80% ethyl acetate / heptane gradient).
  • Oleic acid was converted by the 5,8-diol synthase Z «DS into 13 mM 5,8- dihydroxy-9(Z)-octadecenoic acid and small amount of 8-hydroperoxy-9(Z)-octadecenoic acid ( Figure 5).
  • Estupinan. 2014 Estupinan M, Diaz P, Manresa A. - Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity. Biochim Biophys Acta. 2014;1842(10): 1360-71. 2014 Jun 27. (https://doi.Org/10.1016/j.bbalip.2014.06.010)
  • Jian Xu. 2019 Jian Xu, Yujing Hu, Jiajie Fan, Mamatjan Arkin, Danyang Li, Yongzhen Peng, Weihua Xu, Xianfu Lin, Qi Wu, Light-Driven Kinetic Resolution of Alpha-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase.
  • Sorigue, 2017 Damien Sorigue, Bertrand Legeret, Stephan Cuine, Stephanie Blangy, Solene Moulin, Emmanuelle Billon, Pierre Richaud, Sabine Brugiere, Yohann Coute, Didier Nurizzo, Pavel Miiller, Klaus Brettel, David Pignol, Pascal Amoux, Yonghua Li-Beisson, Gilles Peltier, Fred Beisson - An algal photoenzyme converts fatty acids to hydrocarbons. Science, 2017,
  • the term “substantially”, such as in “substantially consists”, will be understood by the person skilled in the art.
  • the term “substantially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially may also be removed.
  • the term “substantially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.
  • the term “comprise” includes also embodiments wherein the term “comprises” means “consists of’.
  • the term “and/or” especially relates to one or more of the items mentioned before and after “and/or”.
  • a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2.
  • the term “comprising” may in an embodiment refer to “consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species”.
  • enantiomerically pure means that the enantiomeric excess (ee) of the enantiomeric mixture is higher than 99%.
  • the invention further applies to a device comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.
  • the invention further pertains to a method or process comprising one or more of the characterising features described in the description and/or shown in the attached drawings.

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Abstract

A photo enzymatic cascade reaction to transform unsaturated fatty acids into secondary fatty alcohols, and in particular into enantiomerically pure secondary fatty alcohols is disclosed. In a first hydroxyl introduction stage the unsaturated fatty acid is hydrated and/or hydroxylated with a first enzyme to provide a fatty hydroxy acid. The second stage comprises decarboxylation of the intermediate hydroxyacid by a photoactivated decarboxylase. A broad range of (poly)unsaturated fatty acids can be transformed into enantiomerically pure fatty alcohols in a simple one-pot two-step approach.

Description

Photo biocatalytic synthesis of secondary fatty alcohols from unsaturated fatty acids
FIELD OF THE INVENTION
The invention relates to a method for the preparation secondary fatty alcohols.
BACKGROUND OF THE INVENTION
Long-chain (fatty) secondary alcohols may be useful as active ingredients in cosmetic formulations, performance additives in oleochemicals, as building blocks in natural product synthesis, and in organic photosensitisers.
Park and co-workers established the synthesis of dicarboxylic- and w -hydroxy acids from unsaturated fatty acids (Jung, 2015). Also, the selective reduction of the carboxylate group to either the alcohol or aldehyde moiety is possible.
SUMMARY OF THE INVENTION
Envisioning a biobased chemical industry, there is an increasing interest in the transformation of biomass-derived starting materials into chemical building blocks. Natural fatty acids are particularly interesting building blocks; especially, if derived from agricultural wastes or non-edible sources. Interestingly enough, until recently, chemical methodologies for the conversion of fatty acids or their glycerides have been largely restricted to their (trans)esterification for the production of biodiesel or cosmetic esters. Currently, this situation is changing dramatically with various research groups developing new chemistries to valorise fatty acids.
For example, with the discovery of the fatty acid decarboxylase OleT, synthesis of terminal alkenes from fatty acids has come into reach. Also, the hydroxylation of fatty acids using P450 monooxygenases or peroxygenases has been reported.
The established synthetic routes towards long-chain secondary alcohols almost exclusively build on Grignard-type reactions of halide-derived nucleophiles with aldehydes or formic acid esters (Saeki, 2012) thereby necessitating multistep syntheses leading to racemic products and generating significant amounts of salt waste.
The present invention relates to a more practical and environmentally less demanding alternative also yielding secondary fatty alcohols. According to one embodiment the invention provides a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising the steps of (a) a hydroxyl introduction stage comprising hydration and/or hydroxylation of the unsaturated fatty acid with a first enzyme to provide a fatty hydroxy acid; and (b) a decarboxylation stage comprising decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
According to a further embodiment the invention provides a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising enzymatic hydration of the unsaturated fatty acid with a hydratase to form a fatty hydroxy acid and decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
According to a further embodiment the invention provides for a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising comprises enzymatic hydroxylation of the unsaturated fatty acid with a fatty acid hydroxylase and decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
In a preferred embodiment these processes may even result into secondary fatty alcohols of high enantiomeric purity.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The invention provides a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising the steps of (a) a hydroxyl introduction stage comprising hydration and/or hydroxylation of the unsaturated fatty acid with a first enzyme to provide a fatty hydroxy acid; and (b) a decarboxylation stage comprising decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
According to a particular embodiment the present invention relates to a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid, wherein the hydroxyl-introduction stage comprises enzymatic hydration of a double bond.
According to a particular embodiment the present invention relates to a method for the preparation of a-secondary fatty alcohol from an unsaturated fatty acid, comprising the steps of enzymatic hydration of the unsaturated fatty acid to form a fatty hydroxy acid and decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol. The unsaturated fatty acid may contain more than one double bond, such as two, three, four or five double bonds.
The unsaturated fatty acid may further contain one or more hydroxyl groups, such as one, two, three or four hydroxyl groups.
According to a further embodiment the invention provides a method for the preparation of an enantiomerically pure secondary fatty alcohol from an unsaturated fatty acid, wherein an unsaturated fatty acid of the general formula [1] is converted into a fatty hydroxy acid of formula [2] and the fatty hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [3] wherein R1 and R2 independently represent an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
According to a further embodiment the invention provides a method for the preparation of an enantiomerically pure secondary fatty alcohol from an unsaturated fatty acid, wherein an unsaturated fatty acid is first hydroxylated with a fatty acid hydroxylase and the resulting fatty hydroxy acid is decarboxyl ated with a photoactivated decarboxylase to form a secondary fatty alcohol.
Suitable unsaturated fatty acids which can be converted using the method according to the present invention may include for example myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, linoleic acid, docosadienoic acid, a-linolenic acid, pinolenic acid, a-eleostearic acid, mead acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid , adrenic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, docosahexaenoic acid, a-hydroxymyristic acid, b-hydroxymyristic acid, a-hydroxypalmitic acid, b-hydroxypalmitic acid, b- hydroxystearic acid, 12-hydroxy stearic acid, 17-hydroxystearic acid, 3-hydroxy-13- methyltetradecanoic acid, 12-hydroxy-cis-9-octadecenoic acid, ustilic acid, ricinoleic acid, 15- hydroxyl-linoleic acid, 9,16-dihydroxyhexadecanoic acid, 18-hydroxyl octadec-9-enoic acid, 9, 19,18-trihydroxy octadecanoic acid, (5S,12S)-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acid, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid, 12(S)-hydroxy-(5Z,8Z,10E,14Z)- eicosatetraenoic acid, 15(S)-hydroxy-(5Z,8Z,l lZ,13E)-eicosatetraenoic acid, and 20-hydroxy- (5Z,8Z,1 lZ,14Z)-eicosatetraenoic acid.
The introduction of a hydroxyl group into the unsaturated fatty acid to form a fatty hydroxy acid can be carried out for example using a hydratase enzyme from theE.C. class 4.2.1. Without limiting the selection to those exemplified, suitable enzymes may include fumarate hydratase (E.C. 4.2.1.2), aconitate hydratase (E.C. 4.2.1.3), enoyl-CoA hydratase (E.C. 4.2.1.17), maleate hydratase (E.C. 4.2.1.31), oleate hydratase (E.C. 4.2.1.53), 3- hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55), isohexenylglutaconyl-CoA hydratase (E.C. 4.2.1.57), 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (E.C. 4.2.1.59), 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratase (E.C. 4.2.1.60), 3-hydroxypalmitoyl- [acyl-carrier-protein] dehydratase (E.C. 4.2.1.61), hydroperoxide dehydratase (E.C. 4.2.1.92), /raws-feruloyl-CoA hydratase (E.C. 4.2.1.101), linalool dehydratase (E.C. 4.2.1.127), lupan- 3p,20-diol synthase (E.C.4.2.1.128), carotenoid 1,2-hydratase (E.C. 4.2.1.131), 2- hydroxyhexa-2,4-dienoate hydratase (E.C.4.2.1.132), copal-8-ol diphosphate hydratase (E.C.4.2.1.133) and very-long-chain (3i?)-3-hydroxyacyl-CoA dehydratase (E.C.4.2.1.134).
In addition to these hydratases also so-called lipoxygenases (of enzyme class E.C. 1.13.11.x) may be useful enzymes for the transformation of unsaturated fatty acids according to the present invention. Exemplary lipoxygenase enzymes may include: linoleate 13S-lipoxygenase; arachidonate 12-lipoxygenase, arachidonate 15 -lipoxygenase, arachidonate 5 -lipoxygenase, arachidonate 8-lipoxygenase, linoleate 11 -lipoxygenase, linoleate 98- lipoxygenase, linoleate 8R-lipoxygenase, linolenate 9R-lipoxygenase, linoleate 10R- lipoxygenase, oleate 1 OS-lipoxygenase and linoleate 9/13 -lipoxygenase.
Further enzymes suitable for enzymes for the transformation of unsaturated fatty acids into fatty alcohols according to the present invention are 7,10-diol synthases and 5,8-diol synthases.
These enzymes can be obtained from various sources including (but not limited to): Arabidopsis thaliana , Aspergillus species, such as Aspergillus flavus , Aspergillus nidulans, Brassica oleracea var. gemmifera , Casuarina glauca, Cucumis sativus , Datisca glomerata , Elizabethkingia meningoseptica , Fusarium oxysporum , Glycine max , Flomo sapiens , Hordeum vulgare , Lactobaccilus species, Lysinibacillus fusiformis , Macrococcus caseolyticus, Momordica char anti a, Nicotiana attenuata , Nostoc punctiforme , Olea europaea , Oryza sativa, Pseudomonas species, such as Pseudomonas aeruginosa , Solanum lycopersicum , Solanum tuberosumand Stenotrophomonas maltophilia.
Suitable enzymes for the hydratase reaction are for example an oleate hydratase derived from Elizabethkingia meningoseptica (EmO ) (Demming, 2017) (SEQ ID NO: 6) or an oleate hydratase derived from a Lactobacillus species (ZrOH) (SEQ ID NO:2).
According to a particular embodiment the present invention relates to a method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid, wherein the hydroxyl-introduction stage comprises hydroxylation, and wherein the first enzyme comprises a fatty acid hydroxylase.
Suitable enzymes for the hydroxylation reaction of the fatty acid are 7,10-diol synthase from Pseudomonas aeruginosa (Estupinan, 2014), and 5,8-diol synthase from Aspergillus nidulans (Seo, 2014).
More in particular, 5,8-diol synthase can be applied as this bifunctional enzyme adds two instead of only one new OH functionalities into an unsaturated fatty acid by a two- step reaction, according to the following scheme:
The fatty acid according to formula (4) is first converted by the 5,8-diol synthase into an intermediary compound (5) wherein it is further converted by the 5,8-diol synthase into the diol (6) and this fatty di-hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [7] wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
Accordingly, a further embodiment of the method according to the present invention wherein a fatty acid according to formula (4) is converted by the 5,8-diol synthase into the diol (6) and this fatty di-hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [7] wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
A suitable photoactivated decarboxylase for use according to the present invention is for example photoactivated decarboxylase from Chlorella variabilis NC64A (CvFAP) and photoactivated decarboxylase enzymes having a sequence identity of at least 50 %, prefered 65%, more preferred 80%, most preferred 85 % with the reported sequence of decarboxylase from Chlorella variabilis NC64A (CvFAP) (Sorigue, 2017).
This photoactivated decarboxylase from Chlorella variabilis NC64A (CvFAP) was recently reported to enable the synthesis of alkanes from fatty acids or the kinetic resolution of a-substituted acids (Jian Xu, 2019).
An enzyme used in the process according to the present invention may be obtained from the organism which naturally produces such enzyme. Alternatively, the enzyme may be produced by a host cell which is transformed to produce the enzyme or several of the enzymes needed in the process of the invention by recombinant techniques known in the art.
In one embodiment an enzyme may be applied during the process of the invention in the form of whole cells producing the enzyme.
In a further embodiment, an enzyme may be applied during the process of the invention in the form of a lysate of the cells producing the enzyme.
In a further embodiment, an enzyme may be applied during the process of the invention in a more or less purified form, such as in a form essentially free from particulate material from the producing cells. Purification of the enzyme may be performed by any method known in the art.
In a further embodiment, an enzyme used in the process according to the present invention may be a variant or mutant of a naturally occuring enzyme, still having the desired enzymatic activity. Such variant enzyme may have a sequence identity as compared to the amino acid sequence of the naturally occuring enzyme of at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% and more preferably at least 99%.
In the method according to the present invention, the reactions can suitably be carried out in an emulsion of an aqueous phase and a water-immiscible organic phase. Herein, the water-immiscible organic phase may also include a hydrophobic phase and an oil phase. The water-immiscible organic phase may provide both a source of the (unsaturated fatty acid) substrate as well as a sink for the reaction product, whereas the enzymes will substantially reside in the aqueous phase. For this purpose, either a water-in-oil emulsion or an oil-in-water emulsion may be employed.
Accordingly, in one embodiment of the method of the invention the reactions are carried out in an emulsion of an aqueous phase and a water-immiscible organic phase (such as an oil phase), more in particular the reactions may be carried out in a water-in-oil emulsion or an oil-in-water emulsion.
The oil phase may for example comprise triolein. Accordingly, in one embodiment of the method of the invention the reactions are carried out in an emulsion of water and triolein.
For use in the method according to the invention, the unsaturated fatty acids can be derived from a natural source, wherein the fatty acids may be found e.g. as esters, such as triglycerides, phospholipids or cholesterol esters. The unsaturated fatty acids can be used directly as such or may be obtained from their natural source e.g. as amides, thioesters or esters of these unsaturated fatty acids.
In this latter case, the amides, thioesters or esters have to be converted to the unsaturated fatty acids e.g. by an enzymatic reaction such as: double bond can be (E) or (Z)-configured
X = NR'R" amides X = SR' thioester X = OR'ester
Accordingly, in one embodiment of the method of the invention the unsaturated fatty acid is obtained from a corresponding amide, thioester or ester.
Preferred sources of natural unsaturated fatty acids are mono-, di- or triesters of glycerol as present in natural fats and oils. Examples of such glycerides are (but are not limited to): soybean oil, tea seed oil, rapeseed oil, cottonseed oil, olive oil, castor oil, sunflower oil, peanut oil, canola oil, sunflower seed oil, sea buckthorn oil, linseed oil, palm oil or jojoba oil, including their (partially) hydrolysed forms.
As hydrolases for preparing the free unsaturated fatty acid may be used any enzymes from the class of carboxylic acid hydrolases (E.C. 3.x.x.x). A non-exhaustive list of carboxylic acid hydrolases comprises in particular so-called esterases, lipases, peptidases, amidases, thioesterases and glycosidases.
However, also chemical catalysts such as so-called Lewis-acids or -bases or Bronsted acids and bases are suitable for the hydrolysis step in preparing the free unsaturated fatty acids.
Accordingly, in one embodiment of the method of the invention the unsaturated fatty acid is obtained from a corresponding mono-, di- or triester of glycerol
An enzyme used in the process according to the present invention may be obtained from the organism which naturally produces such enzyme. Alternatively, the enzyme may be produced by a host cell which is transformed to produce the enzyme or several of the enzymes needed in the process of the invention by recombinant techniques known in the art.
In one embodiment an enzyme may be applied during the process of the invention in the form of whole cells producing the enzyme.
In a further embodiment, an enzyme may be applied during the process of the invention in the form of a lysate of the cells producing the enzyme.
In a further embodiment, an enzyme may be applied during the process of the invention in a more or less purified form, such as in a form essentially free from particulate material from the producing cells. Purification of the enzyme may be performed by any method known in the art.
In a further embodiment, an enzyme may be applied during the process of the invention in immobilized form. Many useful methods for immobilization of enzymes are known in the art.
In the process of the invention the concentrations of the enzymes may be chosen to be optimal for the enzymatic conversions, and may range between 0.000001 mM and 10 mM, preferably between 0.001 mM and 1 mM, more preferably between 0.001 mM and 0.1 mM.
During the experiments it was found that one-pot one-step procedures (i.e. performing the hydroxyl-introducing step and the decarboxylation reaction at the same time) predominantly yielded the decarboxylation product of the unsaturated fatty acid.
Accordingly, in one embodiment, the process of to the present invention may be carried out as a one-pot two-step procedure. Such one-pot two-step procedure may comprise first performing the hydroxyl-introducing step followed by the addition of the photoactivated decarboxylase and illumination to promote the decarboxylation reaction.
Alternatively, the one-pot two-step procedure may comprise first performing the hydroxyl-introducing step in the presence of both the hydroxyl-introducing enzyme in dark conditions and the photoactivated decarboxylase and later start illumination to promote the decarboxylation reaction. To this end, the illumination will be started when substantially all of the unsaturated fatty acid is converted into the hydroxylated form. Under the reaction conditions (such as temperature, pH, and concentrations) employed for a given unsaturated fatty acid a person skilled in the art will be able to determine this starting point for illumination without undue experimentation. As applied in the photoactivated decarboxylase step of the present invention “illumination” relates to targeting a light source of the reaction medium with a wavelength of between 380 and 500 nm, more preferably near to 450 nm.
In the process of the present invention “dark conditions” may imply substantially the absence of illumination of the reaction medium with a light source at a wavelength of between 380 and 500 nm.
According to another embodiment a flow-chemistry setup may be used. In embodiments, the enzymes may be immobilised. Many methods for immobilisation of enzymes are known in the art.
The enzymes may be immobilized for example by covalent bonding to a suitable surface or a carrier, by adsorption to a suitable surface, by entrapment e.g. in microspheres, by cross-linking e.g. to each other and/or with a suitable matrix material, or by affinity binding.
In one embodiment, in a continuous flow, the reaction medium may be conducted through a compartment containing immobilised hydratases and/or another immobilised hydroxyl-introducing enzyme. Subsequently, the reaction mixture may flow through a compartment containing an immobilised photodecarboxylase. In embodiments, the residence time in each of the compartments may be controlled, for instance to provide for a substantially complete conversion of the starting compound into the end product of the relevant reaction e.g. by regulating the flow. In further embodiments the dimension of the respective compartment may be geared to providing a substantially complete conversion of the starting compound into the end product of the relevant reaction. Alternatively, or additionally, the temperature in each of the compartments may be controlled. In specific embodiments, conditions like temperature and or residence time may be controlled for individual compartments, such that e.g. temperatures may differ between compartments.
In the process of the invention the temperature of the reaction mixture may be chosen to be optimal for the enzymatic conversions, and may vary from 5 to 80 °C, more preferably from 15 to 50 °C, even more preferably from 20 to 40 °C.
In the process of the inventions the concentrations of the unsaturated fatty acids may be chosen to be optimal for the enzymatic conversions and may preferably range between 0.01 mM - 1000 mM; more preferred: 1 mM - 250 mM; even more preferred: 5- 100 mM.
During the experiments with the two-step one-pot process described below, the overall system operated optimally at slightly alkaline pH values, representing a compromise between the alkaline pH optimum for CvFAP and slightly acidic pH for the hydroxyl- introducing enzyme. In preferred embodiments of the process of the present invention, the pH of the reaction medium ranges between 3 and 12 more preferably between 4 and 11, even more preferably between 5 and 10, even more preferably between 5 and 9.
In a further embodiment, the present invention also relates to novel compounds, such as can be produced by the process according to the present invention.
In a further embodiment, the present invention relates to a compound obtainable by the method according to the invention, wherein the compound is selected from the group consisting of compound (3) wherein R1 and R2 independently represent an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and compound (7) wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
In a further embodiment, the present invention relates to a compound obtainable by the method according to the invention, wherein the compound is enantiomerically pure.
More in particular, the present invention relates to the novel compounds (Z)- heptadec-6-en-9-ol; (7R,9R)-heptadecane-7,9-diol; (R,5Z, 1 lZ)-heptadeca-5, 1 l-dien-9-ol; (3Z,6Z)-heptadeca-3,6-dien-9-ol; (Z)-nonadec-6-en-9-ol; (6Z, 12Z)-nonadeca-6, 12-dien-9-ol.
In another embodiment the present invention relates to a flow-through device, especially configured for carrying out the process of the present invention. This device comprises a multitude of compartments, arranged in series. Especially, the compartments are fluidly (especially liquidly) coupled, such that a first compartment is in fluid connection with a second compartment; the second compartment may optionally be configured in fluid connection with an optional third compartment, etc... Each compartment comprises an inlet and an outlet, and wherein the outlet of a first compartment is connected to the inlet of a second compartment. At least two of the compartments of the device may house immobilized enzymes. For example, in a particular embodiment a first compartment may house immobilized enzymes useful for the hydroxyl introduction stage (such as immobilized hydratating and/or hydroxylating enzymes), and a second compartment may house immobilized enzymes useful for the decarboxylation stage (such as immobilized photoactivated decarboxylase). The compartment housing the immobilized photoactivated decarboxylase further comprises means for illuminating the content of the compartment with a suitable light source emitting light with a wavelength within the range as defined above. Hence, the device may further comprise or be functionally coupled to a controlling system. The controlling system may be configured to execute the process with the flow-through device. As indicated above, the controlling system may be configured to control one or more of residence time, temperature, etc. The controlling system may also be configured to control the light source.
The term “controlling” and similar terms like “controlled” and “control” especially refer at least to determining the behavior or supervising the running of an element. Hence, herein “controlling” and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc.. Beyond that, the term “controlling” and similar terms may additionally include monitoring. Hence, the term “controlling” and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element. The controlling of the element can be done with a controlling system, which may also be indicated as “controller”. The controlling system and the element may thus at least temporarily, or permanently, functionally be coupled. The element may comprise the controlling system. In embodiments, the controlling system and element may not be physically coupled. Control can be done via wired and/or wireless control. The term “controlling system” may also refer to a plurality of different controlling systems, which especially are functionally coupled, and of which e.g. one controlling system may be a master controlling system and one or more others may be slave controlling systems. A controlling system may comprise or may be functionally coupled to a user interface.
The system, or apparatus, or device may execute an action in a “mode” or “operation mode” or “mode of operation”. Likewise, in a method an action or stage, or step may be executed in a “mode” or “operation mode” or “mode of operation”. The term “mode” may also be indicated as “controlling mode”. This does not exclude that the system, or apparatus, or device may also be adapted for providing another controlling mode, or a plurality of other controlling modes. Likewise, this may not exclude that before executing the mode and/or after executing the mode one or more other modes may be executed.
However, in embodiments a controlling system may be available, that is adapted to provide at least the controlling mode. Would other modes be available, the choice of such modes may especially be executed via a user interface, though other options, like executing a mode in dependence of a sensor signal or a (time) scheme, may also be possible. The operation mode may in embodiments also refer to a system, or apparatus, or device, that can only operate in a single operation mode (i.e. “on”, without further tunability).
Hence, in embodiments, the controlling system may control in dependence of one or more of an input signal of a user interface, a sensor signal (of a sensor), and a timer. The term “timer” may refer to a clock and/or a predetermined time scheme. For instance, the sensor signal may be the signal of a sensor configured to sense conversion rate and/or conversion efficiency.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Photo enzymatic hydration-decarboxylation of oleic acid.
A: Typical time course [oleic acid] = 7 mM, [L/ΌH @E. coli\ = 15 gL 1, [CvFAP] = 2 mM, Tris-HCl buffer pH 8.0 (lOOmM, with 50 mM of NaCl), illumination with blue light (l = 450 nm; intensity = 13.7 mEL V1): oleic acid (■), 10-hydroxy stearic acid (·),: 9-heptadecanol (¨)·
B: influence of the ZrOH concentration, [L/ΌH@//. coli \ = 2.5-20 gL 1 at pH 8.0 and C: influence of the reaction pH, [L/ΌH@//. coli ] = 10 gL 1. For all the reactions, first the L/ΌH- catalysed hydration reaction was performed for l lh followed by addition of CvFAP and illumination for another 6 h at 30 °C.
Figure 2. Trienzymatic cascade for the transformation of triolein into 9-heptadecanol.
Figure 3. Product scope of the photo enzymatic reaction system according to the invention.
Figure 4. Biotransformation of oleic acid into 5,8-dihydroxy-9(Z)-octadecenoic acid by (A) E. coli BL21(DE3) pET21a-H«DS and (B) E. coli BL21(DE3) pACYC-PelBSS- H«DS. Typical time course [oleic acid] = 15 mM, [4//DS @E. coli ] = 7 gL 1, HEPS buffer pH 7.5 (50mM, with 10 % (v/v) DMSO): oleic acid (■), 8-hydroperoxy-9(Z)-octadecenoic acid (8-HPOME) (O), 5,8-dihydroxy-9(Z)-octadecenoic acid (5,8-diHOME) (·).
Figure 5. Photo enzymatic diol synthesis-decarboxylation of oleic acid. Typical time course [oleic acid] = 15 mM, [d//DS @E. coli\ = 7 gL 1, [CvFAP/®//. coli ] = 3.5 gL 1, HEPS buffer pH 7.5 (50mM, with 10 % (v/v) DMSO), illumination with blue light (l = 450 nm; intensity = 13.7 mEL V1): oleic acid (■), 8-hydroperoxy-9(Z)-octadecenoic acid (8-HPOME)
(O), 5,8-dihydroxy-9(Z)-octadecenoic acid (5,8-diHOME) (·), (Z)-heptadec-8-ene-4,7-diol
(¨). For the reaction, first the d//DS-catalysed diol synthetic reaction was performed for 0.7 h followed by addition of CvFAP and illumination for another 3 h.
Figure 6. Synthesis of 5S,8R-dihydroxy-9(Z)-octadecenoic acid and (4S,7R,Z)-heptadec- 8-ene-4,7-diol from oleic acid in a three-step-one-pot cascade combining 5,8-diol synthase with CvFAP. The absolute configuration is based on the enantioselectivity of the synthase as previously established by Oh and co-workers (Seo, 2015).
EXAMPLES
For the purpose of illustrating the present invention, oleate hydratase from Lactobacillus reuteri (L/ΌH) was chosen as the hydratase enzyme to catalyse the first step of the cascade in Examples 1-4.
Example 5 illustrates the diol synthesis as the first step, using a 5,8-diol synthase of Aspergillus nidulans (d//DS) as the hydroxyl-introducing enzyme.
For the second step in each of the Examples 1-5 we applied the photoactivated decarboxylase from Chlorella variabilis NC64A (CvFAP), which was produced by recombinant expression in E. coli following established protocols and used either as cell-free extracts or in whole cells.
Methods
Preparation of the hydratase biocatalyst.
Oleate hydratase from Lactobacillus reuteri (LrO ) was produced via recombinant expression of E. coli BL21 (DE3) cells harbouring pET28a(+) L/ΌH. To this end the synthetic gene encoding L/ΌH (Accession number: WP 109913811) (SEQ ID NO: 1) was cloned into a pET28 vector and the enzyme was recombinantly expressed in E. coli BL21 (DE3) cells. The lyophilised cells overexpressing ZrOH were used for further reactions. Control experiments with empty E. coli cells (not containing the plasmid for ZrOH) exhibited no hydratase activity.
These cultures were grown overnight in lysogeny broth (LB) medium, containing 30 pg/mL kanamycin. The pre-cultures were used to inoculate large cultures (1000 mL LB + 50 pg/mL kanamycin in 5 L shake flasks). Cells were grown at 37 °C, 180 rpm, until an OD6OO between 0.6-0.8 was reached. Protein production was induced by the addition of 0.5 mM IPTG (final concentration) and the cells were left at 20 °C, 180 rpm, for overnight (18 hours). Cells were harvested by centrifugation (11000 g at 4 °C for 10 min), washed with Tris- HC1 buffer (50 mM, pH 7.5, 100 mM NaCl) and centrifuged again. The cell pellets were collected and stored at -80 °C for further use.
Preparation of the photodecarboxylase biocatalyst.
The fatty acid photo decarboxylase from Chlorella variabilis NC64A (CvFAP) using a nucleotide sequence coding for the decarboxylase according to SEQ ID NO: 3 was produced following a previously established protocol (Huijbers, 2018) In short, 10 mL precultures of E. coli BL21 (DE3) cells harboring the designed pET28a-His-TrxA-CvFAP plasmid were grown overnight in terrific broth (TB) medium, containing 50 pg/mL kanamycin. From these, 500 mL cultures (TB + 50 pg/mL kanamycin in 2 L shake flasks) were prepared (cell growth at 37 °C, 180 rpm, until an ODeoo between 0.7-0.8 followed by induction by the addition of 0.5 mM IPTG). The cultures were incubated at 17 °C, 180 rpm, for another 20 hours. Cells were harvested (centrifugation at 11,000 g, 4 °C for 10 min), washed with Tris- HC1 buffer (50 mM, pH 8, 100 mM NaCl) and centrifuged again. The cell pellet was suspended in the same buffer, and 1 mM PMSF was added. Cells were lysed by passing them twice through a Multi Shot Cell Disruption System (Constant Systems Ltd, Daventry, UK) at 1.5 bar, followed by centrifugation of the cell lysate (38000 g at 4 °C for 1 h). After centrifugation, 5% glycerol (w/v) was added to the soluble fraction, the cell extract was aliquoted, frozen in liquid nitrogen and stored at -80 °C.
The total protein content of the cell extract was determined by a BCA Assay (Interchim), using BSA as a standard. CvFAP production was analysed by SDS-PAGE using a Criterion™ Cell electrophoresis system (Bio-Rad).
Preparation of the 5.8-diol synthase biocatalyst First, we have constructed a recombinant E. coli expressing and sorting the 5,8- diol synthase from Aspergillus nidulans (4//DS) using a nucleotide sequence coding for the diol synthase according to SEQ ID NO:4 into the periplasmic space, because it generates a reaction intermediate (i.e., 8-hydroperoxy-9(Z)-octadecenoic acid) having strong cytotoxicity (Kuhn, 2005). Introduction of the signal sequence of PelB (Lei, 1987; Jung, 2015) into HwDS allowed the enzyme to incorporate into the periplasm. Notably, E. coli BL21(DE3) pACYC- PelBSS-A/rDS displayed approximately 10-fold greater transformation rates and 4-fold higher final product concentration, as compared to the control strain E. coli BL21(DE3) pET21a- H«DS (Figure 4).
General procedures for the cascade reaction with oleate hydratase
2.5-20 mg of lyophilized whole cells of oleate hydratase, and 2.0 mg of oleic acid were added into 980 pL of Tris-HCl buffer (lOOmM, with 50 mM of NaCl). The resultant suspension was stirred at 30 °C for 11 hours. 20 pL of photodecarboxylase (from stock solution with a concentration of 102 pM) was added afterwards and the suspension was illuminated with blue LED light (wavelength 450 nm) and stirred for another 6 hours.
The final reaction conditions were: [substrate]=7mM, [lyophilised ZrOH@ E. coli]= 2.5-20 mg mL 1, [CvFAP]=2 pM, Tris-HCl buffer (pH 8.0, lOOmM, with 50 mM of NaCl), blue light (intensity=13.7 mEL V1), total volume 1.0 mL.
To analyse the product, 1 mL of ethyl acetate (containing 5 mM of 1-octanol) was added to the above reaction suspension (1:1 volume ratio) and vigorously mixed. The organic phase was collected by centrifugation and was dried over MgSCL. The obtained sample was analysed by gas chromatography (Cp sil 5CB, column 50 mx0.53mmx 1.0pm).
General procedure for the cascade reaction with 5.8-diol synthase
The ri//DS-catalysed diol synthetic reaction was combined with the photodecarboxylation by CvFAP. The first and second reactions by H«DS were initiated by adding 15 mM oleic acid into the reaction medium (HEPS buffer pH 7.5 (50mM, with 10 % (v/v) DMSO), containing the recombinant E. coli BL21(DE3) pACYC-PelBSS-/l//DS.
The CvFAP-catalysed photo decarboxylation was next initiated by adding the recombinant A. coli BL21(DE3) pET28a-CvFAP into the 4//DS reaction medium. Preparative-scale synthesis starting from linoleic acid
98 mL of Tris-HCl buffer (pH 8.0, 100 mM, with 50 mM of NaCl) containing 10 mM of substrate and 2 g of lyophilised LrOH@E. coli were mixed in a beaker and stirred at 30 °C for 48 hours. The beaker was sealed by using parafilm. 2 mL of photodecarboxylase (from stock solution with a concentration of 102 uM) was added afterwards and the suspension was illuminated by blue LED and stirred for 48 hours.
The final reaction condition was: [linoleic acid] = 10 mM, [lyophilised Lr01i@E. coli ]= 20 mg mL 1, [CvFAP] = 2 mM, Tris-HCl buffer (pH 8.0, lOOmM, with 50 mM of NaCl), blue light (intensity = 13.7 mEL V1), total volume 1.0 mL.
At the end of the cascade reactions, the mixture was extracted with ethyl acetate (75 mL, 2x). The extraction solvent of the combined phases was removed under reduced pressure. The crude product was purified via flash chromatography (liquid loading) on silica gel using heptane/ethyl acetate 40:1 as eluent for 15 min, followed by a programmed gradient for 10 min (ethyl acetate / heptane (2.5 to 80% ethyl acetate / heptane gradient). 82.5 mg (32.5% isolated yield) of the corresponding alcohol was obtained starting from linoleic acid.
Example 1
Oleate as starting material
To identify the factors influencing the product formation of the photo enzymatic cascade we used oleate as model substrate. Indeed, the cascade reaction proceeded via the proposed sequence: ZrOH catalysed hydration of oleic acid followed by CvFAP catalysed decarboxylation to yield 9-heptadecanol.
Using cell-free preparations of L/ΌH gave only low product formation (0.4 mM of the desired 9-heptadecanol stating from oleic acid). We attribute this to a relatively poor stability of ZrOH under these conditions and therefore focussed using L/ΌH in lyophilised whole cells (ZrOH@A. coli.).
It was noted that one-pot one-step procedures (i.e. performing the hydration and the decarboxylation reaction at the same time) predominantly yielded the decarboxylation product of oleic acid ((Z)-heptadec-8-ene). Therefore, for all further experiments we followed a one-pot two-step procedure, i.e. first performing the hydration reaction followed by the addition of CvFAP and illumination to promote the decarboxylation reaction or first preforming the hydration reaction in dark conditions in the presence of the photoactivated decarboxylase followed by illumination to promote the decarboxylation reaction.
Full hydration of 7 mM oleic acid was achieved within 11 h while the subsequent photo enzymatic decarboxylation was considerably faster (Figure 1A). A systematic variation of the reaction parameters (Figure IB) confirmed our initial assumption that ZrOH represents the limiting factor in the catalytic cascade. Relatively high L/ΌH (lyophilised cells) concentrations (15-20 gL 1) were necessary to obtain full conversion of oleic acid into the desired product within the time frame of the experiment. Moreover, the pH has a significant influence on the performance of both enzymes (Figure 1 C). The overall system operated optimally at slightly alkaline pH values, representing a compromise between the alkaline pH optimum for CvFAP and slightly acidic pH for ZrOH.
While determination of the hydratase activity was not straight forward, the catalytic performance of the photodecarboxylase was estimated to be in the range of 1000 h 1, corresponding well with what we observed previously (Huijbers, 2018).
Example 2
Cascade reaction in two-phase system
To alleviate the poor solubility of fatty acids such as oleic acid under the reaction conditions we used a so-called two liquid phase approach with triolein as organic phase serving both as substrate reservoir and product sink (Figure 2). In such a way 17.4 mM of 9-heptadecanol was obtained starting from 20 mM of oleic acid dissolved in triolein (87% yield, see Supporting Information).
Use of triglycerides as starting material
The next step was to precede the cascade by a hydrolase step to enable triglycerides as starting materials (Figure 2). Using triolein as organic phase the lipase from Candida rugosa (CrLip) catalysed the hydrolysis of the triglyceride while L/ΌH mediated the quasi-irreversible hydration of the C=C-double bond. The lipase was commercially obtained from Sigma-Aldrich (Triacylglycerol acylhydrolase, EC Number 3.1.1.3) and used according to recommendations of the supplier.
After the illumination of the reaction mixture in the presence of CvFAP, 6.9 mM of 9-heptadecanol was observed in the organic phase.
Example 3
Investigation of substrate scope
In this example we investigated the substrate scope of the photo enzymatic cascade reaction. A broad range of (poly)unsaturated fatty acids were converted into the corresponding alcohols (Figure 3).
Reaction conditions: [substrate] = 5 mM, [ZrOH@A. coli , lyophilised cells] = 20 gL 1, [CvFAP] = 2 mM, Tris-HCl buffer (lOOmM, with 50 mM of NaCl), blue light (l = 450 nm; intensity=13.7 mEL V1). The reactions were performed in a two-step fashion: first the L/ΌH-catalysed hydration reaction was performed for 1 lh followed by addition of CvFAP and illumination for another 6h. nd= not determined.
Conversion = [productjfinai x [substrate]initiai l x100%. The enantiomeric excess (e.e.) was determined by 'H NMR analysis after the fatty alcohols were derivatised by fV)-(+)- O-acetylmandelic acid.
A9-un saturated fatty acids showed relative high conversion ranging from 24- 74%. Four of the alcohol products were submitted to O-acylation using ((S)-(+)-0- acetylmandelic acid for NMR analysis to determine their optical purity. All alcohol products were essentially optically pure, which is in line with the reported high stereospecificity of FAHs.
Example 4
Preparative-scale synthesis starting from linoleic acid
Following the study of substrate scope, we selected linoleic acid for a semi preparative scale reaction yielding 82.5 mg (32.5% isolated yield) of the corresponding optically pure alcohol (Figures SI 7-21, details see Supporting Information) though the bienzymatic cascade.
98 mL of Tris-HCl buffer (pH 8.0, 100 mM, with 50 mM of NaCl) containing 10 mM of substrate and 2 g of lyophilised LrOH@E. coli were mixed in a beaker and stirred at 30 °C for 48 hours. The beaker was sealed by using parafilm. 2 mL of photodecarboxylase (from stock solution with a concentration of 102 mM) was added afterwards and the suspension was illuminated by blue LED and stirred for 48 hours.
The final reaction condition was: [linoleic acid] = 10 mM, [lyophilised Lr01i@E. coli ]= 20 mg mL 1, [CvFAP] = 2 pM, Tris-HCl buffer (pH 8.0, lOOmM, with 50 mM of NaCl), blue light (intensity = 13.7 mEL V1), total volume 1.0 mL.
At the end of the cascade reactions, the mixture was extracted with ethyl acetate (75 mL, 2x). The extraction solvent of the combined phases was removed under reduced pressure. The crude product was purified via flash chromatography (liquid loading) on silica gel using heptane/ethyl acetate 40:1 as eluent for 15 min, followed by a programmed gradient for 10 min (ethyl acetate / heptane (2.5 to 80% ethyl acetate / heptane gradient).
82.5 mg (32.5% isolated yield) of the corresponding alcohol was obtained starting from linoleic acid. Example 5
Photo enzymatic diol synthesis-decarboxylation of oleic acid
A three-step cascade by two enzymes was investigated for preparation of (Z)- heptadec-8-ene-4,7-diol from oleic acid (Figure 6).
Oleic acid was converted by the 5,8-diol synthase Z«DS into 13 mM 5,8- dihydroxy-9(Z)-octadecenoic acid and small amount of 8-hydroperoxy-9(Z)-octadecenoic acid (Figure 5).
Remarkably, 5,8-dihydroxy-9(Z)-octadecenoic acid was transformed by CvFAP into 11 mM (Z)-heptadec-8-ene-4,7-diol. The overall conversion yield reached 71% based on GC/MS analysis, whereas the absolute configuration of the product remained to be investigated.
All the results indicated that (Z)-heptadec-8-ene-4,7-diol could be prepared with a high conversion yield from oleic acid by the three-step cascade by two enzymes (Z«DS and CvFAP) expressed in recombinant A. coli cells, respectively.
CITED DOCUMENTS
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Estupinan. 2014: Estupinan M, Diaz P, Manresa A. - Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity. Biochim Biophys Acta. 2014;1842(10): 1360-71. 2014 Jun 27. (https://doi.Org/10.1016/j.bbalip.2014.06.010)
Huiibers. 2018: Mieke M. E. Huijbers, Wuyuan Zhang, Fabio Tonin, Frank Hollmann - Light-Driven Enzymatic Decarboxylation of Fatty Acids - Angewandte Chemie 57 (41),
2018, Pages 13648-13651 (https://doi.org/10.1002/anie.2018071191
Jian Xu. 2019: Jian Xu, Yujing Hu, Jiajie Fan, Mamatjan Arkin, Danyang Li, Yongzhen Peng, Weihua Xu, Xianfu Lin, Qi Wu, Light-Driven Kinetic Resolution of Alpha-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase. Angewandte Chemie 58 (25), 2019, 8474-8478 (https://DOI.org/10.1002/anie.2019Q31651 2015: Sang-Min Jung, Joo-Hyun Seo, Jung-Hoo Lee, Jin-Byung Park, Jin-Ho Seo - Fatty acid hydration activity of a recombinant Escherichia coli-based biocatalyst is improved through targeting the oleate hydratase into the periplasm. Biotechnology Journal 10 (12), 2015, Pages 1887-1893 (https://doi.org/10.1002/biot.20150Q14n
Kuhn. 2005: Hartmut Kuhn, Jan Saam, Sebastian Eibach, Hermann-Georg Holzhiitter, Igor Ivanov, Matthias Walther - Structural biology of mammalian lipoxygenases: Enzymatic consequences of targeted alterations of the protein structure? Biochemical and Biophysical Research Communications 338 (1), 2005, Pages 93-101 (https://doi.Org/10.1016/j.bbrc.2005.08.238)
Lei. 1987: S P Lei, H C Lin, S S Wang, J Callaway, and G Wilcox - Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J Bacteriol. 1987, 169(9): 4379- 4383. (https://doi.Org/10.1128/ib.169.9.4379-4383.1987)
Saeki. 2012: Akinori Saeki, Saya Yoshikawa, Masashi Tsuji, Yoshiko Koizumi, Marina Ide, Chakkooth Vijayakumar†, Shu Seki - A Versatile Approach to Organic Photovoltaics Evaluation Using White Light Pulse and Microwave Conductivity - J. Am. Chem. Soc. 2012 134 (46), pages 19035-19042 (https://doi.org/10.1021/ia309524f)
Sorigue, 2017: Damien Sorigue, Bertrand Legeret, Stephan Cuine, Stephanie Blangy, Solene Moulin, Emmanuelle Billon, Pierre Richaud, Sabine Brugiere, Yohann Coute, Didier Nurizzo, Pavel Miiller, Klaus Brettel, David Pignol, Pascal Amoux, Yonghua Li-Beisson, Gilles Peltier, Fred Beisson - An algal photoenzyme converts fatty acids to hydrocarbons. Science, 2017,
357 (6354), pp. 903-907. (https://doi.org/10.1126/science.aan6349)
DEFINITIONS
As used herein the term “substantially”, such as in “substantially consists”, will be understood by the person skilled in the art. The term “substantially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term “substantially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. The term “comprise” includes also embodiments wherein the term “comprises” means “consists of’. The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".
As used herein, the term “enantiomerically pure” means that the enantiomeric excess (ee) of the enantiomeric mixture is higher than 99%.
Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The devices herein are amongst others described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation.
It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
The invention further applies to a device comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterising features described in the description and/or shown in the attached drawings. The various aspects discussed in this patent can be combined in order to provide additional advantages. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:
1. Method for the preparation of a secondary fatty alcohol from an unsaturated fatty acid comprising:
(a) a hydroxyl introduction stage comprising hydration and/or hydroxylation of the unsaturated fatty acid with a first enzyme to provide a fatty hydroxy acid;
(b) a decarboxylation stage comprising decarboxylation of the fatty hydroxy acid with a photoactivated decarboxylase to form a secondary fatty alcohol.
2. Method according to claim 1, wherein the hydroxyl introduction stage comprises enzymatic hydration of a double bond.
3. Method according to claim 2, wherein an unsaturated fatty acid of the general formula [1] is enzymatically converted into a fatty hydroxy acid of formula [2] and the fatty hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [3] wherein Rl and R2 independently represent an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
4. Method according to claim 2 or 3, wherein the enzymatic hydration is carried out by an enzyme selected from the group consisting of a hydratase enzyme from the E.C. class 4.2.1 and a lipoxygenase of the E.C. class 1.13.11.
5. Method according to any of the claims 2 to 4, wherein the enzymatic hydration is carried out by an oleate hydratase.
6. Method according to claim 5, wherein the oleate hydratase is selected from an oleate hydratase derived from Elizabethkingia meningoseptica (IkvO ) and an oleate hydratase derived from a Lactobacillus species (L/ΌH).
7. Method according to any one of the preceding claims, wherein the hydroxyl introduction stage comprises hydroxylation, and wherein the first enzyme comprises a fatty acid hydroxylase.
8. Method according to claim 7, wherein the fatty acid hydroxylase comprises a 5,8-diol synthase.
9. Method according to claim 8, wherein a fatty acid according to formula (4) is converted by the 5,8-diol synthase into the diol (6) and this fatty di-hydroxy acid is converted by a photoactivated decarboxylase into a secondary fatty alcohol of formula [7] wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
10. Method according to any of the preceding claims wherein the photoactivated decarboxylase is a photoactivated decarboxylase derived from Chlorella variabilis NC64A (CvFAP).
11. Method according to any of the preceding claims wherein the two conversions are carried out in a one-pot two-step procedure, by first performing the hydroxyl-introducing reaction under dark condition followed by illumination to promote the decarboxylation reaction.
12. Method according to any of the preceding claims, wherein the reactions are carried out in an emulsion of an aqueous phase and a water-immiscible organic phase.
13. Method according to claim 12, wherein the reactions are carried out in a water-in-oil emulsion or an oil-in-water emulsion.
14. Method according to any of the preceding claims 12 to 13, wherein the reactions are carried out in an emulsion of water and triolein.
15. Method according to any of the preceding claims wherein the unsaturated fatty acid is obtained from a corresponding amide, thioester or ester.
16. Method according to any of the preceding claims 1-14, wherein the unsaturated fatty acid is obtained from a corresponding mono-, di- or triester of glycerol
17. Method according to any of the preceding claims, wherein the reactions are carried out at a pH in the range between 3 and 12 more preferably between 4 and 11, even more preferably between 5 and 10, even more preferably between 5 and 9.
18. Method according to any of the preceding claims, wherein in a continuous flow a reaction medium is conducted sequentially
(a) through a compartment containing immobilised hydratases and/or other immobilised hydroxyl-introducing enzymes and
(b) through a compartment containing an immobilised decarboxylase.
19. Flow-through device for use in the method according to claim 18 comprising a multitude of compartments, arranged in series and fluidly (especially liquidly) coupled, such that a first compartment is in fluid connection with a second compartment, wherein each compartment comprises an inlet and an outlet, and wherein the outlet of a first compartment is connected to the inlet of a second compartment, wherein at least two of the compartments of the device are housing immobilized enzymes, wherein a first compartment is housing immobilized enzymes useful for the hydroxyl introduction stage (such as immobilized hydratating and/or hydroxylating enzymes), and wherein a second compartment is housing immobilized enzymes useful for the decarboxylation stage (such as immobilized photoactivated decarboxylase).
20. Flow-through device according to claim 19 wherein the second compartment is housing an immobilized photoactivated decarboxylase and wherein this compartment further comprises means for illuminating the content of the compartment with a suitable light source emitting light with a wavelength within the range suitable for the photoactivated decarboxylation reaction.
21. A compound obtainable by the method according to any one of preceding claims, wherein the compound is selected from the group consisting of wherein R1 and R2 independently represent an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and wherein R3 represents an optionally branched hydrocarbon group with 0 to 12 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups, and R4 represents an optionally branched hydrocarbon group with 0 to 8 carbon atoms, optionally containing one or more double bonds and optionally containing one or more hydroxyl groups.
22. The compound according to claim 21, wherein the compound is enantiomerically pure.
23. Compound selected from the group consisting of (Z)-heptadec-6-en-9-ol, (7R,9R)- heptadecane-7,9-diol, (R,5Z,1 lZ)-heptadeca-5,l l-dien-9-ol, (3Z,6Z)-heptadeca-3,6-dien-9-ol, (Z)-nonadec-6-en-9-ol and (6Z,12Z)-nonadeca-6,12-dien-9-ol.
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